Methods for treating progressive multiple sclerosis

Methods of treating progressive multiple sclerosis

The present invention relates to a method for treating patients with progressive multiple sclerosis (MS) and a product with instructions for the use.

This application claims the priority of U.S. Provisional Application No. 61/097,464 filed on September 16, 2008, which is incorporated herein by reference in its entirety.

<i>Multiple sclerosis</i>

Multiple sclerosis (MS) is an inflammatory and demyelinating degenerative disease of the human central nervous system (CNS). It is a disease that exists all over the world, with about 300,000 people in the United States suffering from the disease; it is a disease commonly suffered by young people, of which 70% to 80% develops between the ages of 20 and 40 (Anderson et al.<i>Ann Neurology</i> 31(3):333-6(1992); Noonan et al.<i>Neurology</i> 58:136-8 (2002)). Based on clinical course, magnetic resonance imaging (MRI) scan evaluation, biopsy and pathological analysis of autopsy materials, MS is a heterogeneous disease (Lucchinetti et al.<i>Ann Neurol</i> 47:707-17 (2000)). The disease itself exhibits a large number of possible combinations of defects, including spinal cord, brain stem, cranial nerves, cerebellum, cerebrum, and cognitive syndrome. Especially when under observation for 25 years, most patients with MS eventually develop disability. Half of MS patients require walking sticks within 15 years of the onset of the disease. MS is the main cause of neurological disability in young and middle-aged people, and until the past ten years there is no known effective treatment for it. MS is difficult to diagnose due to non-specific clinical findings, leading to the development of highly structured diagnostic criteria, including several technological advances consisting of MRI scans, evoked potentials, and cerebrospinal fluid (CSF) research. All diagnostic criteria rely on the following general principles: There are scattered lesions in the central white matter that appear at different times and cannot be explained by other causes (such as infection, vascular disease, or autoimmune disease) (McDonald et al.<i>Ann Neurol</i> 50:121-7 (2001)). MS has four disease types: relapsing-remitting MS (RRMS; 80%-85% of cases), primary progressive MS (PPMS, 10%-15% of cases), and progressive relapsing MS (PRMS, 5%) Incident cases) and secondary progressive MS (SPMS) (Kremenchutzky et al.<i>Brain</i> 122(Pt 10):1941-50(1999); Confavreux et al.<i>N Engl J Med</i> 343(20):1430-8(2000)). It is estimated that 50% of patients with RRMS will develop SPMS within 10 years; and up to 90% of RRMS patients will eventually develop SPMS (Weinshenker et al.<i>Brain</i> 112(Pt 1):133-46 (1989)).

Currently, there are four categories of six drugs approved for the treatment of RRMS in the United States, but no drugs are approved for the treatment of PPMS. RRMS treatment includes the following: interferons, IFN-β-1a (<img file="TW201014605A_D0001.tif" />and<img file="TW201014605A_D0002.tif" />) And IFN-β-1b(<img file="TW201014605A_D0003.tif" />); glatiramer acetate (<img file="TW201014605A_D0004.tif" />) (A polypeptide); natalizumab (TYSABRI<img file="TW201014605A_D0005.tif" />); and mitoxantrone (mitoxantrone) (<img file="TW201014605A_D0006.tif" />) (A cytotoxic agent). Other drugs have been used with varying degrees of success, including corticosteroids, methotrexate, cyclophosphamide, azathioprine and intravenous (IV) immunoglobulin. The benefits of currently approved treatments are that, as shown by two comprehensive analyses, the recurrence rate of RRMS is relatively low (about 30%) and the prevention of disability (Filippini et al.<i>Lancet </i>361:545-52(2003))。

Other clinical studies have evaluated other immunomodulators of MS, including tumor necrosis factor alpha inhibitors and modified peptide ligands, which worsen MS rather than improve MS (Lanaercept Multiple Sclerosis Research Group and University of British Columbia MS/MRI (Lenercept Multiple Sclerosis Study Group and the University of British Columbia MS/MRI)<i>Neurology</i> 53:457-65 (1999); Bielekova et al.<i>Nat Med</i> 2000;6:1167-75 (2000), the errata is in<i>Nat Med</i> Published in 6:1412 (2000)).

