Method of treating multiple sclerosis

FIELD: medicine.

SUBSTANCE: anti-Cd20 antibodies are administered in amount of 0.5-4.0 g. 16-60 weeks later administration is repeated. To 4 and more antibody effects are performed. Each effect comprises one or two dosages of antibody. Antibody can be introduced intravenously, subcutaneously or subtunically. The second medicine can be used therewith. A product containing the instructions and a container with 0.5-4.0 g of antibody. In addition, the product can contain the container with the second medicine and the application instructions.

EFFECT: invention allows for prolonged period before stated progression of disease, reduced recurrence rate.

38 cl, 6 dwg, 4 ex

 

The technical field to which the invention relates

This invention relates to methods for treating multiple sclerosis (MS) (MS) in a subject with use of special schemes and protocols of doses and to the product instructions with respect to such use.

The technical field

Multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinizing degenerative disease of the Central nervous system (CNS). It is a disease that is common worldwide, which affects approximately 300,000 persons in the United States; it is a disease of young adults, and in 70-80% of patients, it appears between the ages of 20 and 40 years (Anderson et al. Ann Neurology 31(3):333-6 (1992); Noonan et al. Neurology 58:136-8 (2002)). MS is a heterogeneous disorder, based on clinical course, evaluation of image scanning nuclear magnetic resonance (NMR tomography) and analysis of the pathology material biopsy and autopsy (Lucchinetti et al. Ann Neurol 47:707-17 (2000)). This disease manifestoes in a large number of possible combinations of disorders, including syndromes of the spinal cord, brain stem, cranial nerve, cerebellar, cerebral and cognitive syndromes. Progressive loss of earning capacity is the fate of most clients is impressive with MS, in particular, a 25-year perspective. Half of the patients with MS need to stick to walking within 15 years from the onset of the disease. MS is a major cause of neurological disability in young adults and adults of middle age and up to the last decade had not known of favorable treatment. MS is difficult to diagnose due to non-specific clinical indicators that led to the development of a highly structured diagnostic criteria, which include several technological advances, consisting of scanning NMR diagnosis caused (induced) potentials and studies of cerebrospinal fluid (CSF). All diagnostic criteria are based on the General principles of scattered damage in the white matter of the brain, occurring at different times and not explained by other etiology such as infection, vascular disorders or autoimmune disorder (McDonald et al. Ann Neurol 50:121-7 (2001)). MS has four picture of the disease: relapsing-remitting MS (RRMS; 80-85% of cases), primary progressive MS (PPMS; 10-15% at origination), progressive relapsing MS (PRMS; 5% at origination) and secondary progressive MS (SPMS) (Kremenchutzky et al. Brain 122 (Pt 10):1941-50 (1999); Confavreux et al. N Engl J Med 343(20):1430-8 (2000)). On close assessment 50% patsie the tov with RRMS within 10 years will develop SPMS and in up to 90% of patients with RRMS will eventually develop SPMS (Weinshenker et al. Brain 112(Pt 1):133-46 (1989)).

There are currently six drugs in the four classes are approved in the United States for the treatment of RRMS, while no drugs were not approved for PPMS. Methods of treatment of RRMS include the following: class of interferons, IFN-beta-1A (REBIF® and AVONEX®) and IFN-beta-1b (BETASERON®), glatiramer acetate (COPAXONE®), the polypeptide; natalizumab (TYSABRI®) and mitoxantrone (NOVANTRONE®), a cytotoxic agent. Other drugs used with varying degrees of success, including corticosteroids, methotrexate, cyclophosphamide, azathioprine and intravenous (IV) immunoglobulin. The beneficial effects approved in present methods of treatment are relatively modest (~30%) for the rate of relapse and prevention of disability in RRMS, as suggested by the results of two recent meta-analyses (Filippini et al. Lancet 361:545-52 (2003)).

In other clinical studies have evaluated other immunomodulatory drug for treatment in MS, including inhibitors of tumor necrosis factor α and altered peptide ligands that rather worsened than improved for MS (Lenercept Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Neurology 53:457-65 (1999); Bielekova et al. Nat Med 2000;6:1167-75 (2000), typographical errors are listed in the Nat Med 6:1412 (2000)).

Dominated the prevailing point of view on the pathophysiology of MS, what is spalanie is mediated mainly by CD4 +Th1 T-cells. Therapeutic approaches based on this theory, such as the use of IFN-beta and latinamericana, reduce, but not completely prevent the manifestation of exacerbations or the accumulation of disability.

The existence of a humoral component in MS person unconditionally recognized for decades, as demonstrated by the inclusion of oligoclonal bands CSF and increased vnutriobolochechnoe synthesis of IgG in the diagnostic criteria for MS (Siden A. J Neurol 221:39-51 (1979); McDonald et al., Ann Neurol 50:121-7 (2001); Andersson et al., Eur J Neurol 9:243-51 (2002); O'connor, P. Neurology 59:S1-33 (2002)). The presence of oligoclonal bands, increased free light chains and increased vnutriobolochechnoe synthesis of IgA correlates with the disease activity of MS and may be a prognostic factor for more severe outcome (Rudick et al. Mult Scler 1:150-5 (1995); Zeman et al. Acta Cytol 45:51-9 (2001); Izquierdo et al. Acta Neurol Scand 105:158-63 (2002); J. Wolinsky J Neurol Sci 206:145-52 (2003); Villar et al. Ann Neurol 52:222-6 (2002)).

Antimelanoma antibodies against myelin basic protein (MBP) and myelin oligodendrotsitarnym glycoprotein (MOG)) were detected in the serum of patients with progressive and recurrent forms of MS (Reindl et al. Brain 122:2047-56 (1999); Egg et al. Mult Scler 7(5): 285-9 (2001)). Antimelanoma antibodies were also detected in the CSF of patients with MS (Reindl et al. Brain 122:2047-56 (1999); Egg et al. Mult Scler 7(5): 285-9 (2001); Andersson et al., Eur J Neurol 9:243-51 (2002)). the more types of antibodies, such as antiganglioside antibodies or antimarijuana antibodies, was observed in patients with MS (Mata et al. Mult Scler 5:379-88 (1999); Sadatipour et al. Ann Neurol 44:980-3 (1998)). A recent report has indicated that the presence of serum anti-MOG and anti-MBP antibodies were strong prognostic factor for progression of clinically isolated demyelinating event in a certain RRMS (Berger et al. N Engl J Med 349:139-45 (2003)). Corrected a risk factor in obtaining exacerbation was equal 76,5 for patients who were seropositive for both antibodies, and 31.6 for patients who were seronegative only in respect of anti-MOG antibodies.

At the International consortium for pathology determined that antibodies associated with myelin, are present in most patients with MS, with plasma cells and b-cells, also found in the lesions of MS, which provided additional evidence concerning the humoral role in MS (Prineas and Wright, Lab Invest 38:409-21 (1978); M. Esiri Neuropathol Appl Neurobiol 6:9-21 (1980); Genain et al. Nat Med 5:170-5 (1999); Lucchinetti et al. Ann Neurol 47:707-17 (2000); Wingerchuk et al. Lab Invest 81:263-81 (2001)). B-cells are detectivesyme in the cerebrospinal fluid (CSF) of patients with MS, and the presence of relatively high proportions of b cells may be an indicator of a more severe progression of disability (Cepok et al. Brain 124(Pt 11):2169-76 (2001)).

Rituxin is b, as reported, exhausted peripheral b cells from all subjects with RRMS or syndrome opsoclonus-myoclonus and reduced the number of b-cells in the CSF in some patients (Pranzatelli et al. Neurology 60(Suppl1) PO5.128:A395 (2003); Cross et al. “Preliminary Results from a Phase II Trial of Rituximab in MS” (abstract) Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis ACTRIMS 20-1 (October, 2004)). Cm. also Cree et al. “Tolerability and Effects of Rituximab Anti-CD20 Antibody in Neuromyelitis Optica and Rapidly Worsening Multiple Sclerosis” Meeting of the Am. Acad. Neurol. (April, 2004).

CD20 antibodies and therapy with their application

Lymphocytes are one of many types of cells produced by the bone marrow during the process of hemopoiesis. There are two major populations of lymphocytes: b-lymphocytes (b-cells) and T lymphocytes (T cells). Lymphocytes, which are of particular interest are b cells.

B cells Mature in the bone marrow and leave the bone marrow, expressive antigennegative antibody on their cell surface. When the "untrained" In-cell first encounters with the antigen against which it is a specific membrane-bound antibody, the cell begins to divide, and its progeny differentiate Into cells of immunological memory and effector cells, called plasma cells (plasmacytoma). In-memory cells have a long life and continue to Express memb novasone antibody with the same specificity, as the original parent cell. Plasma cells do not produce membrane-bound antibody, but instead produce antibody in a form that can be secreted. Secreted antibodies are the main effector molecule of the humoral immune system.

The CD20 antigen (also called conservative differentiation antigen of human lymphocytes, VR) 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):11282-11287 (1989); and Einfeld et al. EMBO J. 7(3):711-717 (1988)). This antigen is also expressed on more than 90% of b-cell non Hodgkin's lymphoma (NHL) (Anderson et al. Blood 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 stage (early stage) in the activation process for initiation of the cell cycle and differentiation (Tedder et al. see above) and possibly functions as a channel of calcium ions (Tedder et al. J. Cell. Biochem. 14D:195 (1990)).

When the condition of expression of CD20 in b-cell lymphoma this antigen may serve as a candidate for "targeting" of such lymphomas. Essentially, such targeting may be in General form as follows: antibodies specific to assigned and the surface antigen CD20 b-cells, enter the patient. These anti-CD20 antibodies specifically bind to the antigen CD20 (obviously) both normal and malignant b cells; this antibody is associated with the surface antigen CD20, can lead to the destruction and depletion of neoplastic b-cells. In addition, chemical agents or radioactive labels that have the potential destruction of the tumor, can be conjugated with anti-CD20-antibody, so that this agent specifically “delivered” to the neoplastic b-cells. Regardless of this approach, the primary goal is the destruction of the tumor; the specific approach may be determined by the specific anti-CD20-antibody, and therefore, the available approaches to targeting the CD20 antigen can vary considerably.

Antibody Rituximab (RITUXAN®) is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen. Rituximab is an antibody called “C2B8” in U.S. Patent No. 5736137, issued April 7, 1998 (Anderson et al.). RITUXAN® is indicated for the treatment of patients with relapsed or refractory nizkozameshhennoj or follicular, CD20-positive, b-cell non-Hodgkin's lymphoma. Studies of the mechanism of action in vitro showed that RITUXAN® binds the human complement and analyzes lymphoid b-cell lines through to plement-dependent cytotoxicity (CDC) (Reff et al. Blood 83(2):435-445 (1994)). Additionally, he has significant activity in assays for antibody-dependent cellular cytotoxicity (ADCC). More recently, it was shown that RITUXAN® has antiproliferative action analyses include thymidine treated with tritium, and directly induces apoptosis, whereas other anti-CD19 and CD20 antibodies do not directly induce apoptosis (Maloney et al. Blood 88(10):637a (1996)). Synergies between RITUXAN® and chemotherapy and toxin also observed experimentally. In particular, RITUXAN® sencibilisiruet drug resistant cell line b-cell lymphoma rights to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and ricin (Demidem et al. Cancer Chemotherapy & Radiopharmaceutical 12(3):177-186 (1997)). Preclinical in vivo studies showed that RITUXAN® depletes b cells from the peripheral blood, lymph nodes and bone marrow abacadabra monkeys, presumably through complement - and cell-mediated processes (Reff et al. Blood 83(2):435-445 (1994)).

Rituximab was approved in the United States in November 1997 for the treatment of patients with relapsed or refractory nizkozameshhennoj or follicular CD20+B-cell non-Hodgkin's lymphoma (NHL) at a dose of 375 mg/m2once a week in the form of four doses. In April 2001 the Department for quality control of food products is tov, drugs and cosmetics (FDA) has approved additional claims in relation to the treatment nizkozameshhennoj NHL: re-treatment (once a week in the form of four doses) and an additional scheme of doses (once a week in the form of eight doses). More than 300,000 patients received Rituximab either as monotherapy or in combination with immunosuppressant or chemotherapeutic drugs. Patients were also treated with Rituximab as maintenance therapy for up to 2 years (Hainsworth et al. J Clin Oncol 21:1746-51 (2003); Hainsworth et al. J Clin Oncol 20:4261-7 (2002)).

Rituximab has been studied in various non-malignant autoimmune disorders, in which b cells and autoantibodies seems to play a role in the pathophysiology of the disease (Edwards et al. Biochem Soc Trans 30:824-8 (2002)). It was reported that Rituximab potentially weakens the signs and symptoms of rheumatoid arthritis (RA) (Leandro et al. Ann Rheum Dis. 61:883-8 (2002); Emery et al. Arthritis Rheum 48(9):S439 (2003)), lupus erythematosus (Eisenberg R. Arthritis Res Ther 5:157-9 (2003); Leandro et al. Arthritis Rheum 46:2673-7 (2002)), immune thrombocytopenia (D'arena et al. Leuk Lymphoma 44:561-2 (2003)), autoimmune anemia (Zaja et al., Haematologica 87:189-95 (2002) (typographical errors are listed in Haematologica 87:336 (2002)), autoimmune neuropathy (Pestronk et al. J Neurol Neurosurg Psychiatry 74:485-9 (2003)), paraneoplastic syndrome opsoclonus-myoclonus (Pranzatelli et al. Neurology 60(Suppl1) PO5.128:A395 (2003)) and relapsing-remitting R is ssennoga sclerosis (RRMS) (Cross et al. (abstact) Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis 20-1 (2003)).

Study phase II (WA16291) was performed on patients with rheumatoid arthritis (RA) results 48 days subsequent medical (follow-up) observations on the safety and efficacy of Rituximab (Emery et al. Arthritis Rheum 48(9):S439 (2003); Szczepanski et al. Arthritis Rheum 48(9):S121 (2003)). In General, 161 patients were evenly randomized to four branches of treatment: methotrexate, Rituximab; Rituximab plus methotrexate; Rituximab plus cyclophosphamide (CTX). The scheme of treatment with Rituximab was scheme 1 g, administered intravenously on days 1 and 15. Infusion of Rituximab for patients with RA was well tolerated by most patients, and 36% of patients experienced at least one adverse event during their first infusion (compared to 30% of patients receiving placebo). In General, it was believed that the majority of adverse events were mild - moderate in severity and were well balanced across all treatment groups. In General, there were 19 serious adverse events in all four branches of the study for 48 weeks, which were slightly more common in the Rituximab group/CTX. The infections were well balanced across all groups. The average rate of serious infections in this population of patients with RA was of 4.66 per 100 patient-years, which is is lower than the coefficient of infections requiring hospitalization in patients with RA (to 9.57 per 100 patient-years), reported in an epidemiological study based on community (Doran et al. Arthritis Rheum 46:2287-93 (2002)).

Reported safety profile of Rituximab in a small number of patients with neurological disorders, including autoimmune neuropathy (Pestronk et al. J Neurol Neurosurg Psychiatry 74:485-9 (2003)), the syndrome opsoclonus-myoclonus (Pranzatelli et al. Neurology 60(Suppl1) PO5.128:A395 (2003)) and RRMS (Cross et al. Preliminary results from a phase II trial of Rituximab in MS (abstact) Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis 20-1 (2003)), was similar to the profile reported in cancer or in the treatment of RA. In the ongoing sponsored researcher testing (IST) of Rituximab in combination with interferon-beta (IFN-beta) or latinamerica.com in subjects with RRMS (Cross et al., see above) 1 out of 10 treated subjects were admitted to hospital for overnight observation after he experienced a moderate fever and chills after the first infusion of Rituximab, while 9 subjects completed the scheme with four infusions without any reported adverse events.