The main points of MS pathophysiology support that inflammation is mainly caused by CD4⁺ Th1 T cell mediated. The treatment method is based on theories such as IFN-β and glatiramer acetate reduce (but not completely prevent) the occurrence of deterioration or accumulation of disability.

As evidenced by the presence of CSF oligoselective bands and increased intrathecal IgG synthesis in the diagnostic criteria for MS, it has been defaulted for decades that there is a body fluid component in human MS (Siden A. <i>J Neurol</i> 221:39-51 (1979); McDonald et al.<i>Ann Neurol</i> 50:121-7 (2001); Andersson et al.<i>Eur J Neurol</i> 9:243-51 (2002); O'COnnor, P. <i>Neurology</i> 59: S1-33 (2002)). The presence of oligoptic bands, increased free light chains, and increased intrathecal IgM synthesis are associated with MS disease activity and may be predictors of more severe outcomes (Rudick et al.<i>Mult Scler</i> 1:150-5 (1995); Zeman et al.<i>Acta Cytol</i> 45:51-9 (2001); Izquierdo et al.<i>Acta Neurol Scand</i> 105:158-63 (2002); Wolinsky J. <i>J Neurol Sci</i> 206:145-52 (2003); Villar et al.<i>Ann Neurol</i> 53:222-6(2003))。

Anti-myelin antibodies (myelin basic protein (MBP) and myelin oligodendritic glial cell glycoprotein (MOG)) were detected in the serum of patients with progressive and relapsing MS (Reindl et al.<i>Brain</i> 122:2047-56 (1999); Egg et al.<i>Mult Scler</i> 7(5):285-9 (2001)). Anti-myelin antibodies were also detected in the CSF of MS patients (Reindl et al.<i>Brain</i> 122:2047-56 (1999); Egg et al.<i>Mult Scler</i> 7(5):285-9(2001); Andersson et al.<i>Eur J Neurol</i> 9:243-51 (2002)). Other types of antibodies have been observed in patients with MS, such as anti-neurodesin antibodies or anti-neurociliary antibodies (Mata et al.<i>Mult Scler</i> 5:379-88 (1999); Sadatipour et al.<i>Ann Neurol</i> 44:980-3 (1998)). The report indicates that the presence of serum anti-MOG and anti-MBP antibodies is a strong predictor of the progression from a clinically isolated demyelination event to a clear RRMS (Berger et al.<i>N Engl J Med</i> 349:139-45 (2003)). The adjusted risk ratio for patients who were seropositive for both antibodies to experience worsening was 76.5, and the adjusted risk ratio for patients who were only seropositive for anti-MOG to experience worsening was 31.6.

The International Association of Pathology has found that most patients with MS have antibodies that bind to myelin. Plasma cells and B cells have also been found in MS lesions, providing additional evidence for the presence of humoral effects in MS (Prineas and Wright,<i>Lab Invest</i> 38:409-21 (1978); Esiri M. <i>Neuropathol Appl Neurobiol</i> 6:9-21 (1980); Genain et al.<i>Nat Med</i> 5:170-5 (1999); Lucchinetti et al.<i>Ann Neurol</i> 47:707-17 (2000); Wingerchuk et al.<i>Lab Invest</i> 81:263-81 (2001)). B cells can be detected in the CSF of patients with MS and the presence of a relatively high proportion of B cells can predict the progression to more severe disabilities (Cepok et al.<i>Brain</i> 124(Pt 11): 2169-76 (2001)).

In patients with RRMS or ocular clonic-myoclonus syndrome, rituximab (Rituximab) has been reported to deplete peripheral B cells in all patients and reduce the number of CSF B cells in some patients (Pranzatelli et al.<i>Neurology</i> 60 (Supplement 1) PO5.128:A395 (2003); Cross et al. "Preliminary Results from a Phase II Trial of Rituximab in MS" (Abstract)<i>Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis ACTRIMS</i> 20-1 (October 2003); Cross et al.<i>J</i>.<i> Neuroimmunol.</i> 180:63-70 (2006)). See also Cree et al. "Tolerability and Effects of Rituximab "Anti-CD20 Antibody" in Neuromyelitis Optica and Rapidly Worsening Multiple Sclerosis"<i>Meeting of the Am. Acad. Neurol.</i>(April 2004); Cree et al<i>Neurology</i> 64:1270-2(2005)。

<i>CD20 antibody and therapies using it</i>

Lymphocytes are one of many types of white blood cells produced in the bone marrow during the hematopoiesis process. There are two main lymphocyte populations: B lymphocytes (B cells) and T lymphocytes (T cells). The lymphocytes of particular interest in this article are B cells.