Patents and publications patents related to antibodies against CD20, include U.S. Patent No. 5776456, 5736137, 5843439, 6399061 and 6682734 and US 2002/0197255, US 2003/0021781, US 2003/0082172, US 2003/0095963, US 2003/0147885 (Anderson et al.); U.S. patent No. 6455043, US 2003/0026804 and WO2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); WO 2000/27433 and US 2004/0213784 (Grillo-Lopez and Leonard); WO 2000/44788 (Braslawsky et al.); WO 2001/10462 (Rastetter, W.); WO 01/10461 (Rastetter and White); WO 2001/10460 (White and Grillo-Lopez); US 2001/0018041, US 2003/0180292, WO 2001/34194 (Hanna and Hariharan); US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); US 2002/0012665 and WO 2001/74388 (Hanna, N.); US 2002/0058029 (Hanna, N.); US 2003/0103971 (Hariharan and Hanna); US 2002/0009444 and WO 2001/80884 (Grillo-Lopez, A.); WO 2001/97858 (White, C.); US 2002/0128488 and WO 2002/34790 (Reff, M.); WO 2002/060955 (Braslawsky et al.); WO 2002/096948 (Braslawsky et al.); WO 2002/079255 (Reff and Davies); US Patent No. 6171586 and WO 1998/56418 (Lam et al.); WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, U.S. Patent No. 6194551, U.S. Patent No. 6242195, U.S. Patent No. 6528624 and U.S. Patent No. 6538124 (Idusogie et al.); WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al.); WO 2001/03734 (Grillo-Lopez et al.);US 2002/0004587 and WO 2001/77342 (Miller and Presta); US 2002/0197256 (Grewal, I.); US 2003/0157108 (Presta, L.); WO 04/056312 (Lowman et al.); US 2004/0202658 and WO 2004/091657 (Benyunes, K.); WO 2005/000351 (Chan, A.); US 2005/0032130A1 (Beresini et al.); US 2005/0053602A1 (Brunetta, P.); U.S. Patent№№ 6565827, 6090365, 6287537, 6015542, 5843398 and 5595721, (Kaminski et al.); U.S. patent No. 5500362, 5677180, 5721108, 6120767 and 6652852 (Robinson et al.); U.S. patent No. 6410391 (Raubitschek et al.); U.S. patent No. 6224866 and WO 00/20864 (Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); US2005/0079174A1 (Barbera-Guillem et al.); WO 2000/67795 (Goldenberg); US 2003/0133930 and WO 2000/74718 (Goldenberg and Hansen); US 2003/0219433 and WO 2003/68821 (Hansen et al); W02004/058298 (Goldenberg and Hansen); WO 2000/76542 (Golayetal.); WO 2001/72333 (Wolin and Rosenblatt); U.S. Patent No. 6368596 (Ghetie et al.); U.S. patent No. 6306393 and US 2002/0041847 (Goldenberg, D.); US 2003/0026801 (Weiner and Hartmann); WO 2002/102312 (Engleman, E.); US 2003/0068664 (Albitar et al.); WO 2003/002607 (Leung, S.); WO 2003/049694, US 2002/0009427 and US 2003/0185796 (Wolin et al.); WO 2003/061694 (Sing and Siegall); US 2003/019818 (Bohen et al.); US 2003/0219433 and WO 2003/068821 (Hansen et al.); US 2003/0219818 (Bohen et al.); US2002/0136719 (Shenoy et al.); WO 2004/032828 (Wahl et al.); and WO 2002/56910 (Hayden-Ledbetter). Cm. the U.S. patents 5849898 and EP 330191 (Seed et al.); EP332865A2 (Meyer and Weiss); U.S. Patent No. 4861579 (Meyer et al.); US2001/0056066 (Bugelski et al.); WO 1995/03770(Br et al.); US 2003/0219433 Al (Hansen et al.); WO 2004/035607 (Teeling et al.); US 2004/0093621 (Shitara et al.); WO 2004/103404 (Watkins et al.); WO 2005/000901 (Tedder et al.); US 2005/0025764 (Watkins et al.); WO 2005/016969 and US 2005/0069545 A1 (Carr et al.); WO 2005/014618 (Chang et al.). Some of these include, inter alia, the treatment of multiple sclerosis.

Publications related to therapy with Rituximab include: Perotta and Abuel "Response of chronic relapsing ITP of 10 years duration to Rituximab" Abstract # 3360 Blood 10(1)(part 1-2): p. V (1998); Stashi et al. "Rituximab chimeric anti-CD20 monoclonal antibody treatment for adults with chronic idopathic thrombocytopenic purpura" Blood 98(4):952-957 (2001); Matthews, R. "Medical Heretics"New Scientist(April 7, 2001); Leandro et al. "Clinical outcome in 22 patients with rheumatoid arthritis treated with 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): 5370 (2001); Leandroet al."An open study of lymphocyte depletion in systemic lupus lupus", Arthritis & Rheumatism 46(1):2673-2677 (2002); Edwards and Cambridge "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete lymphocytes In" Rhematology 40:205-211 (2001); Edwardset 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): S197 (2002); Levine and Pestronk "IgM antibody-related polyneuropathies: B-ell depletion chemotherapy using Rituximab" Neurology52: 1701-1704 (1999); DeVitaet al."Efficacy of selective cell blockade in the treatment of rheumatoid arthritis" Arthritis &Rheum 46:2029-2033 (2002); Hidashida et al. "Treatment of DMARDs-Refractory rheumatoid arthritis with Rituximab." Presented at the Annual Scientific Meeting of the American College of Rheumatology; Oct 24-29; New 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; Speckset al."Response of Wegener''s granulomatosis to anti-CD20 chimeric monoclonal antibody therapy" Arthritis & Rheumatism 44(12):2836-2840 (2001); Anoliketal., "B lympocyte Depletion in the Treatment of Systemic Lupus (SLE): a Phase I/II Trial of Rituximab (RITUXAN®) in SLE" Arthritis And Rheumatism, 46(9), S289-S289 Abstract 717 (October, 2002), and Albertet al.,"A Phase I Trial of Rituximab (Anti-CD20) for Treatment of Systemic Lupus Lupus" Arthritis And Rheumatism, 48(12): 3659-3659, Abstract LB9 (December, 2003); Martin and Chan Pathogenic Roles of cells in Human Autoimmunity: Insights from the Clinic Immunity 20:517-527 (2004).

Disclosure of invention

The invention includes, at least partially, the choice of doses for anti-CD20 antibodies, which provides a safe and active treatment of subjects with multiple sclerosis, such as PPMS or RRMS.

Thus, this invention relates to a method of treating multiple sclerosis in a subject, involving the administration of an effective amount of the CD20 antibody to the subject to provide an initial exposure to the antibodies in the amount of about 0.5 to 4 grams followed by a second exposure to antibodies approximately 0.5-4 g, and this is the second effect of antibodies does not hold up to approximately 16-60 weeks after the initial exposure to antibodies, and each of impacts antibodies teach the subject as one or two doses of the antibody.

In addition, this invention relates to a product containing (a) a container containing CD20-antibody; and (b) a package insert with instructions regarding the treatment of multiple sclerosis in the subject, where these instructions indicate that the subject enter the amount of antibody that is effective for carrying out the initial effects of the antibodies in the amount of about 0.5 to 4 grams followed by a second exposure to the antibodies in the amount of approximately 0.5-4 g, and this is the second effect of the antibody does not hold up to approximately 16-60 weeks after the initial exposure to antibodies, and each of impacts antibodies teach the subject in as one or two doses of the antibody.

Brief description of drawings

Figa denotes the mapping of sequences, comparing the amino acid sequence of the variable domain of the light chain (VLeach mouse N (SEQ ID NO:1), gumanitarnogo option 27.v16 (SEQ ID NO:2) and subgroups of I Kappa light-chain man (SEQ ID NO:3). CDR VLN and hu27.v16 are as follows: CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:5) and CDR3 (SEQ ID NO:6).

Figv denotes the mapping of sequences, comparing the amino acid sequence of the variable domain of the heavy chain (VNeach mouse is about N (SEQ ID NO:7), gumanitarnogo option 27.v16 (SEQ ID NO:8) and a consensus sequence of a human subgroup III heavy chain (SEQ ID NO:9). CDR VNN and hu27.v16 are as follows: CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:11) and CDR3 (SEQ ID NO:12).

On figa and figv CDR1, CDR2 and CDR3 in each circuit are enclosed in brackets, frame flanked by regions FR1-FR4, as indicated. N refers to the murine antibody N. Asterisks between two rows of sequences indicate provisions that differ between the two sequences. The numbering of residues corresponds to Kabat et al. Sequences of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), indels, shown as a, b, c, d and E.

Figure 2 shows the amino acid sequence of the light chain Mature 27.v16 (SEQ ID NO:13).

Figure 3 shows the amino acid sequence of the heavy chain Mature 27.v16 (SEQ ID NO:14).

Figure 4 shows the amino acid sequence of the heavy chain Mature 27.v31 (SEQ ID NO:15). L-chain 27.v31 is the same as for 27.v16.

Figure 5 shows the comparison of the light chain Mature 27.v16 and 27.v511 (SEQ ID NO:13 and 16 respectively) with the numbering of Kabat residues of the variable domain and the Eu numbering of residues of the constant domain.

Fig.6 shows the comparison of the heavy chain Mature 27.v16 and 27.v511 (SEQ ID NO:14 and 17, respectively) with the numbering of Kabat residues of the variable domain and the Eu numbering mod is s constant domain.

A detailed description of the preferred embodiments

I. Definitions

B-cell is a lymphocyte that Matures in the bone marrow and includes untrained In-cell In-cell memory or effector B-cell (plasma cell, plasmatic). Here In the cell can be a normal or non-malignant B-cell.

Marker surface of b-cells" or "surface antigen In cells" denotes in this context, the antigen expressed on the surface of b cells that can be targeted by antibodies, which binds with him. Examples of surface markers of b-cells include surface markers of cells CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CDw79, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 (for descriptions, see The Leukocyte Antigen Facts Book, 2ndEdition, 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other surface markers of b-cells include RP105, FcRH2, CR2 b cells, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, at btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA and 239287. The marker At the cell surface, representing a special interest, mainly expressed on b cells compared to other non-b-cell tissues of a mammal and may be expressed as the precursors of b cells and Mature b cells. Preferred surface marker for b-cells is in the estuaries is preferably ü CD20.

The antigen CD20", or "CD20", is deglycosylation postbellum 35 kDa detected on the surface of more than 90% of b cells from peripheral blood or lymphoid organs. CD20 is present on normal b cells and malignant b cells, but not expressed on stem cells. Other names CD20 in the literature include “conservative antigen In lymphocytes and VR”. The CD20 antigen is described, for example, Clark et al. Proc. Natl. Acad. Sci. (USA) 82:1766 (1985).

“Antibody antagonist” refers here antibody, which, after binding to a marker of the surface of b cells to b cells, destroys or depletes b cells in a mammal and/or inhibits one or more functions In cells, for example, reducing or preventing a humoral response induced In the cell. Antibody antagonist preferably is able to Deplete b cells (i.e. to reduce circulating levels of b-cells in the mammal treated with this antibody. This depletion can be achieved through various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), inhibition of proliferation of b-cells and/or induction of death In cells (e.g., via apoptosis).

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" relative to the tsya to cell-mediated reactions, in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., natural killer cells (PC-cells), neutrophils, and macrophages) recognize bound antibody on the target cell and then cause this lysis of target cells. The primary cells for mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 Raveth and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of interest molecules can be analyzed ADCC in vitro, such as described in U.S. Patent No. 5500362 or 5821337. Applicable to such analyses of effector cells include mononuclear cells of peripheral blood (PBMC) and natural killer cells (PC-cells). Alternative or along with this, the ADCC activity of interest molecules can be assessed in vivo, e.g., in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998).

"Effector cells" are leukocytes which Express one or more FcR and perform effector functions. Preferably, these cells Express at least FcγRIII and perform ADCC-effector function. Examples of human leukocytes which mediate ADCC include mononuclear cells of peripheral blood (PBMC), the nature of the major killer cells (PC-cells), monocytes, cytotoxic T cells and neutrophils; and are preferred PBMC and PC cells.

The terms “Fc receptor” or “FcR” is used to describe a receptor that binds to the Fc region of antibodies. Preferred FcR is a native sequence FcR person. Moreover, a preferred FcR is associated with IgG-antibody (a gamma receptor) and includes subclasses of FcγRI, FcγRII and FcγRIII, including allelic variants and alternative splanirowannya forms of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains the motif of the activation immunoreceptor-based tyrosine (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRII contains the motif inhibition of immunoreceptor-based tyrosine (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR are reviewed in Ravetch and Kinet, Ann. Rev. Immunol. 9:457-921 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). FcR, including those that must be identified in the future, included here under the term "FcR". This term also includes the receptor newborn, FcRn, which is responsible the figures for the transfer of maternal IgG to the fetus (Guyer et al. J. Immunol. 117:587 (1976) and Kim et al. J. Immunol. 24:249 (1994)).

"Complement-dependent cytotoxicity" or "CDC" refers to the ability of the molecule to lyse target in the presence of complement. Path activation of the complement begins with the binding of the first component of the complement system 1q with a molecule (e.g. antibody)in complex with cognate antigen. To assess activation of complement can be performed CDC analysis, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996).

"Inhibiting growth of antibodies" are antibodies that prevent or reduce proliferation of cells expressing the antigen is associated with this antibody. For example, the antibody can prevent or reduce proliferation of b-cells in vitro and/or in vivo.

Antibodies which induce apoptosis, are antibodies that induce programmed cell death, such as b cells, as determined by standard assays of apoptosis, such as binding of annexin V, fragmentation of DNA, shrinkage of cells, the expansion of the endoplasmic reticulum, the fragmentation of the cells and/or formation of membrane vesicles (called apoptotic bodies).

The term “antibody” is used in this context in the broadest sense of the word, and it specifically includes monoclonal antibodies, polyclonal antibodies, m is sitespecifically antibodies (for example, bespecifically antibodies)formed from at least two intact antibodies, and antibody fragments, as they exhibit the desired biological activity.

"Antibody fragments" include a portion of an intact antibody, preferably containing region of binding antigen. Examples of fragments of antibodies include Fab fragments, Fab', F(ab')2and Fv; dyatel; linear antibodies; single-chain molecule antibodies and multispecific antibodies formed from fragments of antibodies.

For the purposes of this invention “intact antibody” refers to an antibody containing the variable domains of the heavy and light chains, as well as Fc-region.

“Native antibodies” are usually heterotetrameric glycoproteins of approximately 150000 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 isotypes of immunoglobulins. Each heavy and light chain also has disulfide bridges within the chain at regular distances from each other. 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 con is e (V L) and a constant domain at its other end; the constant domain of the light chain is associated with the first constant domain of the heavy chain and the variable domain of the light chain is associated with the variable domain of the heavy chain. It is believed that specific amino acid residues form a boundary surface between the variable domains of the light chain and heavy chain.

The term “variable” refers to the fact that some parts of the variable domains largely differ in their sequence among antibodies and are used in the binding and specificity of each particular antibody against the specific antigen. However, this variability is not evenly distributed on the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in the variable domains, both light chain and heavy chain. More highly conserved part of the variable domains are called the frame regions (FR). The variable domains of native heavy and light chains each contain four FR, receiving substantially the configuration of the β-folds, connected by three hypervariable regions, which form loops connecting this β-structure, and in some cases forming part of this β-structure. Hypervariable regions in each chain are held together with top quality construction is in close proximity to these FR and with the hypervariable regions from the other chain, contribute to the formation of antigennegative site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The papain cleavage of antibodies produces two identical antigenspecific fragment, called "Fab"fragments, each of which has a single antigennegative website, and the rest of the "Fc"fragment, whose name reflects its ability to easily crystallize. Treatment with pepsin yields F(ab')2-the fragment that has two antigenspecific site and still able to cross-link the antigen.

"Fv" is the minimum antibody fragment which contains a complete antigen-recognizing and antigennegative website. This region consists of a dimer variable domain of one heavy chain and one light chain in a tight, non-covalent linkages. In this configuration three hypervariable region of each variable domain interact to define antigennegative site on the surface of the VH-VL-dimer. Together, these six hypervariable regions reported antigennegative the specificity of the antibody. One is about even a single variable domain (or half of an Fv, containing only three hypervariable region, specific against the antigen are able to recognize and bind antigen, although at a lower affinity than the entire binding site.

Fab-fragment also contains the constant domain of the light chain and the first constant domain (SN) the heavy chain. Fab'fragments differ from Fab fragments by the addition of a few residues to the carboxyl end of the heavy chain CN domain, including one or more cysteine residues of the hinge region of the antibody. Fab'-SH is the designation of Fab'in which the residue (remainder) of cysteine constant domains have at least one free thiol group. Fragments of antibodies F(ab')2were originally obtained as pairs of Fab'-fragments, which have between them cysteine hinge region. There are also other chemical combinations of fragments of antibodies.

The "light chains" of antibodies (immunoglobulins) from any vertebrate can be attributed to one of two clearly distinct types, called Kappa (κ) and lambda (λ), 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 and IgM, and several of them can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and Da. The constant domains of the heavy chain, which correspond to these different classes of antibodies are called α, δ, ε, γ and µ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Single-chain Fv" or "scFv"-fragments of the antibodies contain VHand VLdomains of antibodies, where these domains are present in a single polypeptide chain. Preferably this an Fv polypeptide further comprises a polypeptide linker between domains VHand VLthat enables the scFv to form the desired structure for antigen binding. In the review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diately" refers to small fragments of antibodies with two antihistamine sites, and these fragments contain the variable domain of the heavy chain (VH)connected to the variable domain light chain (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 complementary domains of another chain and create two antigenspecific site. Diately described more fully, for example, in EP 404097; WO 93/11161 and Holinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term "monoclonal antibody" refers in this context to an antibody obtained from a population essentially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may occur while obtaining monoclonal antibodies, and these options are usually present in minor amounts. In contrast to the preparations of polyclonal antibodies, 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, monoclonal antibodies are valuable due to the fact that they are not contaminated by other immunoglobulins. The definition of “monoclonal” indicates the character of the antibody as being obtained from an essentially homogeneous population of antibodies, and should not be understood as requiring receipt of this antibody by any particular method. For example, monoclonal antibodies for use in accordance with this invention can be obtained hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be obtained by means of recombinant DNA (see, for example, the Patent is SHA No. 4816567). "Monoclonal antibodies" may also be isolated from phage libraries of antibodies using methods described, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Biol. Chem., 222:581-597 (1991).

Monoclonal antibodies specifically described here include "chimeric" antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with the corresponding sequences or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, whereas the rest of the chain (chain) is identical with the corresponding sequences or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, as they exhibit the desired biological activity (U.S. Patent No. 4816567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Representing interest here antibody include "primaryservername" antibodies containing antigennegative sequence of the variable domain obtained from a Primate, non-human (e.g., the lower narrow-nosed monkeys, such as baboon, macaque-rhesus or abacadabra monkey), and sequence of the constant region of human (N is an awning U.S. No. 5693780).

"Humanitarianism" forms of antibodies, non-human (e.g. murine)are chimeric antibodies that contain minimal sequence derived from an immunoglobulin that is not owned by the person. For the most part humanitarianism 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 species other than human (donor antibody)such as mouse, rat, rabbit, or Primate, non-human, having the desired specificity, affinity and efficacy. In some cases, remnants of the framework region (FR) of a human immunoglobulin replace the corresponding residues of the frame region of immunoglobulin that does not belong to man. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications produced to further improve the efficacy of the antibodies. Usually humanitariannet antibody will contain essentially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of immunoglobulin that does not belong to man, and all or su is estu all of these FR is FR sequence of human immunoglobulin, except for the replacement (replacement) FR, as noted above. Humanitariannet antibody is also not necessary to include at least a portion of constant region of immunoglobulin, usually part of a constant region of human immunoglobulin. For further details, see Jones et al., 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" refers in this context to amino acid residues of an antibody which are responsible for binding to the antigen. Hypervariable region contains amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and/or residues from a “hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Frame" the remains or the remains of the "FR" are remnants of the variable domain, other than the remains of the hypervariable region defined here.

"Unbound antibody" is an antibody (defined here), which is not conjugated with hetero is ulichney molecule, such as the cytotoxic portion of the molecule, or a radioactive label.

Examples of antibodies which bind the CD20 antigen include: "SV", which is now called "Rituximab" ("RITUXAN®") (U.S. Patent No. 5736137); the yttrium-[90]-labeled mouse antibody V designated "Y2B8" or "Ibritumomab Tiuxetan" (ZEVALIN®), commercially available from IDEC Pharmaceuticals, Inc. (U.S. patent No. 5736137; W deposited ATSS under access number NV June 22, 1993); murine IgG2 "V1", also called "Tositumomab", not necessarily labeled131I obtaining antibodies131I-B1" or "iodine I131 tositumomab" (BEXXAR™), commercially available from Corixa (see also U.S. Patent No. 5595721); murine monoclonal antibody "1F5" (Press et al., Blood 69(2):584-591 (1987) and its variants, including "having "foci" framework region" or humanitarianly 1F5 (WO03/002607, Leung, S.; ATCC Deposit HB-96450); murine antibody N and chimeric antibody N (U.S. Patent No. 5677180); humanitariannet antibody N; huMax-CD20 (Genmab, Denmark, WO2004/035607); AME-133 (Applied Molecular Evolution); A20 antibody or variants, such as chimeric or humanitariannet antibody A20 (sa, hA20, respectively) or IMMU-106 (U.S. Patent 2003/0219433, Immunomedics); CD20-binding antibodies, including epitope-depleted Leu-16, 1H4 or V, optionally conjugated with IL-2, described in U.S. Patent 2005/A and WO 2005/16969 (Carr et al.); bespecifically antibody that binds to CD22 is CD20, for example hLL2xhA20 (WO2005/14618, Chang et al.); monoclonal antibodies L27, G28-2, 93-B, B-C1 or NU-B2 from the International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)); 1H4 (Haisma et al. Blood 92:184 (1998)); conjugate anti-CD20-auristatin E. (Seattle Genetics); anti-CD20-IL2 (EMD/Biovation/City of Hope); anti-CD20-MAb-therapeutic (EpiCyte) and anti-CD20-antibody TRU 015 (Trubion).

The terms "Rituximab" or "RITUXAN®" refers in this context to the genetically engineered chimeric mouse/human monoclonal antibody directed against the CD20 antigen and named "SV" in U.S. Patent No. 5736137, including its fragments that retain the ability to bind CD20. Rituximab commercially available from Genentech.

Only for the purposes of this invention and unless otherwise indicated, the term "humanitarianly N" refers to humanitarianlaw the antibody that binds CD20 person, or his antigennegative fragment, where the antibody is effective in depleting b-cells of primates in vivo, and this antibody contains a variable near its N-chain (VHat least the sequence of the CDR-H3 of SEQ ID NO:12 (Fig. 1B) of antibodies against CD20 human and essentially consensus remains framework region (FR) of the heavy chain subgroup III person (VHIII). In the preferred embodiment, this antibody further comprises a sequence of CDR H1 H-chain SEQ ID NO:10, the sequence of the CDR-H2 of SEQ ID NO:11, and more preferably further comprises a CDR-L1 L-chain SEQ ID NO:4, the sequence of the CDR-L2 of SEQ ID NO:5, a sequence of CDR L3 SEQ ID NO:6 and is essentially a consensus residues framework region (FR) light chain subgroup I human (VI), where VHthe area may be attached to the constant region of the chain of human IgG, where the area may be, for example, IgG1 or IgG3. In the preferred embodiment, this antibody contains a sequence of VHSEQ ID NO:8 (v16, as shown in Fig. 1B), optionally also contains a sequence VLSEQ ID NO:2 (v16, as shown in figa), which may have amino acid substitutions D56A and N100A in the H chain and S92A in L-chain (v96). Preferably, this antibody is an intact antibody containing the amino acid sequence of the light and heavy chains of SEQ ID NO:13 and 14 respectively, as shown in figure 2 and 3. In another preferred embodiment, this antibody is the antibody 27.v31 containing the amino acid sequence of the light and heavy chains of SEQ ID NO:13 and 15 respectively, as shown in figure 2 and 4. This antibody may further comprise at least one amino acid substitution in the Fc region that improves ADCC and/or CDC activity, such as an antibody, in which these amino acid substitutions are S298A/E333A/K334A, more preferably 27.v31 having the amino acid sequence of the heavy chain SEQ ID NO:15 (shown in figure 4). is uboe of these antibodies may further comprise at least one amino acid substitution in the Fc region, which reduces the CDC activity, for example, containing at least the replacement CA. Cm. U.S. patent No. V (Idusogie et al.).

Preferred humanized N is intact antibody or antibody fragment that contains a variable sequence of the light chain:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO:2)

and variable sequence of the heavy chain:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID NO:8).

If humanitariannet antibody N is intact antibody, preferably it contains the amino acid sequence of light chain:

and amino acid sequence of the heavy chain:

or amino acid sequence of the heavy chain:

In a preferred embodiment of the present invention variable region variants based on N version 16 will have the amino acid sequence v16, except for the sequences in positions of amino acid substitutions, which are shown in the table below. If there are no other indications, options N will have the same light chain, which light chain v16.

Version Nchange light chain (VL)Change Fc
31--S298A, E333A, K334A
96D56A, N100AS92A
114D56A, N10M32L, S92AS298A, E333A, K334A
115D56A, N100AM32L, S92AS298A, E333A, K334A, E356D, M358L

“Isolated” antibody is an antibody that has been identified and separated and/or removed from a component of its natural environment. Contaminant components of its natural environment are substances that could interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments, the implementation of this antibody will be purified (1) to more than 95 wt.% antibodies, as determined by the Lowry method, and most preferably more than 99 wt.%, (2) to the extent d is sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, using sequencing machine with a rotating Cup or (3) to homogeneity by means of electrophoresis in LTO-SDS page under reducing or non conditions using the dye Kumasi blue or, preferably containing silver media. The selected antibody includes the antibody in situ within recombinant cells, since at least one component of the natural environment antibodies will not be present. Typically, however, the selected antibody is obtained using at least one stage of cleaning.

A “subject” referred to in this context of the subject-person. Usually this subject is suitable for treatment about multiple sclerosis. For the purposes of this invention, a suitable subject is a subject who has experienced or is a risk that tests one or more signs, symptoms, or other indicators of multiple sclerosis was diagnosed as having multiple sclerosis, regardless of, for example, whether diagnosed it (with the “new advent” MS), whether diagnosed previously with a new relapse or exacerbation, whether diagnosed earlier and is in remission, etc. and/or has the risk of developing multiple sclerosis. A subject suffering from multiple sclerosis or with the risk of multiple sclerosis, can bilinearization identified as subject, which was subjected to screening for elevated levels of CD20-positive b-cells in serum, cerebrospinal fluid (CSF) and/or damage (damage) of the MS and/or subjected to screening using analysis for detection of autoantibodies, evaluated qualitatively, and preferably quantitatively. Examples of such autoantibodies associated with multiple sclerosis include antibodies against myelin basic protein (MBP)antibodies against myelin oligodendrotsitarnym glycoprotein (MOG)antibodies against ganglioside and/or antibodies against neurofilament. Such autoantibodies can be detected in the serum of the subject, cerebrospinal fluid (CSF) and/or damage caused MILLISECONDS. Under the "high" level (levels) antibodies or b cells in this context mean the level (s) of such antibodies or b cells, which significantly exceed the level (s) of the individual that does not have MS.

"Treatment" of a subject refers here to both therapeutic treatment, prophylactic or preventive measures. Persons in need of treatment include those already with multiple sclerosis, as well as individuals who have multiple sclerosis should be prevented. Thus, the subject can be diagnosed as having multiple sclerosis or can be Pedroso is its or susceptible to multiple sclerosis.

"Symptom" MS refers to any abnormal event or deviation from the normal in structure, function, or sensation, experienced by this subject and which is the indicator of MILLISECONDS.

"Multiple sclerosis" refers to a chronic and often leads to disability, a disease characterized by progressive dysfunction of myelin. There are four internationally recognized forms of MS, primary progressive multiple sclerosis (PPMS), and relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS) and progressive relapsing multiple sclerosis (PRMS).

"Primary progressive multiple sclerosis" or "PPMS" is characterized by the gradual development of this disease from its appearance without overlapping each other relapses and remissions. It may be the smoothing periods of disease activity and it can be good and bad days or weeks. PPMS is different from RRMS and SPMS fact that his appearance usually occurs in the late age of thirty or forty years later, he develops both men and women, and the initial activity of the disease is often in the spinal cord, not the brain. PPMS often migrates to the brain, but is less likely to damage of the brain than RRMS or SPMS; for example, people with PPMS less of veroia the ability to develop cognitive problems. PPMS is a subtype of MS, which is less likely to detect inflammatory (with the increased accumulation of gadolinium) damage on the scanning image nuclear magnetic diagnostics (MRI). This primary progressive form of the disease affects 10-15% of all people with multiple sclerosis. PPMS may be determined in accordance with the criteria described in McDonald et al. Ann Neurol 50:121-7 (2001). Subject with PPMS, being treated in this context, is usually the subject with probable or definite diagnosis of PPMS.

"Relapsing-remitting multiple sclerosis or RRMS is characterized by relapses (also known as exacerbations), during which new symptoms to recur or worsen the old symptoms. For relapses followed by periods of remission, during which the subject is fully or partially recovered from nedostatochnosty acquired during a relapse. Relapses can last for several days, weeks or months, and recovery may be slow and gradual or instantaneous. The vast majority of people manifesting MS, first diagnosed as having RRMS. This occurs when they are at the age of twenty-thirty years, although known diagnoses in much earlier or later age. Twice as many women than men appear who t this MS subtype. During relapses myelin, a protective insulating sheath around nerve fibers (neurons) in the areas of the white matter of the Central nervous system (CNS), may be damaged in the inflammatory response of your own immune system. This causes a large variety of neurological symptoms, which vary depending on which areas of the Central nervous system are damaged. Directly after relapse of inflammatory reaction disappears, and a special type of glial cells in the Central nervous system (called oligodendrocytes) organizes remyelinization - the process by which myelin sheath around the axon can be restored. This remyelinization may be responsible for remission. Approximately 50% of patients with RRMS turn into patients with SPMS within 10 years from the appearance of the disease. After 30 years, this figure reaches 90%. At any point in time relapsing-remitting form of the disease is responsible for approximately 55% of all cases in people with MS.

"Secondary progressive multiple sclerosis" or "SPMS" is characterized by a steady progression of clinical neurological damage from overlapping each other relapses and minor remissions and plateaus or without them. People who develop SPMS, previously experienced periods of RRMS, which length is between two to forty years or more. Any overlapping each other relapses and remissions tend to fade over time. Since the introduction of the secondary progressive phase of the disease disability begins to progress more quickly than it has progressed during RRMS, although this progression can still be slow in some individuals. After 10 years 50% of people with RRMS will have developed SPMS. For 25-30 years, this figure will rise to 90%. SPMS tends to be associated with lower levels of education inflammatory lesions than RRMS, but the overall severity of the disease continues to progress. At any point in time SPMS is responsible for approximately 30% of all people with multiple sclerosis.

"Progressive relapsing multiple sclerosis" or "PRMS" is characterized by a steady progression of clinical neurological damage from overlapping each other by relapses and remissions. There is a substantial recovery directly after relapse, but between relapses there is a gradual worsening of symptoms. PRMS affects approximately 5% of all people with multiple sclerosis. Some neuroscientists believe that PRMS is a kind of PPMS.

The expression "effective amount" refers to the number of antibodies (or other lekarstvennoj the tools) which is effective to prevent, reduce the intensity of symptoms or treatment of multiple sclerosis. Such an effective amount will typically lead to an improvement in the signs, symptoms, or other indicators of MS, such as the reduction in the rate of relapse, prevention of disability, reducing the number and/or volume MRI of brain damage, better timed walks of length 25 feet, prolongation of time to disease progression (for example, using a scale developed Status disability (Expanded Disability Status Scale, EDSS)), etc.

The term "impact antibody" denotes here the contact with the antibody or the effects of the antibodies in one or more doses, administered over a period of time approximately 1-20 days. These doses can be administered once or at fixed or irregular time intervals during this period of exposure of the antibody. Initial and final (for example, second or third) the effect of antibodies is separated in time from one another, as described here.

The term "immunosuppressive agent"as used here for adjuvant therapy refers to substances that act for the suppression or mask the immune system of a mammal, which provide the. This term includes substances which inhibit the production of cytokines that negatively regulate or inhibit the expression of autoantigen or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4665077); non-steroidal anti-inflammatory drug (NSAID); ganciclovir, tacrolimus, glucocorticoids, such as cortisol or aldosterone, anti-inflammatory agents, such as an inhibitor of cyclooxygenase, the inhibitor of 5-lipoxygenase or antagonist of leukotriene receptor; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; parlodel; danazol; Dapsone; glutaric aldehyde (which masks the MHC antigens, as described in U.S. patent No. 4120649); antiidiotypic antibodies for MHC antigens and fragments of MTL; cyclosporin a; steroids such as corticosteroids or glucocorticosteroids or analogues of glucocorticoids, such as prednisone, methylprednisolone, and dexamethasone; inhibitors digidrofolatreduktazy, such as methotrexate (oral or subcutaneous); hydroxychloroquine; sulfasalazin; Leflunomide; cytokine or receptor antagonists of cytokines, including anti-interferon-alpha, -beta, or gamma antibodies, antibodies against tumor necrosis factor (infliximab is whether adalimumab), anti-TNF-alpha immunoadhesin (etanercept), antibodies against tumor necrosis factor beta, anti-IL-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CD11 - and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous antilimfocitarnyi globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4-antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published 7/26/90); streptokinase; TGF-beta; streptodornase; RNA or DNA from the host; FK506; RS-61443; desoxypeganine; rapamycin; T-cell receptor (Cohen et al., U.S. patent No. 5114721); fragments of the T-cell receptor (Offner et al., Science, 251: 430 to 432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989) and WO 91/01133) and antibodies against T-cell receptor (ER 340109), such as TV.

The term "cytotoxic agent" refers in this context to the substance that inhibits or prevents the function of cells and/or causes destruction of cells. This term includes radioactive isotopes (e.g., At211I131I125, Y90That Re186That Re188Sm153Bi212, R32and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

"Chemotherapeutic agent" is Henichesk the e connection applicable in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® (cyclophosphamide; alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, Carbogen, matureup and uredepa; ethylenimines and methylmelamine, including altretamin, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenin (in particular, bullatacin, bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); cryptophycin ( particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and SW-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard analogues, such as chlorambucil, chlornaphazine, halophosphate, estramustine, ifosfamide, mechlorethamine, hydrochloride mechlorethamine, melphalan; novemberin, finestein, prednimustine, trofosfamide, uracil-similar to mustard gas; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, in particular calicheamicin gamma 1I and calicheamicin omega I1 (see, for example, Angew. Chem. Intl. Ed. Engl., 33: 183-18 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; spiramycin; and neocarzinostatin chromophore and related chromophores chromoproteins enediynes antibiotics), alkalinity, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, carcinotron, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN® (including morphosyntactical, cyanomethaemoglobin, 2-pyrrolidinecarbonyl and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycophenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogs, such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiotensin, such as aminoglutaric the ID, mitotane, trilostane; DOPOLNITEL folic acid, such as prolinnova acid; Eagleton; aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; slipline acetate; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; londini; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; podophyllin acid; 2-acylhydrazides; procarbazine; PSK® polysaccharide complex (JHS Natural Produts, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trihlortrietilamin; trichothecenes (in particular, the toxin T-2, verrucarin And, roridin and unguided); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid, for example, paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™, does not contain cremophor, albumin-engineered composition of the nanoparticles of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel TAXOTERE® (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine GEMZAR®; 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mit is Cintron; vincristine; vinorelbine NAVELBINE®; Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine and pharmaceutically acceptable salts, acids or derivatives of any of the above medicines.