B cells mature in the bone marrow and leave the bone marrow, expressing antigen-binding antibodies on the surface of their cells. When a native B cell first encounters an antigen specific to its membrane-bound antibody, the cell begins to divide rapidly, and its offspring differentiate into memory B cells and effector cells called "plasma cells". Memory B cells have a longer lifespan and will continue to express membrane-bound antibodies with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibodies but instead produce antibodies in a secretable form. The secreted antibodies are the main effector molecules of humoral immunity.

CD20 antigen (also known as human B lymphocyte restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of about 35kD located on pre-B lymphocytes and mature B lymphocytes (Valentine et al.<i>J. Biol. Chem.</i> 264(19):11282-11287 (1989); and Einfeld et al.<i>EMBO J.</i> 7(3):711-717 (1988)). The antigen is also expressed in more than 90% of B-cell non-Hodgkin's lymphomas (NHL) (Anderson et al.<i>Blood</i> 63(6):1424-1433 (1984)), but not found on hematopoietic stem cells, progenitor B cells, normal plasma cells or other normal tissues (Tedder et al.<i>J. Immunol.</i> 135(2):973-979(1985)). CD20 regulates the early stages of the activation process of cell cycle initiation and differentiation (Tedder et al., supra) and may act as a calcium ion channel (Tedder et al.<i>J. Cell. Biochem.</i> 14D:195 (1990)).

Given that CD20 is expressed in B-cell lymphoma, this antigen can be a candidate for "targeting" this type of lymphoma. Essentially, the targeting can be summarized as follows: the patient is administered an antibody specific for the CD20 surface antigen of B cells. These anti-CD20 antibodies specifically bind to the CD20 antigen of normal B cells and malignant B cells (on the surface); antibodies that bind to the CD20 surface antigen can cause the destruction and depletion of neoplastic B cells. In addition, chemical agents or radiolabels that have the potential to destroy tumors can be combined with anti-CD20 antibodies to specifically "deliver" the agents to neoplastic B cells. Regardless of the method used, the main purpose is to destroy the tumor; the specific method can be determined by the specific anti-CD20 antibody used and therefore the method that can be used to target the CD20 antigen can be significantly different.

Rituximab (RITUXAN<img file="TW201014605A_D0007.tif" />) The antibody is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen. Rituximab is an antibody called "C2B8" in US Patent No. 5,736,137 (Anderson et al.) issued on April 7, 1998. RITUXAN<img file="TW201014605A_D0008.tif" />Designated for the treatment of patients with relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin's lymphoma. In vitro studies of the mechanism of action show that RITUXAN<img file="TW201014605A_D0009.tif" />Binding to human complement and lysing lymphoid B cell lines via complement-dependent cytotoxicity (CDC) (Reff et al.<i>Blood</i> 83(2):435-445 (1994)). In addition, it has significant activity in the antibody-dependent cellular cytotoxicity (ADCC) assay. Recently, RITUXAN has been shown<img file="TW201014605A_D0010.tif" />In the tritiated thymidine incorporation assay, it has an anti-proliferative effect and directly induces cell apoptosis, while other anti-CD19 and CD20 antibodies do not have this effect (Maloney et al.<i>Blood</i> 88(10):637a (1996)). RITUXAN has also been observed in experiments<img file="TW201014605A_D0011.tif" />There is a synergy with chemotherapy and toxins. In detail, RITUXAN<img file="TW201014605A_D0012.tif" />Sensitize drug-resistant human B-cell lymphoma cell lines to the cytotoxic effects of cranberry (doxorubicin), CDDP, VP-16, diphtheria toxin and ricin (Demidem et al.<i>Cancer Chemotherapy &amp; Radiopharmaceuticals</i> 12(3):177-186 (1997)). In vivo preclinical research show RITUXAN<img file="TW201014605A_D0013.tif" />The B cells in peripheral blood, lymph nodes and bone marrow of macaques may be consumed through complement and cell-mediated processes (Reff et al.<i>Blood</i> 83(2):435-445(1994))。