This definition also includes antihormonal agents that act regulatory or inhibitory effect on hormone action on tumors such as antiestrogens and selective modulators of estrogen receptors (SERM), including, for example, tamoxifen (including NOLVADEX tamoxifen®), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON; aromatase inhibitors that inhibit the enzyme aromatase, which regulates the production of estrogen in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutetimid, megestrol acetate MEGASE®, exemestane AROMASIN®, formestane, fadrozole, vorozole RIVISOR®, letrozole, FEMARA® and anastrozole ARMIDEX®; and anti-androgens such as flutamide, nilutamide, bikalutamid, leuprolide, goserelin; and troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, particularly oligonucleotides that inhibit expression of genes in the transmission of signals, uchastvuya is in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; vaccines such as vaccines, gene therapy, for example a vaccine ALLOVECTIN®vaccine, LEUVECTIN® vaccine VAXID®; rIL-2 PROLEUKIN®; topoisomerase inhibitor 1 LUPROTECAN®; rmRH ABARELIX® and pharmaceutically acceptable salts, acids or derivatives of any of the above medicines.

The term “cytokine” is a generic term for proteins released by one population of cells, which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini; interleukins (IL)such as IL-1, IL-1α, 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-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). In this context, the term cytokine includes proteins from natural sources or from a culture of recombinant cells and biologically active equivalents of the cytokines with the natural sequence, including synthetically derived low-molecular particles and their pharmaceutically acceptable derivatives and salts.

The term “hormone” refers to a polypeptide hormones that are secreted mainly glandular organs with streams. In the term hormones included, for example, growth hormone such as human growth hormone, N-methionyl-human growth hormone and bovine growth hormone; parathyroid hormone; Tyr is Xin; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyrostimulin hormone (TSH), and luteinizing hormone (LH); prolactin; placental lactogenic, mouse gonadotropin-associated peptide, inhibin; activin; mullerova inhibitory substance and thrombopoetin. In this context, the term hormone includes proteins from natural sources or from a culture of recombinant cells and biologically active equivalents of the hormone with the natural sequence, including synthetically derived low-molecular particles and their pharmaceutically acceptable derivatives and salts.

The term "growth factor" refers to proteins that stimulate the growth and include, for example, the growth factor of the liver; fibroblast growth factor; vascular endothelial growth factor; nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGF)such as TGF-α and TGF-β; insulinopenia growth factors I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and-γ, colony stimulating factors (CSF), such as macrophage CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF). In this context, the term growth factor includes proteins from natural sources or from the culture of R is combinant cells and biologically active equivalents of the hormone with the natural sequence, including synthetically derived low-molecular particles and their pharmaceutically acceptable derivatives and salts.

The term "integrin" refers to a receptor protein that allows cells to contact with the extracellular matrix and to respond to the extracellular matrix and is involved in various cellular functions, such as wound healing, cell differentiation, "homing" tumor cells and apoptosis. They are part of a large family of cell adhesion receptors that are involved in interactions of cell-extracellular matrix and cell-cell. Functional integrins consist of two transmembrane glycoprotein subunits, called alpha and beta, which are ecovalence related. Alpha-subunit, all have some homology with respect to each other, as well as beta-subunit. These receptors always contain one alpha chain and one beta chain. Examples include Alfaretta, Alfaretta, Alfaretta, LFA-1, alpha-4-integrin, etc. In this context, the term integrin includes proteins from natural sources or from a culture of recombinant cells and biologically active equivalents of the hormone with the natural sequence, including synthetically derived low-molecular particles and their pharmaceutically acceptable derivatives and salts.

Examples "and what of taganito or antibodies, integrins include here antibody against LFA-1, such as Efalizumab (RAPTIVA®)commercially available from Genentech; antibody against alpha-4-integrin, such as natalizumab (TYSABRI®)available from Biogen; diabeticheskiy derivatives of phenylalanine (WO 2003/89410); derivatives of phenylalanine (WO 2003/70709, WO 2002/28830, WO 2002/16329 and WO 2003/53926); derivatives of phenylpropionic acid (WO 2003/10135); enamine derivatives (WO 2001/79173); propanoic acid derivatives (WO 2000/37444); derivatives alanovoy acid (WO 2000/32575); substituted phenylpropane (U.S. Patent numbers 6677330 and 6348463); derivatives of aromatic amines (U.S. Patent No. 6369229) and the polypeptide domain disintegrin ADAM (U.S. Patent 2002/0042368), antibodies to integrin alfaretta (EP 633945); United Aza-bridge connection bicyclic derivatives of amino acids (WO 2002/02556) etc.

For the purposes described here, the term "tumor necrosis factor alpha (TNF-alpha)" refers to a molecule TNF-alpha man, containing the amino acid sequence described in Pennica et al., Nature, 312:721 (1984) or Aggarwal et al., JBC, 260:2345 (1985).

"Inhibitor of TNF-alpha" is in this context, an agent which inhibits, to some extent, the biological function of TNF-alpha, usually by binding to TNF-alpha and neutralizing its activity. Examples of TNF inhibitors, specifically rassmatrivaemykh here are Etanercept (ENBREL®), Infliximab (REMICADE®) and Adalimumab (HUMIRA™).

Examples of the modifying C is bolovanje Antirheumatic drugs or DMARDs include hydroxychloroquine, sulfasalazin, methotrexate, Leflunomide, etanercept, infliximab (plus oral and subcutaneous methotrexate, azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, immunoadsorption protein And stafilokok, including their salts and derivatives, etc.

Examples of "non-steroidal anti-inflammatory drugs or NSAIDs are aspirin, ibuprofen, naproxen, indomethacin, sulindac, tolmetin, including their salts and derivatives, etc.

"Corticosteroid" refers to any of several synthetic or naturally occurring substances with the General chemical structure of steroids that mimic or enhance the actions of the naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone, dexamethasone, a glucocorticoid and betamethasone.

"Package insert" is the instructions are usually included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, introduction, contraindications, other therapeutic products that can be combined with the product packaging, and/or warnings concerning the use of such therapeutic products, etc.

II. Therapy

This image is the buy provides a method of treating multiple sclerosis in a subject, suffering from it, introducing an effective amount of an antibody that binds to a surface marker of b-cells (preferably antibodies against CD20), this subject. MS, which should be treated, includes primary progressive multiple sclerosis (PPMS), and relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS) and progressive relapsing multiple sclerosis (PRMS), but the preferred option implementation here is PPMS or RRMS.

According to the preferred Protocol doses of this method requires the introduction of an effective amount of the antibody against CD20 subject with MS to provide initial exposure to antibodies in the amount of about 0.5 to 4 g, preferably 1.5 to 2.5 grams), followed by a second exposure to the antibodies in the amount of about 0.5 to 4 g, preferably 1.5 to 2.5 grams), and this is the second effect of the antibody does not hold up to approximately 16-60 weeks after the initial exposure to antibodies. For the purposes of this invention, the second effect of antibodies is the next time when this subject is administered an antibody against the CD20 after the initial exposure to antibodies, and between the initial and second impacts antibodies no intermediate treatment or sub the full effects of antibodies against CD20.

The interval between the initial and the second or subsequent impacts antibodies can be measured either from the first or second dose of the initial impact of antibodies, but preferably it is measured from the first dose of the initial impact of antibodies.

In preferred embodiments, the implementation of these impacts antibodies are separated from one another by approximately 24 weeks or 6 months, or about 48 weeks, or 12 months.

In one embodiment, a second exposure to the antibody does not hold, until about 20-30 weeks from the initial exposure to antibodies, with subsequent optional third effect of the antibodies in the amount of about 0.5 to 4 grams (preferably about 1.5 to 2.5 grams), and this is the third effect of the antibody does not hold up to approximately 46-60 weeks (preferably about 46 to 54 weeks) after the initial impact, and then preferably not spend the additional impact of antibodies to at least about 70-75 weeks after the initial impact.

In an alternative embodiment, a second exposure to the antibody does not hold up to approximately 46-60 weeks after the initial impact, and subsequent exposure to antibodies, if they have not done before 46-60 weeks after previous exposure to antibodies.

In one embodiment, the subject is subjected to at least three or at least four effects of antibodies, for example approximately 3-60 effects and more preferably approximately 3-40 influences, most preferably about 3-20 effects of antibodies. Preferably such exposure is performed in the intervals, in each case, approximately 24 weeks or 6 months or 48 weeks or 12 months. In one embodiment, each exposure of the antibody is carried out in a single dose of antibody. In Altern the tive embodiment, each exposure antibodies are in the form of two doses of antibody. However, not every action of antibodies should be provided in a single dose or as two doses.

This antibody can be unbound antibody, or it may be conjugated with another molecule, such as a cytotoxic agent such as a radioactive compound. The preferred antibody is Rituximab, humanitariannet antibody N (for example, containing sequence of the variable domain that is represented in SEQ ID NO:2 and 8), or humanitariannet antibody N containing the sequence of the variable domain that is represented in SEQ ID NO:23 and 24, or huMax-CD20 (Genmab).

In one embodiment, the subject has never been treated with the medication (drugs), such as immunosuppressive agents, for the treatment of multiple sclerosis and/or have never been treated with antibody to the marker surface of b-cells (for example, never previously treated with antibody against CD20).

This antibody is administered by any suitable means, including parenteral, local, subcutaneous, intraperitoneal, intra-lungs, intranasal introduction and/or the introduction of damage. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Also discusses vnutriobolochechnoe introduction (see, for example, an Application for PA is UNT U.S. No. 2002/0009444, Grillo-Lopez, A, concerning vnutriobolochechnoe delivery of antibodies against CD20). In addition, this antibody can be conveniently introduced by pulse infusion, e.g., with decreasing doses of antibodies. Preferably the dose is administered intravenously, subcutaneously or vnutriobolochechnoe, most preferably intravenous infusion.

Although the antibody against CD20 may be the only drug, administered to a subject to treat multiple sclerosis, you do not need to enter a second drug, such as a cytotoxic agent, chemotherapeutic agent, an immunosuppressive agent, cytokine, cytokine antagonist or antibody against a cytokine, growth factor, hormone, integrin, integrin antagonist or antibody against integrin (e.g., antibody against LFA-1, such as efalizumab (RAPTIVA®)commercially available from Genentech, or an antibody against alpha-4-integrin, such as natalizumab (TYSABRI®)available from Biogen) and so, with the antibody that binds to a surface marker of b-cells (for example, an antibody against CD20).

In a preferred embodiment, the combination therapy is antibody combined with drug class interferons, such as IFN-beta-1A (REBIF® and AVONEX®) or IFN-beta-1b (BETASERON®), Oligopeptide, such as latinamerica (COPAXONE®); cytotoxic agent, such as mitoxan is he (NOVANTRONE®), methotrexate, cyclophosphamide, chlorambucil, azathioprine; intravenous immunoglobulin (gamma globulin), lymphocyte-depleting therapy (e.g., mitoxantrone, cyclophosphamide, Campton, anti-CD4, cladribine, irradiation of the whole body, bone marrow transplantation); a corticosteroid (e.g., methylprednisolone, prednisone, dexamethasone or glucorticoids), including systemic corticosteroid therapy; not depleting lymphocytes immunosuppressive therapeutic agent (e.g., mycophenolate-mofetil (MMF) or cyclosporine); drug of the class of statins", lowering cholesterol, which includes tseriwastatina (BAYCOL®), fluvastatin (LESCOL®), atorvastatin (LIPITOR®), lovastatin (MEVACOR®), pravastatin (PRAVACHOL®), simvastatin (ZOCOR®); estradiol; testosterone (not necessarily at higher doses; Stuve et al. Neurology 8:290-301 (2002)); hormone replacement therapy; treatment of symptoms secondary or related to MS (e.g., muscle spasticity, incontinence, pain, fatigue); TNF inhibitor; disease modifying Antirheumatic drug (DMARDs); non-steroidal anti-inflammatory drug (NSAID); plasmapheresis; levothyroxine; cyclosporin A; a somatostatin analogue; a cytokine or a receptor antagonist qi is Okina; the antimetabolite; immunosuppressive agent; rehabilitation surgery, radioactive iodine; thyroidectomy; another antagonist/antibody surface of b-cells, etc.

The second drug is administered with the initial exposure and/or later influences CD20 antibodies, the combined introduction includes co-administration, using separate tracks or a single pharmaceutical composition, and the consequent introduction in any order, preferably where there is a period of time during which both agent or active agents simultaneously exhibit their biological activity.

Apart from the introduction of antibodies to a subject, the application considers the introduction of antibodies by gene therapy. This introduction of nucleic acid encoding this antibody is included in the expression the introduction of an “effective amount” of the antibody. See, for example, WO96/07321, published March 14, 1996, regarding the application of gene therapy to generate intracellular antibodies.

There are two main approaches to the delivery of the nucleic acid (optionally contained in a vector) into cells of the subject: in vivo and ex vivo. For delivery in vivo nucleic acid is injected directly into the subject, usually in the place where you want this antibody. For treatment of ex vivo cells of the subject is extracted, Amu is andnew acid is introduced into these bins and these modified cells are administered to a subject, either directly or, for example, encapsulated within porous membranes which are implanted into the subject (see, for example, U.S. Patent numbers 4892538 and 5283187). There are various methods available for the introduction of nucleic acids into viable cells. These methods vary depending on, carry whether nucleic acid in cultured cells in vitro or in vivo in the cells of the intended host. Methods suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextranase method, the method of deposition of calcium phosphate, etc. Commonly used vector for gene delivery ex vivo is a retrovirus.

The currently favored methods of transfer of nucleic acids in vivo include transfection with viral vectors (such as adenovirus, herpes simplex I or adeno-associated virus) and lipid based (applicable lipids for mediated lipid transport genes are, for example, DOTMA, DOPE and DC-Chol). In some situations, it is advisable to ensure the source of the nucleic acid with an agent that targets the target cells, such as antibody specific against a membrane protein found on the cell surface or in the target cell, a ligand for a receptor on the target cell and d When using liposomes proteins that are associated with membrane protein cell surface 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 the bloodstream, and proteins that target intracellular localization and enhance intracellular half-period of existence. The way of receptor-mediated endocytosis is described, for example, Wu et al., J. Biol. Chem. 262:4429-4432 (1987) and Wagner et al., Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990). In relation to currently known protocols tagging of genes and gene therapy, see Anderson et al., Science 256:808-813 (1992). Cm. also WO 93/25673 and cited in its reference.

III. Antibodies

The methods and products of this invention utilize or include an antibody that binds to a surface marker of b-cells, in particular marker that binds to CD20. Thus, there will be described methods of obtaining such antibodies.

Marker surface of b-cells applicable for obtaining antibodies or to screen for antibodies, can be, for example, a soluble form of token or part thereof, containing the desired epitope. Alternatively, or along with this, cells expressing the marker on their cell surface the STI, can be used to generate antibodies or to screen for antibodies. Other forms of surface marker of b-cells applicable for generating antibodies will be apparent to specialists with expertise in this field.

What follows is a description of examples of methods for producing antibodies used in accordance with this invention.

(i) polyclonal antibodies

Polyclonal antibodies are preferably propagated in animals multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful conjugation of the relevant antigen to a protein that is immunogenic in the species to be of immunization, such as hemocyanin fissurella, serum albumin, bovine thyroglobulin, or trypsin inhibitor soybean, using a bifunctional or derivatizing agent, for example maleimidophenylmethacrylates ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2or R1N=C=NR, where R and R1are different alkyl groups.

Animals subjected to immunization against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively the public) with 3 volumes complete adjuvant's adjuvant and injected this solution intradermally in multiple places. After one month these animals again subjected to immunization 1/5-1/10 the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. After 7-14 days in these animals take blood and serum analyzed for antibody titer. Animals repeatedly subjected to immunization until you see the plateau of the title. Preferably the animal is subjected to immunization with the conjugate of the same antigen, but conjugated to a different protein and/or using different cross-linking reagent. The conjugates can also be obtained in the culture of recombinant cells in the form of a fused protein. To enhance the immune response also use an aggregating agents such as alum.

(ii) Monoclonal antibodies

From the population essentially homogeneous receive antibodies monoclonal antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that arise in obtaining monoclonal antibodies, such wananty usually present in minor amounts. Thus, the definition of "monoclonal" indicates the character of the antibody as a non-discrete mixture or polyclonal antibodies.

For example, monoclonal antibodies can be obtained using hybridomas the way it was first described Kohler et al., Nature, 256:495 (1975), or may be obtained by means of recombinant DNA (U.S. Patent No. 4816567).

In the hybridoma method, a mouse or other suitable animal host are subjected to immunization, as described above, for the induction of lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, the lymphocytes may be immunized in vitro. Then lymphocytes merge with myeloma cells using a suitable merge agent such as polyethylene glycol, to obtain the hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

Thus obtained hybridoma cells were seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival nesmith, source myeloma cells. For example, if the original myeloma cells lack the enzyme hypoxanthineguanine (HGPRT or HPRT), the culture medium for these hybridomas typically will include gipoksantin, aminopterin and thymidine environment (NAT), moreover, these substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are cells that effectively merge, maintain a stable produc is of the high level of antibodies selected antitelomerase cells and are sensitive to the environment, as a NAT environment. Among them, preferred myeloma cell lines are murine myeloma lines, such as lines derived from tumors of mice JUICE-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP cells-2 or H-Ag8-653, available from the American type culture Collection, Rockville, Maryland USA. Myeloma cell lines of human and heteromyinae mouse-human cell lines have been described to obtain monoclonal human 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 is grown cells examined for the production of monoclonal antibodies directed against this antigen. Preferably, the binding specificity of monoclonal antibodies produced hybridoma cells is determined by immunoprecipitation or analysis of binding in vitro, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can be determined, for example, analysis of Scatchard Munson et al., Anal. Biochem., 107:220 (1980).

After identification of hybridoma cells that produce antibodies of the desired specificity, affinity and/or activity, the clones may be subcloned procedures limiting dilutions and grown article is startname methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, medium (D-MEM or medium RPMI-1640. In addition, the hybridoma cells may be grown in vivo as ascitic tumors in the animal.

Monoclonal antibodies secreted by the subclones, appropriately isolated from the culture medium, ascitic fluid or serum-established procedures purification of immunoglobulins, such as, for example, chromatography on protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding these monoclonal antibodies, easy to produce and is sequenced using conventional procedures (e.g., by using oligonucleotide probes that are able to bind specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. After selecting this DNA may be placed into expression vectors, which are then transferout in cell host, such as E. coli cells, the cells COS monkey cells Chinese hamster ovary (Cho or myeloma cells that otherwise do not produce the protein of the immunoglobulin, to obtain the synthesis of monoclonal antibodies in the recombinant cell host. Overview hundred the year on recombinant expression in bacteria of DNA, encoding this antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

In the following embodiment, antibodies or antibody fragments can be isolated from phage libraries of antibodies generated using the methods 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 allocation of mouse antibodies and human antibodies, respectively, using phage libraries. The following publications describe the obtaining of high affinity (nm range) human antibodies by the shuffling circuit (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 methods represent a viable alternative to conventional hybridoma methods monoclonal antibodies for isolation of monoclonal antibodies.

This DNA can also be modified, for example, by introducing the coding sequence of the constant domains of the heavy and light chains of the person instead of the homologous murine sequences (U.S. Patent No. 4816567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)) or covalent joining the coding sequence of the immunoglobulin whole or part of the coding of posledovatel the activity of the polypeptide of non-immunoglobulin.

Typically, such polypeptides non-immunoglobulin replace the constant domains of an antibody or they replace the variable domains of one antigennegative site of an antibody to create a chimeric bivalent antibody containing one antigennegative site having specificity against a single antigen, and the other antigennegative site having specificity against another antigen.

(iii) Humanized antibodies

Methods of humanizing antibodies, not owned by the person described in this field. Preferably humanitariannet antibody has one or more amino acid residues introduced into it from a source that is not a person. These amino acid residues not belonging to the person, often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed basically according to the method of Winter et al. (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 replacing sequences of the hypervariable region corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4817567), which is essentially less than an intact human variable domain has been replaced by the corresponding sequence is Yu, not a person. In practice humanitarianism antibodies are typically human antibodies in which a portion of residues of the hypervariable region and, possibly, some FR residues replaced by residues from analogous sites in rodent antibodies.

The choice of the variable domains of a man, as light and heavy, for use in obtaining humanized antibodies is very important to reduce antigenicity. In accordance with the so-called method of "best fit" the sequence of the variable domain of a rodent antibodies are screened against the entire library of known sequences of the variable domain of the human. The sequence of the person that is closest to the sequence of the rodent, then take as a frame region (FR) for gumanitarnogo 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 a consensus sequence of all human antibodies of a particular subgroup of the variable regions of light and heavy chains. The same frame area may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

In addition, it is important that antibodies were humanitarian with keeping high the affinity towards the antigen and other useful biological properties. To solve this problem in accordance with the preferred method humanized antibodies produced by way of analysis of the source sequences and various speculative humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulins are usually available and known to experts in this field. Accessible and computer programs, which illustrate and display probable three-dimensional conformational structures of selected candidate sequences of immunoglobulins. Examination of these images makes possible an analysis of the likely role of these 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. This way, FR residues can be selected and combined from the sequences of the recipient and import sequences so that achieves the desired characteristics of the antibodies, such as increased affinity against the target antigen (antigen-targets). Usually the remains of the hypervariable region directly and most substantially involved in influencing antigen binding.

(iv) human Antibodies

Alternatively humanise the tion can be obtained from human antibodies. For example, it is now possible to obtain transgenic animals (e.g. mice)that are capable, after immunization, of producing a full range of human antibodies in the absence of endogenous production of immunoglobulin. For example, it has been described that the homozygous deletion of the gene of the connecting region of the heavy chain (JH) antibodies in chimeric mice and mice with a mutation in the germ line results in complete inhibition of endogenous production of antibodies. The transfer of some genes of the immunoglobulin germline of a human being to such a mouse with a mutation in the germ line will lead to the production of antibodies after stimulation by antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Measurement., 7:33 (1993) and U.S. Patent numbers 5591669, 5589369 and 5545807.

Alternatively, the technology of phage display (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and fragments of antibodies in vitro from a spectrum of genes of variable (V) domains of the immunoglobulin from unimmunized donors. According to this method, genes V-domain clone in frame readout either in the primary or in the minor envelope protein of filamentous bacteriophage, such as M13 or fd, and exhibited in the form of functional fragments of the antibodies on the surface ragovoy particles. Since this filamentous hour the ICA contains a copy of the single-stranded DNA genome of the phage, selections based on the functional properties of this antibody also lead to the selection of this gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties In cells. 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 segments V-genes can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) identified a diverse range anticloning antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Range of V genes from unimmunized human donors can be constructed and antibodies to a diverse range of antigens (including proteins) could be determined using methods described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). Cm. the U.S. patents numbers 5565332 and 5573905.

Human antibodies can also be generated by in vitro activated b-cells (see U.S. Patent numbers 5567610 and 5229275).

(v) antibody Fragments

Developed different ways to obtain fragments of antibodies. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan e al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant cell host. For example, antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternatively, Fab'-SH fragments can be directly extracted from E. coli and chemically bonded with the formation of F(ab')2fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2fragments can be isolated directly from a culture of the recombinant host cells. Other methods of obtaining fragments of antibodies will be apparent to experts in this field. In other embodiments, implementation of the preferred antibody is a single-chain Fv fragment (scFv). Cm. WO 93/16185; U.S. Patent No. 5571894 and U.S. Patent No. 5587458. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Patent 5641870. These linear fragments of antibodies can be a monospecific or bespecifically.

(vi) Bespecifically antibodies

Bespecifically antibodies are antibodies that have the binding specificity against at least two different epitopes. Examples bespecifically antibodies can bind to two different epitopes of the marker surface of b-cells. Other such antibodies may bind the marker to the surface of b cells and complement what Ino to link a second different surface marker of b-cells. Alternatively, the shoulder bind marker surface of b-cells antibodies may be combined with an arm that binds a molecule run on a leukocyte such as a molecule of T-cell receptor (e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)so as to focus the protective mechanisms In the cell. Bespecifically antibodies can also be used to localize cytotoxic agents In the cage. These antibodies have shoulder-binding marker surface of b-cells and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinylchloride, a chain of ricin a, methotrexate or hapten with a radioactive isotope). Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., F(ab')2-bespecifically antibodies).

Methods of obtaining bespecifically antibodies known in the field. Traditional getting a full-sized bespecifically antibodies is based on the co-expression of two pairs of heavy chain-light chain immunoglobulins, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of heavy and light chains of immunoglobulins these hybridoma (quadroma) form a potential mixture of 10 different antibody molecules, of which only the one has to correct bespecifically structure. Purification of the correct molecule, which is usually performed by stages affinity chromatography, is quite difficult, and outputs the product are low. Similar procedures described in WO 93/08829 and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to another approach, the variable domains of the antibodies with the desired specificnosti binding (antibody-antigennegative sites) merge sequences of the constant domains of immunoglobulins. This merger is preferably merge with the constant domain of the heavy chain of immunoglobulin containing at least part of the hinge region, CH2 and CH3-areas. It preferably has a first constant region of the heavy chain (SN)containing the site necessary for binding to the light chain is present at least in one of the mergers. DNA encoding the fusion heavy chain of immunoglobulins and, if desirable, the light chain immunoglobulin is inserted into separate expression vectors and cotransfected in a suitable organism, the host. This provides greater 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 outputs. However, you can embed the coding sequences for two or all three polypep is the breaking of the chains in one expression vector, when the expression of at least two polypeptide chains in equal ratios results in high outputs or when these ratios are of no particular value.

In the preferred embodiment, this approach bespecifically antibodies are of the hybrid heavy chain immunoglobulin with a first binding specificity in one arm and a pair of heavy chain-light chain hybrid immunoglobulins (providing a second binding specificity) in the other shoulder. It was found that this asymmetric structure facilitates the separation of the desired especifismo connections from unwanted combinations of chains of immunoglobulins, as the presence of light chain immunoglobulin only half of this bespecifically molecules provides an easy way branch. This approach is described in WO 94/04690. For further details get bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Patent No. 5731168, can be constructed surface section between a pair of antibody molecules to maximize the percentage of heterodimers that extract their culture of recombinant cells. The preferred surface of the partition contains at least the part With theN3-domain constant domain of the antibody. In this way one or a few is to small amino acid chains from the interface of the first antibody molecules replace the large side chains of amino acids (for example, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size with respect to these large side chains are on the surface section of the second antibody molecule by replacing large side chains of amino acids less (for example, alanine and threonine). This provides a mechanism to increase the output of heterodimer regarding unwanted end-products such as homodimers.

Bespecifically antibodies include sewn or heteroconjugate" antibodies. For example, one of the antibodies in this heteroconjugate may be associated with Avidya, and the other with Biotin. Such antibodies have, for example, proposed to target immune system cells to unwanted cells (U.S. Patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be produced using any convenient method of stitching. Suitable cross-linking agents are well known in this field and are described in U.S. Patent No. 4676980 together with a number of ways stitching.

Methods of obtaining bespecifically antibodies, fragments of antibodies have also been described in the literature. For example, bespecifically antibodies may be produced using chemical education links. Brennan et al., Science, 229:81 (1985) describe a procedure in which the intact antibody proteoliticeski split the OBR is using F(ab') 2-fragments. These fragments regenerate in the presence of ditercalinium agent sodium arsenite to stabilize neighboring dithioles and prevent the formation of intermolecular disulfide bonds. Then, the resulting Fab'-fragments are converted into derivatives of dinitrobenzoate (TNB). Then one of the derivatives of Fab'-TNB turn back to the Fab'-thiol by restoring mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB education especifismo antibodies. Received bespecifically antibodies can be used as agents for the selective immobilization of enzymes.

Were also described different ways of obtaining and allocating bespecifically antibodies from the culture of recombinant cells. For example, bespecifically antibodies were obtained using latinovich lightning. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Peptides latinboy lightning from proteins Fos and Jun were linked to the Fab'portions of two different antibodies by fusion of genes. Homodimeric antibodies were restored in the hinge region to form monomers and then re-oxidized to education heterodimeric antibodies. This method can be used to obtain homodimeric antibodies. Technology "datel"described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)has provided an alternative mechanism for obtaining fragmentability antibodies. These fragments contain the variable domain of the heavy chain (VH)attached to the variable domain of the light chain (VL) a linker that is too short to allow pairing between the two domains on the same chain. Thus, the domains of the VHand VLone fragment are forced to pair with the complementary domains of the VLand VHanother fragment, from education through this two antigenspecific sites. It was also reported another strategy to obtain bespecifically fragments of antibodies using dimers of single-chain Fv (sFv). Cm. Gruber et al., J. Immunol., 152:5368 (1994).

It also considers antibodies with more than two valencies. For example, can be obtained thespecification antibodies. Tutt et al. J. Immunol. 147:60 (1991).

IV. Conjugates and other modifications antibodies

The antibody used in the methods or included in the products described here are not necessarily kongugiruut with a cytotoxic agent. For example, the antibody can be conjugated with the drug, as described in WO 2004/032828.

Chemotherapeutic agents applicable in the generation of such conjugates antibody-cytotoxic agent have been described above.

Conjugates of the antibody and one or more low molecular weight toxins such as calicheamicin, maytansine (Pat the t U.S. No. 5208020), trichoton and SS, are also discussed here. In one embodiment of the present invention the antibody is conjugated with one or more molecules maytansine (for example, approximately 1 to approximately 10 molecules maytansine molecule antibodies). Maytansine may be, for example, turned into a Mau-SS-Me which may be restored in the Mau-SH3 and brought into reaction with modified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) to generate the conjugate maytansinoid-antibody.

Alternatively, this antibody can be conjugated with one or more molecules calicheamicin. Family of antibiotics calicheamicin able to produce double-stranded DNA breaks at subpicomolar concentrations. Structural analogues calicheamicin, which can be used include, but are not limited to, γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θ1I(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 that can be used include a chain of diphtheria toxin, neisvaziuosiu active fragments of diphtheria toxin a chain of exotoxin a (from Pseudomonas aeruginosa), a chain of ricin, a chain abrina, chain And modeccin, alpha sarcin, proteins Aleurites fordii proteins of diantin, Belk is Phytolaca americana (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, krotin, inhibitor Sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. See, for example, WO 93/21232, published October 28, 1993.

Further, this invention considers antibody conjugated with connection nucleotidase activity (e.g., a ribonuclease or a DNA endonuclease such as desoksiribonukleaza; Ncasa).

A variety of radioactive isotopes are available for radioconjugates antibodies. Examples include At211I131I125, Y90That Re186That Re188Sm153Bi212, R32and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be obtained using a variety of bifunctional binding protein agents, such as N-Succinimidyl-3-(2-(pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glyceraldehyde), bis-etidocaine (such as bis-(p-azidobenzoyl)hexanediamine), derivatives of bis-page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active compounds of the Torah (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is an example of chelat forming agent for conjugation of the radionuclide to the antibody. Cm. WO 94/11026. This linker can be "otdalennym linker"facilitating release of the cytotoxic drug in the cell. For example, can be used colorability linker sensitive peptidase linker, dimethylselenide linker or dyslipidaemias linker (Chari et al. Cancer Research 52: 127-131 (1992)).

Alternatively, it may be obtained protein containing the antibody and cytotoxic agent, for example, by recombinant methods or peptide synthesis.

In another embodiment, the antibody may be conjugated to a “receptor” (such as streptavidin) for utilization in advanced targeting the tumor, where the conjugate of the antibody-receptor is administered to a subject, followed by removal of unbound conjugate from the blood flow with the use of agent clearance and then the introduction of a “ligand” (e.g., avidin), which anywhereman with a cytotoxic agent (e.g. a radionuclide).

Antibodies according to this invention may also be conjugated with a prodrug activating enzyme, which is the first turns of the prodrug (e.g., pipidinny chemotherapeutic agent, see WO81/01145) to an active anti-cancer agent. See, for example, WO 88/07378 and U.S. Patent No. 4975278.

The enzyme component of such conjugates includes any enzyme capable of acting on a prodrug in such a way that it converts the prodrug into its more active, cytotoxic form.

Enzymes, which are applicable in the method according to this invention, include, but are not limited to, alkaline phosphatase, applicable for converting phosphate-containing prodrugs into free drugs; arylsulfatase applicable for turning sulfidogenic prodrugs into free drugs; citizendiumissue applicable for converting non-toxic 5-fertilizin in anti-cancer drug, 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins b and L), which are applicable for the conversion of petitcodiac prodrugs into free drugs; D-alanismorissette, applicable for converting prodrugs that contain D-amino acid substituents; splitting carbohydrates enzymes, such as β-galactosidase and neuraminidase, applicable for converting glycosylated prodrugs into free drugs; β-Lac is amaze, applicable for making medicines, derivatizing with β-lactams, into free drugs; and penicillinases, such as penicillin V-amidase or penicillin G-amidase applicable for the conversion of medicines, derivatizing on the nitrogen atoms of their amino groups phenoxyacetyl or phenylacetylene groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known as the "abzyme", can be used to convert the prodrugs of this invention into free active drugs (see, for example, Massey, Nature 328: 457-458 (1987)). Conjugates of the antibody-Abim can be obtained as described herein for delivery of abzyme in the population of tumor cells.

The enzymes of this invention can be covalently linked to the antibody by methods well known in the field, such as the use of heterobifunctional crosslinking reagents discussed above. Alternatively, the fused proteins containing at least antigennegative district antibodies of the present invention, associated with at least a functionally active portion of the enzyme according to this invention, can be constructed using methods of recombinant DNA, are well known in the art (see, e.g., Neuberger et al., Natue, 312: 604-608 (1984)).

Discussed here and other modifications of the antibody. For example, the antibody may be associated with one of the various non-protein polymers, such as polyethylene glycol (PEG), polypropyleneglycol, polyoxyalkylene or copolymers of polyethylene glycol and polypropylenglycol. Antibody fragments such as Fab', associated with one or more PEG molecules, are particularly preferred embodiment of the present invention.

Antibodies described herein can also be prepared in the form of liposomes. Liposomes containing the antibody are obtained by methods known in this field, 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. Patent numbers 4485045 and 4544545 and WO97/38731, published on 23 October 1997. Liposomes with enhanced circulation time is described in U.S. Patent No. 5013556.

Especially applicable liposomes can be obtained by the method of reverse-phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-PE). Liposomes ekstragiruyut through filters with defined pore size to obtain liposomes of the desired diameter. Fab'-fragments of the antibodies according to this invention can be conjugated with these liposomes as described in Martin et al. J. Biol. Chem. 257:286-288 (1982), by Rea is the disulfide exchange. Chemotherapeutic agent is optionally contained within the liposome. Cm. Gabizon et al. J. National Cancer Inst. 81(19):1484 (1989).