Rituximab was approved in the United States in November 1997 at 375 mg/m per week²Four doses are administered to treat patients with relapsed or refractory low-grade or follicular CD20⁺ B-cell non-Hodgkin's lymphoma (NHL). In April 2001, the Food and Drug Administration (FDA) approved other claims for the treatment of low-grade NHL: retreatment (four times a week dosing) and other dosing schedules (eight times a week dosing). More than 300,000 patients have received rituximab monotherapy or combination therapy with immunosuppressive drugs or chemotherapy drugs. Patients have also been treated with rituximab maintenance therapy for up to 2 years (Hainsworth et al.<i>J Clin Oncol</i> 21:1746-51 (2003); Hainsworth et al.<i>J Clin Oncol</i> 20:4261-7(2002))。

Rituximab has also been studied in a variety of non-malignant autoimmune disorders, in which B cells and autoantibodies seem to play a role in the pathophysiology of the disease (Edwards et al.<i>Biochem Soc Trans</i> 30:824-8 (2002)). It has been reported that rituximab may relieve the signs and symptoms of the following diseases: rheumatoid arthritis (RA) (Leandro et al.<i>Ann Rheum Dis</i>. 61:883-8 (2002); Emery et al.<i>Arthritis Rheum</i> 48(9):S439(2003)); Lupus (Eisenberg R. <i>Arthritis Res Ther</i> 5:157-9 (2003); Leandro et al.<i>Arthritis Rheum</i> 46:2673-7 (2002)); Immune Thrombocytopenia (D'Arena et al.<i>Leuk Lymphoma</i> 44:561-2 (2003)); autoimmune anemia (Zaja et al.<i>Haematologica</i> 87:189-95 (2002) (The errata is in<i>Haematologica</i> 87:336 (published in 2002)); autoimmune neuropathy (Pestronk et al.<i>J Neurol Neurosurg Psychiatry</i> 74:485-9 (2003)); paraneoplastic ocular clonus-myoclonus syndrome (Pranzatelli et al.<i>Neurology</i> 60 (Supplement 1) PO5.128:A395 (2003)); and relapsing-remitting multiple sclerosis (RRMS) (Cross et al. (Abstract) The Eighth Annual Meeting of the American Committee for Research and Treatment of Multiple Sclerosis (Eighth Annual) Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis) 20-1 (2003)).

A phase II study (WA16291) was conducted on patients with rheumatoid arthritis (RA), providing 48-week follow-up data on the safety and efficacy of rituximab (Emery et al.<i>Arthritis Rheum</i> 48(9):S439(2003); Szczepanski et al.<i>Arthritis Rheum</i> 48(9): S121(2003)). A total of 161 patients were randomized to four treatment groups on average: methotrexate, rituximab only, rituximab plus methotrexate, rituximab plus cyclophosphamide (CTX) . The treatment regimen of rituximab is to administer 1 g intravenously on the 1st and 15th days. In most patients with RA, infusion of rituximab is well tolerated by most patients, and 36% of patients experienced at least one adverse reaction during their first infusion (compared to those who received a placebo). 30% of patients). All in all, the severity of most adverse reactions is considered mild to moderate and is well balanced among all treatment groups. Within 48 weeks, there were a total of 19 serious adverse reactions in the four groups, slightly more frequent in the rituximab/CTX group. The incidence of infection was well balanced among all groups. The average rate of severe infections in this group of RA patients is 4.66 per 100 patients per year, which is lower than the rate of infections requiring hospitalization among RA patients reported in community epidemiological studies (9.57 per 100 patients per year) (Doran et al.<i>Arthritis Rheum</i> 46:2287-93(2002))。