Discusses the modification of amino acid sequence. For example, it may be desirable to improve the affinity of binding and/or other biological properties of the antibody. Variants of the amino acid sequence of the antibody produced by introducing appropriate nucleotide changes into a nucleic acid, antibody or peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequences of this antibody. Produce any combination of deletions, insertions, or substitutions for the final design, provided that the final construct possesses the desired characteristics. Replacement of amino acids can also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

Applicable method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called “leninskoyi by mutagenesis”as described by Cunningham and Wells, Science, 244:1081-1085 (1989). Here identify a residue or group of residues of target proteins (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced with a neutral or negative the positive charged amino acid (most preferably alanine or polyalanine) to affect the interaction of these amino acids with antigen. Then the position of the amino acids, demonstrating functional sensitivity to the substitutions, improve the introduction of advanced these or other options in these sites change, or instead of these sites replacement. Thus, while the site for introducing variations in the amino acid sequence is predetermined, the nature of the mutation per se is not predetermined. For example, to analyze the performance of a mutation at a specific site spend Leninskoye or random mutagenesis of the codon-target or area target and expressed variants of the antibodies are screened for the desired activity.

Insertions of amino acid sequences include amino - and/or carboxyl-terminal fusions, in the range of length from one residue to polypeptides containing a hundred or more residues, as well as insertions, inside a sequence of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionine residue or the antibody fused with a cytotoxic polypeptide. Other options insertion of antibody molecules include the fusion to the N-end or the end of the antibody of an enzyme or a polypeptide which increases the half-life existence in the serum of this antibody.

Another type of variant is the variant with the substitution of amino acids. These options, which the ants have at least one amino acid residue in the antibody molecule, replaced by another residue. The sites of greatest interest for the replacement mutagenesis of antibodies (antibodies), include the hypervariable regions, but also examined changes FR. Conservative substitutions are shown in table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, may be more significant substitutions, called “exemplary substitutions” in table 1 or described below in reference to classes of amino acids, and these products can be subjected to screening.

Table 1
The original balanceApproximate replacementPreferred replacement
Ala (A)Val; Leu; IleVal
Arg (R)Lys; Gln; AsnLys
Asn (N)Gln; His; Asp; Lys; ArgGln
Asp (D)Glu; AsnGlu
Cys (C)Ser; AlaSer
Gln (Q)Asn; GluAsn
Glu (E)Asp; GlnAsp
Gly (G)AlaAla
His (H)Asn; Gln; Lys; ArgArg
Ile (I)Leu; Val; Met; Ala; Phe; norleucineLeu
Leu (L)Norleucine; Ile; Val; Met; Ala; PheIle
Lys (K)Arg; Gln; AsnArg
Met (M)Leu; Phe; IleLeu
Phe (F)Trp; Leu; Val; Ile; Ala; TyrTyr
Pro (P)AlaAla
Ser (S)ThrThr
Thr (T)Val; SerSer
Trp (W)Tyr; PheTyr
Tyr (Y)Trp; Phe; Thr; SerPhe
Val (V)Ile; Leu; Met; Phe; Ala; norleucineLeu

Substantial modifications in the biological properties of the antibody is performed by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide skeleton in the area of the substitution, for example, in the form of a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (C) the volume of the side chain. Amino acids can be grouped according to similarities in the properties of their side chains (in A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala(A)Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M);

(2) uncharged polar: Gly (G), Ser (S)Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);

(3) acidic: Asp (D)Glu (E);

(4) basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues may be divided into groups based on common properties of the side chains:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail the replacement of a member of a class a member of one of these each the x class.

Any cysteine residue not involved in maintaining the proper conformation of the antibody may be substituted, generally with serine, to improve the oxidative stability of this molecule and prevent aberrant crosslinking. On the contrary, communication (communication) cysteine can be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

Particularly preferred substituted variant includes a substitution of one or more residues of the hypervariable region of the initial antibody. Usually the best approach or received options selected for further development will have improved biological properties relative to the original antibody from which they were obtained. Convenient way for generating such containing replacement options is affinity maturation using phage of diplay. Briefly, multiple sites hypervariable region (e.g., 6-7 sites) mutate to obtain all possible amino acid substitutions at each site. Thus obtained variants of the antibodies are shown monovalent way of particles of filamentous phage in the form of mergers with the product of the gene III of M13 packaged within each particle. These are then displayed by phage variants is subjected to screening for their Messiah. biologicheskuyu activity (for example, the binding affinity of), as described here. To identify candidate sites in hypervariable region for the modification can be performed Leninskoye mutagenesis to identify residues of the hypervariable region, contributing significantly to antigen binding. Alternative or in addition it may be useful to analyze the crystal structure of the complex antigen-antibody to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for replacement in accordance with the methods developed here. After generating such variants, the panel of variants is subjected to screening as described herein and antibodies with improved properties in one or more relevant assays may be selected for further development.

Another type of amino acid variant antibody has a change in the source distribution glycosylation of antibodies. This change involves the deletion of one or more carbohydrate parts of the molecule to be detected by this antibody, and/or adding one or more glycosylation sites that are not present in the antibody.

Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate side to side the howling chain asparagine residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are the recognition sequences for enzymatic joining carbohydrate portion to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, most often serine or threonine, although can be also used 5-hydroxyproline or 5-hydroxylysine.

Addition of glycosylation sites to the antibody is convenient to perform a change in amino acid sequence such that it contains one or more of the above Tripeptide sequences (for N-linked glycosylation sites). This change can also be done by adding or replacing one or more residues of serine or threonine to the sequence of the original antibody (for O-linked glycosylation sites).

If the antibody contains a Fc region, the carbohydrate attached to it, can be changed. For example, antibodies with a Mature carbohydrate structure that lack fucose attached to the Fc region of antibodies, op is Sana'a in the Application for U.S. patent number US 2003/0157108 A1, Presta, L. Cm. also U.S. Patent 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd) in respect of the composition of the anti-CD20 antibodies. Antibody with bisected N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in WO03/011878, Jean-Mairet et al. and U.S. Patent No. 6602684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region of the antibodies disclosed in WO97/30087, Patel et al. Cm. also WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.) in respect of antibodies with altered carbohydrate attached to its Fc-region.

The preferred variant glycosylation of the present invention contains a Fc region, in which the carbohydrate structure attached to the Fc region lacks fucose. Such options had improved ADCC (antibody-dependent cellular cytotoxicity). Optional, this Fc region further comprises one or more amino acid substitutions, which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues). Examples of publications related to "deforsirovannym" or "fucose-deficient" antibodies include: Application for U.S. patent number US 2003/0157108 A1, Presta, L.; WO 00/A; WO01/A; US2003/A; US2002/A; US2004/A; US2004/A; US2004/A; US2004/A; US2004/A; WO03/A; WO03/A; WO 2005/035778; WO2005/035586 (describing the inhibition of RNA (RNAi) fokusirovanie); Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines producing deoksigenirovanii antibodies include cells Lec13 Cho, insufficient in fokusirovanie proteins (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); Application for U.S. patent number US 2003/0157108 A1, Presta, l; and WO2004/056312 A1, Adams et al., in particular, in Example 11) and a cell line with a knockout, such as cells SNO with a knockout of the gene alpha-1,6-fucosyltransferase, FUT8 (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Molecules of nucleic acids encoding amino acid sequence variants of antibodies, receive a variety of ways known in this field. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis previously obtained variant or variant version of the antibody.

It may be desirable modification of the antibodies according to this invention in respect to effector function, for example, in such a way as to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent (CDC) cytotoxicity of antibodies. This can be achieved by introducing one or more amino acid substitutions in the Fc-Paradise is n antibodies. Alternative or in addition, the cysteine residue (cysteine residues) can be introduced into the Fc region, which makes it possible to form between the chains disulfide bond in the area. Thus obtained homodimeric antibody may have improved the ability of internalization and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Cm. 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 obtained using heterobifunctional cross-linking of the linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, it may be engineered antibody that has dual Fc regions and may thereby have an increased complement lysis and ADCC efficiency. Cm. Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

WO00/42072 (Presta L.) describes antibodies with enhanced ADCC function in the presence of effector cells, where these antibodies contain amino acid substitutions in their Fc region. Preferably this antibody with enhanced ADCC contains substitutions at positions 298, 333, and/or 334 of the Fc region. Preferably the altered Fc region is an Fc region human IgG1 containing substitutions at one, two or three of these provisions or consisting of one, two or three of these provisions.

p> Antibodies with altered C1q binding and/or complement-dependent cytotoxicity (CDC) are described in WO99/51642, U.S. Patent No. V, U.S. Patent No. V, U.S. Patent No. V and U.S. Patent No. 6538124 (Idusogie et al.). These antibodies contain amino acid substitution at one or more positions 270, 322, 326, 327, 329, 313, 333 and/or 334 of their Fc region.

To increase the half-period of the existence of serum antibodies can include epitope recycling binding receptor is an antibody (in particular, an antibody fragment)as described, for example, in U.S. Patent 5739277. In this context, "epitope recycling binding receptor" refers to an epitope of the Fc region of the IgG molecule (e.g., IgG1, IgG2, IgG3or IgG4), which is responsible for the increase of the half-period of existence in serum in vivo IgG molecules. Antibodies with substitutions in their Fc region and increased half-periods of existence in serum is described in WO00/42072 (Presta, L.).

Also considered engineered antibodies with three or more (preferably four) functional binding sites of the antigen (Application for U.S. patent US2002/0004587 A1, Miller et al.).

V. Pharmaceuticals

Therapeutic antibody preparations used in accordance with this invention are prepared for storage by mixing the antibody having the desired degree pure is s, with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of lyophilised preparations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include buffers such as phosphate, citrate and containing other organic acids; antioxidants including ascorbic acid and methionine; preservatives, such as chloride of octadecyltrimethoxysilane; chloride hexadecane; benzalkonium chloride, chloride benzene; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechin; 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; hepatoblastoma agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; metal complexes (e.g., complexes of Zn-BAA is OK); and/or nonionic surfactants such as WEEN™, PLURONICS™ or polyethylene glycol (PEG).

Examples of drugs anti-CD20 antibodies described in WO98/56418. This publication describes a liquid mnogorazovyj preparation containing 40 mg/ml Rituximab, 25 mm acetate, 150 mm trehalose, 0.9% of benzyl alcohol of 0.02% Polysorbate 20 at pH 5.0 that has a minimum shelf life of two years at 2-8aboutC. Other interest in the preparation of anti-CD20 antibodies contains 10 mg/ml Rituximab in 9.0 mg/ml sodium chloride, of 7.35 mg/ml sodium citrate, 0.7 mg/ml Polysorbate 80, and sterile water for injection, pH 6.5.

Lyophilized preparations adapted for subcutaneous administration are described in U.S. Patent No. 6267958 (Andya et al.). Such lyophilized preparations can be restored with a suitable solvent to a high concentration of protein, and this restored the drug may be injected subcutaneously to a mammal, which should heal them.

Discusses also crystallized form of the antibody or antibodies. See, for example, US 2002/0136719 A1 (Shenoy et al.).

This drug may also contain more than one active connection required for a particular subject to treatment condition, preferably compounds with complementary activities that do not adversely effect each other. For example, the can is to be desirable additional collateral cytotoxic agent; chemotherapeutic agent; immunosuppressive agent, cytokine, cytokine antagonist or antibody against cytokines; growth factors; hormones; integrin, integrin antagonist or antibody against integrin (e.g., antibodies against LFA-1, such as efalizumab/RAPTIVA, commercially available from Genentech, or antibodies against alpha-4-integrin, such as natalizumab/TYSABRI®)available from Biogen); drug class interferons, such as IFN-β-1A (REBIF® and AVONEX®) or IFN-β-1b (BETASERON®); the oligopeptides, such as latinamerica (COPAXONE®); a cytotoxic agent, such as mitoxantrone (NOVANTRONE®), methotrexate, cyclophosphamide, hlorambuzila or azathioprine; intravenous immunoglobulin (gamma globulin); - depleting lymphocytes drugs (e.g., mitoxantrone, cyclophosphamide, Kampala, anti-CD4, cladribine); not depleting lymphocytes immunosuppressive drugs (e.g., mycophenolate-mofetil (MMF) or cyclosporine); drugs of the class of statins", lowering cholesterol; estradiol; testosterone; means for hormonal substitution therapy; drugs, which treats symptoms secondary or related to the relatively MS (e.g., muscle spasticity, incontinence, pain, fatigue); TNF inhibitor; Antirheumatic drugs modifying disease (DMARDs); non-steroidal anti-inflammatory drug (NSAID), corticosteroids (eg, methylprednisolone, prednisone, dexamethasone or glucorticoid); levothyroxine; cyclosporine A; analogue of somatostatin; cytokine antagonist; antimetabolite; immunosuppressive agent; integrin antagonist or antibody against integrin (e.g., antibodies against LFA-1, such as efalizumab, or antibodies against alpha-4-integrin, such as natalizumab); or antagonist/antibody against the marker surface of b-cells; etc. in this preparation. The type and effective amounts of such other agents depends, for example, the amount of antibodies present in the product, the type of multiple sclerosis, subject to treatment, and clinical parameters of the subjects. They are usually used in the same doses and with the same techniques that were used previously, or about 1-99% of the previous dose.

The active ingredients may be enclosed in microcapsules obtained, for example, ways koatservatsii or interfacial polymerization, for example hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in the colloidal drug-delivery systems (e.g. liposomes, albumen the microspheres, microemulsions, nanoparticles, and n is lokapala) or microemulsion. Such methods are described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980).

Can be cooked preparations prolonged release. Suitable examples of sustained release formulations release include a semi-permeable matrices of solid hydrophobic polymers containing the antibody, and these matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of matrices prolonged release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or poly(vinyl alcohol), polylactide (U.S. Patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, degradiruem the ethylene vinyl acetate, degradiruete copolymers of lactic acid and glycolic acid, such as the LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid and glycolic acid and leuprolide), and poly-D-(-)-3-hydroxybutiric acid.

Preparations for use introduction in vivo must be sterile. This is easily accomplished by filtration through sterile membrane filter.

IV. Product

In another embodiment, provided with the product containing substances that are applicable for the treatment of multiple sclerosis, as described above. Preferably this product contains (a) a container containing the composition, with the holding antibody which binds to a surface marker of b-cells (for example, the antibody against CD20) and a pharmaceutically acceptable carrier or diluent within the container; and (b) a package insert with instructions regarding the introduction of this composition to a subject suffering from multiple sclerosis, to provide initial exposure to antibodies approximately 0.5 to 4 grams followed by a second exposure to antibodies approximately 0.5-4 g, and this second effect is not to provide approximately 16-60 weeks from the initial exposure; or instructions in respect of the introduction of this composition to a subject suffering from PPMS.

This product includes a container and a label or package insert on the container or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be made of various materials, such as glass or plastic. The container holds and contains a composition that is effective for the treatment of multiple sclerosis, and may have a sterile access hole (for example, the container may be a bag of intravenous solution or vial having a stopper, sharp needle for hypodermal injection). At least one active agent in the composition is an antibody. The label or package insert indicates that the composition used in isoamsa for the treatment of multiple sclerosis in the subject, suffering from it, with specific guidance in respect of the number of doses and intervals antibodies and any other secured medicines. This product may further comprise a second container containing a pharmaceutically acceptable diluent, a buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution and dextrose. This product may additionally include other materials desirable from a commercial point of view and from the point of view of the user, including other buffers, diluents, filters, needles and syringes.

Optional this product further comprises a container containing an agent other than an antibody, for the treatment and optionally containing instructions for treatment of a mammal such agent, and such agent is preferably a chemotherapeutic agent or immunosuppressive agent, an interferon, such as IFN-beta-1A (REBIF® and AVONEX®) or IFN-beta-1b (BETASERON®); Oligopeptide, such as latinamerica (COPAXONE®); a cytotoxic agent such as mitoxantrone (NOVANTRONE®), methotrexate, cyclophosphamide, hlorambuzila or azathioprine; intravenous immunoglobulin (gamma globulin), lymphocyte-depleting therapy (e.g., mitoxantrone, cyclophosphamide, Campton anti-CD4, cladribine); not depleting lymphocytes immunosuppressive drug (e.g., mycophenolate-mofetil (MMF) or cyclosporine); drug of the class of statins", lowering cholesterol; estradiol; means for hormone replacement therapy; medicine that treats symptoms secondary or related to MS (e.g., muscle spasticity, incontinence, pain, fatigue); TNF inhibitor; disease modifying Antirheumatic drug (DMARDs); non-steroidal anti-inflammatory drug (NSAID), corticosteroids (eg, methylprednisolone, prednisone, dexamethasone or glucorticoids); levothyroxine; cyclosporin A; a somatostatin analogue; a cytokine or a receptor antagonist of a cytokine; an antimetabolite; immunosuppressive agent; integrin antagonist or antibody against integrin (e.g., an antibody against LFA-1, such as efalizumab, or an antibody against alpha-4-integrin, such as natalizumab) and an antibody against the marker surface of b-cells, etc.

Next, the details of this invention are illustrated by the following non-limiting Examples. All citations in this description is specifically incorporated herein by reference.

EXAMPLE 1

Treatment of primary progressive multiple IC is Erosa (PPMS)

Subject diagnosed with PPMS defined by McDonald et al. Ann Neurol 50:121-7 (2001), treated with anti-CD20-antibody in this example.

Rituximab, commercially available from Genentech, prepare for IV administration in the form of a sterile product in 9.0 mg/ml sodium chloride, 0.7 mg/ml Polysorbate 80, of 7.35 mg/ml dihydrate sodium citrate and Sterile Water for Injection (pH 6.5).

The first course of treatment will consist of a dose of 1 g intravenous (IV) Rituximab, introduced into each of days 1 and 15. Subjects will receive acetaminophen (1 g) and diphenhydramine-HCl (50 mg), or equivalent, for 30-60 minutes before each infusion.

Subsequent treatment courses will be held, beginning at week 24 (day 169), week 48 (day 337) and week 72 (day 505). The second infusion of these subsequent courses of treatment will be performed after 14+1 days after the first infusion.

Subjects who experienced a first relapse, can get an auxiliary treatment with IV or oral corticosteroids. Systemic corticosteroids can be administered using the administration scheme, which does not exceed the impact or duration of treatment, suitable for MS relapse. Relapse is defined as the presence of all the following:

- sharp appearance of neurological abnormalities, which lasts for at least 24 hours;

- change, which is not testi to fever, infection, trauma, concomitant medications, or other etiology;

event objective changes in the test inquirer blind researcher, including a minimum change of 1 point (point) in one of the following scales FS: pyramidal, cranial nerves, cerebellar, sensory, vision changes or gait.

Can be used the following scheme corticosteroids: 1 g IV methylprednisolone once daily for 3 days, then 60 mg of prednisone once a day for 5 days and reduced to 10 mg each day thereafter. If IV-methylprednisolone is not available, you can replace 150 mg IV dexamethasone once daily for 3 days. For exacerbation should be entered only a single course of corticosteroids. Subsequent immunological studies and MRI scans should be obtained after at least 4 weeks after completion of the scheme of administration of corticosteroid. Can be used corticosteroid inhalers (for oral and nasal administration) or intra-articular injection.

Additional regimens, combined with optional anti-SO-antibody include IFN-β, latinamerica, methotrexate, cyclophosphamide or mitoxantrone.

The primary criterion of the efficiency is the time to confirmed progresser the of the disease. Disease progression is defined as an increase of ≥1.0 point from the background Scale Developed Status Disability (EDSS) (Kurtzke J. Neurology 33 (11): 1444-52 (1983)), if the background EDSS equal to ≥5.5 points (inclusive)for which the change cannot be attributed to other etiologies (eg, fever, concomitant disease, relapse or exacerbation of MS or the effects of concomitant drug). Confirmation of progression of the disease can occur at regularly scheduled visit, which is at least 12 weeks (84 days) after the initial progression.

Secondary criteria of the efficiency include the following :

- Change from background to week 96 in total volume of T2 lesions on magnetic resonance tomograms of the brain.

- Change from background to week 96 in brain volume using magnetic resonance tomography MRI.

Optional improvement in any one or more of the following options.

- Functional Composite Scale of Multiple Sclerosis (MSFCS).

- EDSS (Scale Developed Status Disability).

The proportion of subjects with confirmed disease progression at week 96 defined using the EDSS.

Function of the upper extremities, measured on the 9-Hole Peg test (podskali MSFCS).

- Ability to move, as measured by the Timed 25-Foot Walk (timed walking 25 feet) (podskali MSFCS).

- Cognition, as measured by the Paced Auditory Serial Addition Test (only 3 seconds; podskali MSFCS).

- Total volume of T2 lesions on brain magnetic resonance tomograms (weeks 48 and 122).

- Total volume of T1-lesions on brain magnetic resonance tomograms.

- Cross-sectional area of the cervical spinal cord on magnetic resonance tomograms.

- Volume of brain magnetic resonance tomograms (weeks 48 and 122).

The subject treated with Rituximab, as described here, you will discover improvement in the signs, symptoms, or other indicators of PPMS in accordance with any one or more of the above criteria result.

EXAMPLE 2

The treatment of relapsing-remitting multiple sclerosis subject diagnosed with RRMS defined by McDonald et al. Ann Neurol 50: 121-7 (2001), treated with anti-D20-antibody in this example, where the effect of the antibody is carried out with an interval of approximately 6 months.

Rituximab, commercially available from Genentech, prepare for IV administration in the form of a sterile product in 9.0 mg/ml sodium chloride, 0.7 mg/ml Polysorbate 80, of 7.35 mg/ml dihydrate sodium citrate and Sterile Water for Injection (pH 6.5).

The first course of treatment will consist of the eskers 1 g intravenous (IV) Rituximab, enter in each of days 1 and 15. Subjects will receive acetaminophen (1 g) and diphenhydramine-HCl (50 mg), or equivalent, for 30-60 minutes before each infusion.

Subsequent treatment courses will be held, beginning at week 24 (day 169), week 48 (day 337) and week 72 (day 505). The second infusion of these subsequent courses of treatment will be performed after 14±1 days after the first infusion.

Preferably Rituximab is the only agent introduced for the treatment of RRMS. However, subjects may not be necessary to obtain IV or oral corticosteroids, IFN-β, latinamerica, methotrexate, cyclophosphamide or mitoxantrone.

Subjects who experienced a first relapse, can get an auxiliary treatment with IV or oral corticosteroids. Systemic corticosteroids can be administered using the administration scheme, which does not exceed the impact or duration of treatment, suitable for MS relapse. Relapse is defined in this Example as the emergence of new or recurrent neurological symptoms consistent with MS, lasting for more than 48 hours, in the subject, which was in a relatively stable or improving neurological condition within at least 30 days. Changing neurological symptoms should be accompanied by objective neural the environmental deterioration, consistent with the increase in at least half of the stage on the EDSS, or 2 pips (dots) on one of the appropriate points of the functional system (FSS), or 1 point (1 point) two or more appropriate FSS. This change must be confirmed by an inquirer researcher and should affect the selected scale FS (i.e. pyramidal, gait, cerebellar, brainstem, sensory, or associated with vision). The symptoms must persist for ≥ 24 hours and must be symptoms that cannot be attributed to the confusing clinical factors (e.g., fever, infection, injury, adverse reactions concomitant drugs). The only attack of paroxysmal symptoms (e.g., tonic spasm) is not a relapse, but a new occurrence of multiple cases of paroxysmal symptoms for at least 24 hours may be a relapse if it is accompanied with new, relevant objective manifestations. Sensory symptoms without changes in clinical examination, fatigue, mood changes, or the urge bladder or bowel or urinary, will be insufficient to establish a relapse.

The primary criterion of the efficiency is the endpoint MRI gadolinium-enhancing lesions, or time to podtverzhdennogo the progression of the disease, defined as an increase of ≥1.0 point from the background Scale Developed Status Disability (EDSS) (Kurtzke J. Neurology 33(11): 1444-52 (1983)). The primary endpoint of efficacy may not be the total number of gadolinium-enhancing T1 lesions observed on serial magnetic resonance tomograms of the brain at weeks 12, 16, 20 and 24.

Secondary criteria of the efficiency include the recurrence rate; a change from background to week 96 in total volume of T2 lesions on magnetic resonance tomograms of the brain (for example, the change in total volume of T2 lesions on magnetic resonance tomograms of the brain from screening to weeks 24 and 36); change from background to week 96 in brain volume using magnetic resonance tomograms of the brain; Functional Composite Scale of Multiple Sclerosis (MSFCS) and its podskalak; upper extremity impairment, as measured by the 9-Hole Peg test (podskali MSFCS); ability to move, as measured by the Timed 25-Foot Walk (timed walking 25 feet) (podskali MSFCS); knowledge, as measured by the Paced Auditory Serial Addition Test (podskali MSFCS); questionnaire of Quality of Life in Multiple Sclerosis-54 (MSQOL-54); the total volume of T1-lesions on brain magnetic resonance tomograms (for example, the total number of gadolinium-enhancing T1 lesions observed on CE the applications of magnetic resonance tomograms of the brain at weeks 20, 28 and 36); the cross-sectional area of the cervical spinal cord on magnetic resonance tomograms; the proportion of subjects with recurrent week 24 (i.e., between week 0 and week 24) and 36 (i.e., between week 0 and week 36); criterion combined Unique Activity at week 24 and 36.

The patient treated with Rituximab, as described above, to detect an improvement in any one of the above criteria result.

EXAMPLE 3

The treatment of relapsing-remitting multiple sclerosis

Subject diagnosed with RRMS defined by McDonald et al. Ann Neurol 50:121-7 (2001), treated with anti-CD20-antibody. In this example, the effects of the antibody is carried out with an interval of approximately 1 year.

Rituximab, commercially available from Genentech, prepare for IV administration in the form of a sterile product in 9.0 mg/ml sodium chloride, 0.7 mg/ml Polysorbate 80, of 7.35 mg/ml dihydrate sodium citrate and Sterile Water for Injection (pH 6.5).

The first course of treatment will consist of a dose of 1 g intravenous (IV) Rituximab, introduced into each of days 1 and 15. Subjects will receive acetaminophen (1 g) and diphenhydramine-HCl (50 mg), or equivalent, for 30-60 minutes before each infusion.

Subsequent treatment courses will be held, beginning at week 48, week 96. The second infusion of these subsequent courses of treatment will be performed after 14±1 days after what erway infusion.

Preferably Rituximab is the only agent introduced for the treatment of RRMS. However, subjects may not be necessary to obtain IV or oral corticosteroids, IFN-β, latinamerica, methotrexate, cyclophosphamide or mitoxantrone.

Subjects who experienced a first relapse, can get an auxiliary treatment with IV or oral corticosteroids. Systemic corticosteroids can be administered using the administration scheme, which does not exceed the impact or duration of treatment, suitable for MS relapse. Relapse is defined in this Example as the emergence of new or recurrent neurological symptoms consistent with MS, lasting for more than 48 hours, in the subject, which was in a relatively stable or improving neurological condition within at least 30 days. Changing neurological symptoms should be accompanied by objective neurological impairment, consistent with the increase in at least half of the stage on the EDSS, or 2 pips (dots) on one of the appropriate points of the functional system (FSS), or 1 point (1 point) two or more appropriate FSS. This change must be confirmed by an inquirer researcher and should affect the selected scale FS (i.e. pyramidal, gait, cerebellar, stem golovnogo the brain, touch or associated with vision). The symptoms must persist for ≥24 hours and must be symptoms that cannot be attributed to the confusing clinical factors (e.g., fever, infection, injury, adverse reactions concomitant drugs). The only attack of paroxysmal symptoms (e.g., tonic spasm) is not a relapse, but a new occurrence of multiple cases of paroxysmal symptoms for at least 24 hours may be a relapse if it is accompanied with new, relevant objective manifestations. Sensory symptoms without changes in clinical examination, fatigue, mood changes, or the urge bladder or bowel or urinary, will be insufficient to establish a relapse.

The primary criterion of the efficiency is the endpoint MRI gadolinium-enhancing lesions, or the time to confirmed disease progression, defined as an increase of ≥1.0 point from the background Scale Developed Status Disability (EDSS) (Kurtzke J. Neurology 33(11): 1444-52 (1983)). The primary endpoint of efficacy may not be the total number of gadolinium-enhancing T1 lesions observed on serial magnetic resonance tomograms of the brain at weeks 12, 16, 20 and 2.

Secondary criteria of the efficiency include the recurrence rate; a change from background to week 96 in total volume of T2 lesions on magnetic resonance tomograms of the brain (for example, the change in total volume of T2 lesions on magnetic resonance tomograms of the brain from screening to weeks 24 and 36); change from background to week 96 in brain volume using magnetic resonance tomograms of the brain; Functional Composite Scale of Multiple Sclerosis (MSFCS) and its podskalak; upper extremity impairment, as measured by the 9-Hole Peg test (podskali MSFCS); ability to move, as measured by the Timed 25-Foot Walk (timed walking 25 feet) (podskali MSFCS); knowledge, as measured by the Paced Auditory Serial Addition Test (podskali MSFCS); questionnaire of Quality of Life in Multiple Sclerosis-54 (MSQOL-54); the total volume of T1-lesions on brain magnetic resonance tomograms (for example, the total number of gadolinium-enhancing T1 lesions observed on serial magnetic resonance tomograms of the brain at weeks 20, 28 and 36); the cross-sectional area of the cervical spinal cord on magnetic resonance tomograms; the proportion of subjects with recurrent week 24 (i.e., between week 0 and week 24) and 36 (i.e., between week 0 and week 36); criterion combined Unique Activity in n matter of 24 and 36.

The patient treated with Rituximab, as described above, to detect an improvement in any one of the above criteria result.

EXAMPLE 4

Humanized variants N

This example describes humanized variants of antibodies N for use in the ways described here. Humanitariannet antibody N preferably contains one, two, three, four, five or six of the following CDR sequences.

CDR L1 sequence RASSSVSYXH, where X denotes M or L (SEQ ID NO:18), for example SEQ ID NO:4 (Fig. 1A),

CDR-L2 sequence of SEQ ID NO:5 (Fig. 1A),

CDR-L3 sequence QQWXFNPPT, where X denotes S or A (SEQ ID NO:19), for example SEQ ID NO:6 (Fig. 1A),

CDR H1 sequence of SEQ ID NO:10 (Fig. 1B),

CDR H2 sequence AIYPGNGXTSYNQKFKG, where X denotes a D or A (SEQ ID NO:20), for example SEQ ID NO:11 (Fig. 1B), and

CDR H3 sequence VVYYSXXYWYFDV, where X at position 6 denotes N, A, W, or D, and X in position 7 denotes S or R (SEQ ID NO:21), for example, SEQ ID NO:12 (Fig. 1B).

The above CDR sequences normally present in variable light and variable heavy frame sequences in humans, such as essentially the consensus FR-remnants of the human light chain subgroup Kappa (VL6I) of a person and in essence the consensus FR-remnants of the human heavy chain subgroup III (VHIII) person. Cm. also WO 2004/05631 (Lowman et al.).

Variable region of the heavy chain can be attached to the constant region of IgG, where the area may be, for example, IgG1 or IgG3, which includes natural sequence and variant const areas.

In the preferred embodiment, this antibody contains the sequence of the variable domain of the heavy chain SEQ ID NO:8 (v16, shown in Fig. 1B), optional contains the sequence of the variable domain of the light chain of SEQ ID NO:2 (v16, shown in Fig. 1A), and this antibody is optionally contains one or more amino acid substitutions at positions 56, 100 and/or 100A, for example D56A, N100A or N100Y and/or S100R, variable domain of the heavy chain and one or more amino acid substitutions at positions 32 and/or 92, for example M32L and/or S92A, variable domain of the light chain. Preferably, this antibody is an intact antibody containing the amino acid sequence of the light chain of SEQ ID NO:13 or 16 and amino acid sequence of the heavy chain SEQ ID NO:14, 15, 17, 22, or 25.

Preferred humanized antibody N is ocrelizumab (Genentech).

This antibody may further comprise at least one amino acid substitution in the Fc region that improves ADCC activity, for example, so that the amino acid substitutions are at positions 298, 333, and 334, predpochtitelno S298A, E333A, and K334A, using Eu numbering of residues of the heavy chain. Cm. also U.S. Patent No. L, Presta.

Any of these antibodies may contain at least one substitution in the Fc region that improves FcRn-binding or the period of existence in serum, for example a substitution at position 434 of the heavy chain, such as N434W. Cm. also U.S. Patent No. L, Presta.

Any of these antibodies can further comprise at least one substitution in the Fc region that improves the activity of the CDC, for example, at least a substitution at position 326, preferably K326A or 326W. Cm. also U.S. Patent No. V (Idusogie et al.).

Some preferred humanized variants N are cases that contain a variable domain light chain SEQ ID NO:2 and the variable domain of the heavy chain SEQ ID NO:8, including variants with substitutions or without substitutions in the Fc-region (if present), and variants containing the variable domain of the heavy chain with changes N100A; or D56A and N100A; or D56A, N100Y and S100aR; SEQ ID NO:8, and the variable domain of the light chain with changes M32L; or S92A; or N32L and S92A; SEQ ID NO:2.

M in the variable domain of the heavy chain 27.v16 was identified as a potential source of stability antibodies and is another potential candidate for replacement.

In summarizing some of the various preferred versions of the invention, variabilisation options based 27.v16 contains the amino acid sequence v16, except for the provisions of amino acid substitutions, which are shown in the table below. Unless otherwise noted, these options N will have the same light chain, which light chain v16.

Examples options gumanitarnogo antibodies N
Version NChange the heavy chain (VH)Change light chain (VL)Change Fc
16 for links-
31--S298A, E333A, K334A
73N100AM32L
75N100AM32LS298A, E333A, K334A
96D56A, N100AS92A
114D56A, N100AM32L, S92AS298A, E333A, K334A
115D56A, N100AM32L, S92AS298A, E333A, K334A, E356D, M358L
116D56A, N100AM32L, S92AS298A, E334A, K322A
138D56A, N100AM32L, S92AS298A, E333A, K334A, K326A
477D56A, N100AM32L, S92AS298A, E333A, K334A, K326A, N434W
375--K334L
588--S298A, E333A, K334A, K326A
511D56A, N100Y, S100aRM32L, S92AS298A, E333A, K334A, K326A

One preferred humanitariannet antibody N contains the sequence of the variable domain of the light chain 27.v16:

and the sequence of the variable domain of the heavy chain 27.v16:

When humanitariannet antibody 27.v16 is intak the major antibody it can contain the amino acid sequence of light chain:

and amino acid sequence of the heavy chain SEQ ID NO:14, or

Another preferred humanitariannet antibody N contains the sequence of the variable domain of the light chain 27.v511:

and the sequence of the variable domain of the heavy chain 27.v511:

When humanitariannet antibody 27.v511 is intact antibody, it may contain the amino acid sequence of light chain:

and amino acid sequence of the heavy chain SEQ ID NO:17 or:

1. A method of treating multiple sclerosis in a subject, involving the administration of an effective amount of the antibody against CD20 this entity to conduct the initial effects of the antibodies in the amount of about 0.5 to 4 grams followed by a second exposure to the antibodies in the amount of about 0.5 to 4 grams, and this second exposure is not carried out until approximately 16-60 weeks after initial exposure, and each of these influences antibodies teach the subject as one or two doses of the antibody.