The reported safety profile of rituximab has been reported in a small number of patients with the following diseases: neurological disorders, including autoimmune neuropathy (Pestronk et al.<i>J Neurol Neurosurg Psychiatry</i> 74:485-9 (2003)); ocular clonus/myoclonus syndrome (Pranzatelli et al.<i>Neurology</i> 60 (Supplement 1) PO5.128:A395 (2003)); and RRMS (Cross et al. Preliminary results from a phase II trial of Rituximab in MS (Abstract) The 8th Annual Meeting of the American Committee on Multiple Sclerosis Research and Treatment ( Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis) 20-1 (2003)). In the ongoing researcher-initiated trial (IST) of rituximab in combination with interferon beta (IFN-beta) or glatiramer acetate in individuals with RRMS (Cross et al., Same as above), one of the 10 treated individuals experienced moderate fever and chills after the first infusion of rituximab and was hospitalized overnight for observation, while the other 9 individuals completed four courses of infusion without reporting any adverse reactions.

Patents and patent publications on CD20 antibodies, CD20 binding molecules and autoantigen vaccines include: US 5,776,456, 5,736,137, 5,843,439, 6,399,061 and 6,682,734 and US 2002/0197255, US 2003/0021781, US 2003/0082172, US 2003/0095963 , US 2003/0147885, US 2005/0186205 and WO 1994/11026 (Anderson et al.); US 6,455,043, US 2003/0026804, US 2003/0206903 and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); US 2004/0213784 and WO 2000/27433 (Grillo-Lopez And Leonard); WO 2000/44788 (Braslawsky et al.); WO 2001/10462 (Rastetter, W.); WO 2001/10461 (Rastetter and White); WO 2001/10460 (White and Grillo-Lopez); US 2001/ 0018041, US 2003/0180292, US 2002/0028178, WO 2001/34194 and WO 2002/22212 (Hanna and Hariharan); US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); US 2002/0012665, US 2005/ 0180975, WO 2001/74388 and US 6,896,885 (Hanna, N.); US 2002/0058029 (Hanna, N.); US 2003/0103971 (Hariharan and Hanna); US 2005/0123540 (Hanna et al.); US 2002/0009444 and WO 2001/80884 (Grillo -Lopez, A.); WO 2001/97858; US 2005/0112060, US 2002/0039557 and US 6,846,476 (White, C.); US 2002/0128448 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 6,171,586 and 6,991,790 and WO 1998/56418 (Lam et al.); US 2004/0191256 and WO 1998/ 58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, US 6,194,551, US 6,242,195, 6,528,624 and 6,538,124 (Idusogie et al.); US 7,122,637, US 2005/0118174, US 2005/0233382, US 2006/0194291, US 2006/0194290, US 2006/0194957 and WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al.); WO 2001/03734 (Grillo-Lopez et al.); US 2002/0004587, US 2006/0025576 and WO 2001/77342 (Miller and Presta); US 2002/0197256 and WO 2002/ 078766 (Grewal, I.); US 2003/0157108 and WO 2003/035835 (Presta, L.); US 5,648,267, 5,733,779, 6,017,733 and 6,159,730 and WO 1994/11523 (Reff et al., on performance technology); US 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398 and 5,595,721 (Kaminski et al.); US 5,500,362, 5,677,180, 5,721,108, 6,120,767, 6,652,852 and 6,893,625 and WO 1988/04936 (Robinson et al., US); 2000/20864 (Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); WO 2000/67795 (Goldenberg); US 7,074,403 (Goldenberg and Hansen); US 7,151,164 (Hansen et al.); US 2003/0133930; WO 2000/74718 and US 2005/0191300A1 (Goldenberg and Hansen); US 2003/0219433 and WO 2003/68821 (Hansen et al.); WO 2004/058298 (Goldenberg and Hansen) ); WO2000/76542 (Golay et al.); WO 2001/72333 (Wolin and Rosenblatt); US 6,368,596 (Ghetie et al.); US 6,306,393 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 2003/0219818 (Bohen et al.); US 2003/0219433 and WO 2003/068821 (Hansen et al.); US 2003/0219818 (Bohen et al.); US 2002/0136719 (Shenoy et al.); WO 2004/032828 and US 2005/0180972 (Wahl et al.); and WO 2002/56910 (Hayden-Ledbetter). See also US 5,849,898 and EP 330,191 (Seed et al.); EP 332,865A2 (Meyer and Weiss); US 4,861,579 (Meyer et al.); US 2001/0056066 (Bugelski et al.); WO 1995/03770 (Bhat et al.); US 2003/0219433 A1 (Hansen et al.); WO 2004/035607 and US 2004/167319 (Teeling et al. Human); WO 2005/103081 (Teeling et al.); US 2006/0034835, US 2006/0024300 and WO 2004/056312 (Lowman et al.); US 2004/0093621 (Shitara et al.); WO 2004/103404 (Watkins et al.) Human); WO 2005/000901 (Tedder et al.); US 2005/0025764 (Watkins et al.); US 2006/0251652 (Watkins et al.); WO 2005/016969 (Carr et al.); US 2005/0069545 (Carr et al. Human); WO 2005/014618 (Chang et al.); US 2005/0079174 (Barbera-Guillem and Nelson); US 2005/0106108 (Leung and Hansen); US 2005/0123546 (Umana et al.); US 2004/0072290 (Umana et al.); US 2003/0175884 (Umana et al.); and WO 2005/044859 (Umana et al.); WO 2005/070963 (Allan et al.); US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and Ledbetter); US 2005/136049 (Ledbetter et al.); US 2003/118592 (Ledbetter et al.); US 2003/133939 (Ledbetter And Hayden-Ledbetter); US 2005/0202012 (Ledbetter and Hayden-Ledbetter); US 2005/0175614 (Ledbetter and Hayden-Ledbetter); US 2005/0180970 (Ledbetter and Hayden-Ledbetter); US 2005/0202028 (Hayden-Ledbetter) And Ledbetter); US 2005/0202023 (Hayden-Ledbetter and Ledbetter); WO 2005/017148 (Ledbetter et al.); WO 2005/037989 (Ledbetter et al.); US 6,183,744 (Goldenberg); US 6,897,044 (Braslawski et al.); WO 2006/005477 (Krause et al.); US 2006/0029543 (Krause et al.); US 2006/0018900 (McCormick et al.); US 2006/0051349 (Goldenberg and Hansen); WO 2006/ 042240 (Iyer and Dunussi-Joannopoulos); US 2006/0121032 (Dahiyat et al.); WO 2006/064121 (Teillaud et al.); US 2006/0153838 (Watkins), CN 1718587 (Chen et al.); WO 2006/084264 ( Adams et al.); US 2006/0188495 (Barron et al.); US 2004/0202658 and WO 2004/091657 (Benynes, K.); US 2005/0095243, US 2005/0163775, WO 2005/00351 and WO 2006/068867 (Chan, A.); US 2006/0135430 and WO 2005/005462 (Chan et al.); US 2005/0032130 and WO 2005/017529 (Beresini et al.); US 2005/0053602 and WO 2005/023302 (Brunetta, P.); US 2006/0179501 and WO 2004/060052 (Chan et al.); WO 2004/060053 (Chan et al.); US 2005/0186206 and WO 2005/060999 (Brunetta, P.); US 2005/0191297 and WO 2005/061542 (Brunetta, P.); US 2006/0002930 and WO 2005/115453 (Brunetta et al.); US 2006/ 0099662 and WO 2005/108989 (Chuntharapai et al.); CN 1420129A (Zhongxin Guojian Pharmaceutical); US 2005/0276803 and WO 2005/113003 (Chan et al.); US 2005/0271658 and WO 2005/117972 (Brunetta et al.); US 2005/0255527 and WO 2005/11428 (Yang, J.); US 2006/0024295 and WO 2005/120437 (Brunetta, P.); US 2006/0051345 and WO 2005/117978 (Frohna, P.); US 2006/0062787 and WO 2006/012508 (Hitraya, E.); US 2006/0067930 and WO 2006/ 31370 (Lowman et al.); WO 2006/29224 (Ashkenazi, A.); US 2006/0110387 and WO 2006/41680 (Brunetta, P.); US 2006/0134111 and WO 2006/066086 (Agarwal, S.); WO 2006/069403 (Ernst and Yansura); US 2006/0188495 and WO 2006/076651 (Dummer, W.); WO 2006/084264 (Lowman, H.); WO 2006/093923 (Quan and Sewell); WO 2006/ 106959 (Numazaki et al.); WO 2006/126069 (Morawala); WO 2006/130458 (Gazit-Bornstein et al.); US 2006/0275284 (Hanna, G.); US 2007/0014785 (Golay et al.); US 2007/0014720 (Gazit-Bornstein et al.); and US 2007/0020259 (Hansen et al.); US 2007/0020265 (Goldenberg and Hansen); US 2007/0014797 (Hitraya); US 2007/0224189 (Lazar et al.); WO 2007/014238 (Bruge and Bruger); and WO 2008/003319 (Parren and Baadsgaard). Some of these especially include the treatment of multiple sclerosis.