2. The method according to claim 1, where the second exposure is not carried out until approximately 20-30 weeks after the initial impact.

3. The method according to claim 1, where the initial and second the impact of antibodies, each carried out in amounts of about 1.5 to 2.5 grams.

4. The method according to claim 2, additionally providing for the introduction to that subject an effective amount of the antibody against CD20 for the third exposure to the antibodies in the amount of about 0.5 to 4 grams, and the third is the impact of not hold up to approximately 46-60 weeks after the initial impact.

5. The method according to claim 4, where the third effect of the antibody is carried out in the amount of about 1.5 to 2.5 grams.

6. The method according to claim 4, where the third effect of h is to spend approximately 46 to 54 weeks after the initial impact.

7. The method according to claim 4, where the additional impact of antibodies does not hold up to at least about 70-75 weeks after the initial impact.

8. The method according to claim 1, where the second exposure is not carried out until approximately 46-60 weeks after the initial impact.

9. The method according to claim 1, where each of the impacts antibodies teach the subject in a single dose of the antibody.

10. The method according to claim 1, where each of the impacts antibodies teach the subject in the form of two doses of antibodies, where these two doses are first dose and second dose.

11. The method according to claim 10, where the second dose is administered over a period of about 3-17 days after the first dose.

12. The method according to claim 11, where the second dose is administered over a period of about 6 to 16 days after the first dose.

13. The method according to item 12, where the second dose is administered over a period of about 13-16 days after the first dose.

14. The method according to claim 10, where the first and second dose of antibodies, each of approximately 0.5 to 1.5 grams.

15. The method according to claim 10, where the first and second dose of antibodies, each approximately 0.75 to 1.3 grams.

16. The method according to claim 1, where the subject performed three or more of the effects of antibodies.

17. The method according to claim 1, where the subject performed four or more effects of antibodies.

18. The method according to any one of claims 1 to 17, where the second drug is administered with the initial exposure or later impacts, the rich, the antibody against CD20 is the first medicine.

19. The method according to p, where the second drug selected from the group consisting of interferon, latinamericana, a cytotoxic agent, chemotherapeutic agent, mitoxantrone, methotrexate, cyclophosphamide, hlorambuzila, azathioprine, gamma-globulin, Kampala, anti-CD4, cladribine, corticosteroids, mycophenolate-mofetil (MMF), cyclosporine, drug class of statins, lowering cholesterol, estradiol, testosterone, drugs hormone replacement therapy, TNF inhibitor, disease modifying Antirheumatic means (DMARDs), non-steroidal anti-inflammatory drugs (NSAID), levothyroxine, cyclosporin a analogue somatostatin, a cytokine or a receptor antagonist of cytokine, antimetabolite, an immunosuppressive agent, an integrin antagonist or antibody against integrin, antibodies against LFA-1, efalizumab, antibodies against alpha-4-integrin, natalizumab and antibodies against another surface marker of b-cells.

20. The method according to any one of claims 1 to 17, where multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS).

21. The method according to any one of claims 1 to 17, where MS is primary progressive multiple sclerosis (PPMS).

22. The method according to any one of claims 1 to 17, where the subject has never been treated with antibody against CD20.

23. The method according to any one of claims 1 to 17, in which the antibody is unbound antibody.

24. The method according to any one of claims 1 to 17, where the antibody is conjugated with another molecule.

25. The method according to paragraph 24, where the other molecule is a cytotoxic agent.

26. The method according to any one of claims 1 to 17, where the antibody is administered intravenously.

27. The method according to p, where the antibody is injected for each exposure to antibodies.

28. The method according to any one of claims 1 to 17, where the antibody is administered subcutaneously.

29. The method according to any one of claims 1 to 17, where the antibody is administered vnutriobolochechnoe.

30. The method according to any one of claims 1 to 17, where the antibody against CD20 is the only drug, administered to a subject to treat multiple sclerosis.

31. The method according to any one of claims 1 to 17, where this antibody is rituximab.

32. The method according to any one of claims 1 to 17, in which the antibody is humanized N containing the sequence of the variable domain that is represented in SEQ ID NO: 2 and 8.

33. The method according to any one of claims 1 to 17, in which the antibody is humanized N containing the sequence of the variable domain that is represented in SEQ ID NO: 23 and 24.

34. The method according to any one of claims 1 to 17, where the subject has elevated levels (elevated levels of antibodies against myelin basic protein (MBP)antibodies against myelin oligodendrotsitarnym glycoprotein (MOG), anti the ate against ganglioside and/or antibodies against neurofilament.

35. The method according to any one of claims 1 to 17, where elevated levels of b-cells are present in the cerebrospinal fluid (CSF), the damage due to multiple sclerosis, or serum of the subject.

36. Product contains:
(a) a container containing an antibody against the CD20; and
(b) a package insert with instructions regarding the treatment of multiple sclerosis in the subject, where these instructions indicate that the subject enter the amount of antibody that is effective to provide an initial exposure to the antibodies in the amount of about 0.5 to 4 grams followed by a second exposure to the antibodies in the amount of about 0.5 to 4 grams, and this is the second effect of the antibody does not hold up to approximately 16-60 weeks after initial exposure, and each of impacts antibodies teach the subject as one or two doses of the antibody.

37. Product by p, optionally containing container containing a second drug, where the antibody against CD20 is the first medicine, and optionally containing instructions on the package insert treatment of the subject of the second drug.

38. The product according to clause 37, where the second drug selected from the group consisting of interferon, latinamericana, a cytotoxic agent, chemotherapeutic agent, mitoxantrone, meth is trexate, cyclophosphamide, hlorambuzila, azathioprine, gamma-globulin, Kampala, anti-CD4, cladribine, corticosteroids, mycophenolate-mofetil (MMF), cyclosporine, drug class of statins, lowering cholesterol, estradiol, testosterone, drugs hormone replacement therapy, TNF inhibitor, disease modifying Antirheumatic means (DMARDs), non-steroidal anti-inflammatory drugs (NSAID), levothyroxine, cyclosporin a, an analogue of somatostatin, cytokine or receptor antagonist cytokine, antimetabolite, immunosuppressive agent and antibodies against another surface marker of b-cells.



 

Same patents:

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: substance of the invention involves sterile liquid or dry specifically active F(ab')2-fragments of anti-anthrax antibodies containing (35±5) mg·cm3 of protein and at least 96% of F(ab')2-fragments of antibodies recovered from liquid equine anti-anthrax immunoglobulin prepared of blood serum of horses preliminary immunised with strains B anthracis "СТИ"-1 and Ichtiman, and also a anthrax toxin produced by the Kohn's spirit deposition method.

EFFECT: lower reactogenicity and improved immunogenicity.

1 tbl

FIELD: medicine.

SUBSTANCE: aqueous pharmaceutical preparation contains therapeutically effective amount of Cetuximab or EMD 72000, or one of the appropriate murine, humanised or chimeric analogues of an antibody, a buffer of pH 5.2 to 6.0, amino acid chosen from the group containing L-arginin, glycine and L-methionine, and a surface-active substance chosen from the group containing polyethylene sorbitan fatty acid ester and polyoxyethylene-polyoxypropylene copolymer provided that it does not contain sugar.

EFFECT: offered aqueous preparation high temperatures, and can be used for treatment of tumours.

10 cl, 5 ex, 1 tbl

Therapy of mycoses // 2380116

FIELD: medicine.

SUBSTANCE: invention covers a composition applied in therapy of infections caused by fungi Aspergillus in human and animals. The composition contains an antibody or its antigen binding fragment specific at least to one epitope hsp90 from an organism Aspergillus, and at least one antimycotic means chosen from the group containing itraconazole, posaconazole and voriconazole. Epitope is presented by peptides with certain amino acid sequences presented in the description. There is a combined preparation based on the composition and intended for simultaneous, separate or consecutive application in treating said infection. The invention shows possibility to use the composition in implementing the method for preparing a drug of said appointment. There is also described therapy of aspergillosis with using the composition and also a set comprising the drug described above, used in treating the infections caused by fungi Aspergillus.

EFFECT: invention allows administering lower doses of drugs without reducing clinical effectiveness, and improving clinical effectiveness in standard doses with reducing herewith undesired by-effects.

29 cl, 4 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns methods and compositions for inducing apoptosis of cancer cells. Substance of the invention includes the modified antibodies-DR-5 agonists which in combination with apoptosis-inducing agents, synergetically induce apoptosis of cancer cells.

EFFECT: intensified antineoplastic activity.

19 cl, 51 ex, 2 tbl, 35 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely oncology and can be used in treatment of tumours. The method involves introduction of an autologous vaccine representing heat-shock proteins recovered from tumour tissue of the patient in a complex with tumour antigens. Thus treatment with the autologous vaccine is preceded with introduction of an oncolytic attenuated Newcastle disease virus as an adjuvant.

EFFECT: application of the invention allows improving clinical effectiveness in tumours ensured by formation of a vaccine-induced specific immune response against tumour and additional stimulation of nonspecific immunity combined with cytolytic activity of the adjuvant.

2 cl, 4 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns a prophylactic drug for vasculitis. Substance of the invention includes a preventive and/or therapeutic drug for vasculitis containing an antibody to an interleukine-6 (IL-6) receptor as an active ingredient and also method for vasculitis prevention and/or treatment.

EFFECT: development of the new drug expressing therapeutic effectiveness in vasculitis treatment.

39 cl, 4 ex, 6 dwg

FIELD: medicine.

SUBSTANCE: invention includes description of novel pharmaceutical combination, pharmaceutical composition, which includes said combination, application of said combination for obtaining medication and commercial packing. Combination includes (a) at least one anti-inflammatory agent, selected from group, which includes inhibitors of topoisomerase I and inhibitors of topoisomerase II, and (b) epothilone B derivative of formula (I), and optionally at least one pharmaceutically acceptable carrier, for simultaneous, separate or successive application.

EFFECT: obtaining of effective combination for treatment of proliferative disease, namely disease connected with considerable tumor and resistant to other medications.

12 cl

FIELD: veterinary science.

SUBSTANCE: invention relates to veterinary virology and biotechnology. The vaccine contains an active substance and a target additive. The vaccine active substance is represented by a mixture of avirulent refined antigen material from the VNIIZZh strain of cattle rednose virus, Herpesviridae family, Varicellavirus genus, collection of Federal State Institution "Russian National Research Institute for Control, Standardisation and Certification of Veterinary Preparations", "VNIIZZh-DEP", and of avirulent refined antigen material from the VNIIZZh strain of cattle coronavirus, Coronaviridae family, Coronavirus genus, collection of Federal State Institution "Russian National Research Institute for Control, Standardisation and Certification of Veterinary Preparations", "VNIIZZh-DEP", taken at a ratio of 1:1 and in quantities ensuring protective immune activity of each antigen in the animal organism after receipt of the target product injection. With the animals having been immunised the vaccine induces a high level of antigens against the pathogens of cattle rednose and coronoviral infection and is capable to protect varied age and sex group cattle livestock against the above diseases.

EFFECT: with the animals having been vaccinated immunity is generated within 10-15 days after the vaccine re-injection and sustains for at least 6 months.

12 cl, 6 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely oncology, and aims at inducing the immune response in patients with higher levels of soluble receptors of tumour necrosis factor (sTNFRl, sTNFR2) and soluble receptors of interleukine-2 (sIL2R). Therefor, the patient's blood purpose circulates through a filter or a column wherein contacts to effective amount of binding partners. Said binding partners represent immobilised TNFRI, TNFR2 and IL2 or immobilised antibodies binding with sTNFRl, sTNFR2 and sIL2. The blood or plasma levels of receptors are reduced until they become below the normal. It involves at least 12 procedures. The amount of treated plasma per one procedure is approximately equal to one volume of extracellular fluid.

EFFECT: invention allows for selective removal of soluble cytokine receptors with inducing the immune response and ensuring remission of the diseases characterised by high production of sTNFRl, sTNFR2 and sIL2 including malignant tumours.

10 cl, 4 tbl, 1 ex, 5 dwg

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: medicine, pharmaceutical industry and technology, pharmacy.

SUBSTANCE: invention relates to a composition eliciting an antiviral effect. The composition comprises hydrophilic conglomerate of immunoglobulins consortium adsorbed with polyethylene glycol 4000-6000, recombinant interferon-α2 and a special additive taken among the following substances: glycine, glucose, maltose, sodium chloride taken in the definite ratio of components. Invention provides elevating solubility of composition eliciting an antiviral effect and enhanced release of biologically active substances to solution.

EFFECT: valuable medicinal properties of composition.

5 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a composition eliciting an antibacterial effect. Composition comprises hydrophilic conglomerate of immunoglobulins consortium adsorbed with polyethylene glycol 4000-6000 and a special additive taken among the following substances: glycine, glucose, maltose, sodium chloride taken in the definite ratio of components. Invention provides sufficient desorption of biologically active substances in resuspending the composition eliciting an antibacterial effect and comprising consortium of immunoglobulins.

EFFECT: valuable medicinal properties of composition.

5 ex

FIELD: immunology.

SUBSTANCE: the innovation deals with new immunogenic conjugates of beta-propionamide-bound polysaccharide and N-propionamide-bound oligosaccharide with protein, and the method to obtain these conjugates has been suggested, as well. Conjugates should be applied to obtain vaccines against infectious diseases and cancer that enables to broaden the number of preparations applied in treating the above-mentioned diseases.

EFFECT: higher efficiency.

1 dwg, 2 ex, 8 tbl

FIELD: microbiology and immunology, in particular immunodiagnosis.

SUBSTANCE: atypical strain of melioidose Burkholderia pseudomallei-111-6-1 with altered phenotype defected with respect to synthesis of 8 antigen and acting as immunosuppressor is used as antigen for animal immunization. Immune serum is obtained after 2 immunization cycles of animal-producer with titer in gel immunodiffusion reaction not less than 1:128.

EFFECT: immune serum with increased specific activity.

2 tbl, 2 ex

FIELD: medicine and immunology, in particular treatment and prevention immunodeficiency conditions and diseases associated with bacterial or viral aggression.

SUBSTANCE: claimed method includes administration to a patient immunoglobulin drug (e.g., pharmaceutical composition containing 6-12 % of specific heterologous secreted immunoglobulin A, isolated from milk or foremilk of immunized ungulates). Administration is performed parenterally wherein single dose is at least 10 IU/kg of patient weight for treatment or at least 5 IU/kg for prophylaxis; or perorally in dose of 0.2-0.5 g and/or topically one-two times per day for 1-5 days. Method of present invention makes it possible to decrease dose of administrating immunoglobulin due to prolonged retention of its high titers in body fluids.

EFFECT: enlarged range of application and assortment of immunoglobulin drugs.

4 cl, 5 ex

FIELD: pharmaceutics.

SUBSTANCE: the present innovation deals with cryoprotective ointment containing recombinant interferon-α2. The suggested cryoprotective ointment contains recombinant interferon-α2, glycerol, polyethylene glycol 300-6000, polyglucin, buffered 0.02%-Trilon B solution at pH of 5.5-7.0 and ointment foundation at a certain content of components per 1.0 g ointment. Additionally, cryoprotective ointment could contain glycine 3,7-bis(dimethylamino)phenothiazonium chloride, dry immunoglobulin preparation or dry immunoglobulin preparation for enteral application. Ointment foundation of cryoprotective ointment could contain water-free lanolin, Vaseline and Vaseline oil, at the following ratio of components: 2.5;3.5:1 - 6.5:0.5:1. The innovation provides maximal safety of recombinant interferon-α2 activity in cryoprotective ointment at multiple alteration of positive and negative environmental temperature and at keeping cryoprotective ointment under these conditions.

EFFECT: higher efficiency of application.

8 cl, 8 ex

FIELD: medicine, pharmaceutics, pharmacology.

SUBSTANCE: one should apply mammalian anti-HBP-antibodies. The ways are being suggested to identify monoclonal antibody bound, at least, with one epitope upon native HBP (heparin-binding protein) and methods to detect whether a mammal produces HBR being bound with a monoclonal antibody and, also, the kits for the above-mentioned purpose. The present innovation provides the opportunity to apply the mentioned antibodies in preventing and treating disorders associated with bradykinin releasing.

EFFECT: higher efficiency of application.

25 cl, 11 dwg, 3 ex, 1 tbl

FIELD: veterinary science.

SUBSTANCE: animals should be introduced with antihistamine serum (AHS) subcutaneously at the dosage of 4.0-5.0 ml in combination with myxoferon at the quantity of 60-75 dosages and vitamin C at the dosage of 1.0-1.5 ml/animal, once daily, thrice at interval of 5-7 d. Application of AHS in combination with myxoferon and ascorbic acid provides active stimulation of immunological reactivity, increases total body resistance I animals and causes no toxic effects and allergic reactions.

EFFECT: higher efficiency of correction.

3 tbl

FIELD: immunology, biotechnology, medicine.

SUBSTANCE: invention relates to antiidiotypical monoclonal antibody or fragment thereof for BSW17 antibody effecting on LgE Cε3-region bonding to high affinity LgE receptor. Amino acid sequence is as described in specification. antiidiotypical antibody is useful as pharmaceutical composition ingredient for LgE-mediated disease treatment. Invention make in possible to prevent allergic disorders and inflammations due to inhibiting interaction between LgE Cε3-region with high affinity receptor by claimed antibody.

EFFECT: new agent for allergic and inflammation disorder treatment.

7 cl, 32 dwg, 5 tbl, 10 ex

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