Public cases regarding the use of rituximab therapy include: Perotta and Abuel "Response of chronic relapsing ITP of 10 years duration to Rituximab" Abstract No. 3360 <i>Blood</i> 10(1) (Parts 1-2): Page 88B (1998); Stashi et al. "Rituximab chimeric anti-CD20 monoclonal antibody treatment for adults with chronic idopathic thrombocytopenic purpura"<i>Blood</i> 98(4):952-957(2001); Matthews, R. "Medical Heretics"<i>New Scientist</i>(April 7, 2001); Leandro et al. "Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion"<i>Ann Rheum Dis</i> 61:833-888 (2002); Leandro et al. "Lymphocyte depletion in rheumatoid arthritis: early evidence for safety, efficiency and dose response. <i>Arthritis and Rheumatism</i> 44(9):S370(2001); Leandro et al. "An open study of B lymphocyte depletion in systemic lupus erythematosus",<i>Arthritis &amp; Rheumatism</i> 46(1):2673-2677(2002); Edwards and Cambridge "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes"<i>Rhematology</i> 40:205-211 (2001); Edwards et al. "B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders"<i>Biochem. Soc. Trans.</i> 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. <i>Arthritis and Rheumatism</i> 46(9):S197(2002); Levine and Pestronk "IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using Rituximab"<i>Neurology</i> 52:1701-1704 (1999); DeVita et al. "Efficacy of selective Bcell blockade in the treatment of rheumatoid arthritis"<i>Arthritis &amp; Rheum</i> 46:2029-2033 (2002); Hidashida et al. "Treatment of DMARD-Refractory rheumatoid arthritis with Rituximab." At the American Academy of Rheumatology Annual Scientific Meeting (<i>Annual Scientific Meeting of the American College of Rheumatology</i>); October 24-29; Published in New Orleans, LA 2002; Tuscano, J. "Successful treatment of Infliximab-refractory rheumatoid arthritis with Rituximab" at the American Academy of Rheumatology Annual Scientific Meeting (<i>Annual Scientific Meeting of the American College of Rheumatology</i>); October 24-29; Published in New Orleans, LA 2002; Specks et al. "Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy"<i>Arthritis &amp; R</i>heumatism<i></i>44(12):2836-2840(2001); Anolik et al. "B lympocyte Depletion in the Treatment of Systemic Lupus(SLE): Phase I/II Trial of Rituximab(RITUXAN<img file="TW201014605A_D0014.tif" />)in SLE"<i>Arthritis And Rheumatism</i>, 46(9), S289-S289 Abstract 717 (October 2002) and Albert et al. "A Phase I Trial of Rituximab (Anti-CD20) for Treatment of Systemic Lupus Erythematosus"<i>Arthritis And Rheumatism</i>,48(12):3659-3659, abstract LB9 (December 2003); Martin and Chan "Pathogenic Roles of B cells in Human Autoimmunity: Insights from the Clinic"<i>Immunity</i> 20:517-527 (2004); Cree et al. "An open label study of the effects of rituximab in neuromyelitis optica."<i>Neurology</i> 64(7):1270-2(2005): Cross et al. "Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients." J <i>Neuroimmunol</i>,180(1-2):63-70(2006); Bar-Or A. et al. "Safety,pharmacodynamics,and activity of Rituximab in patients with relapsing-remitting multiple sclerosis:a phase I,multicentre,open-label clinical trial."<i>Ann Neurol</i> 63(3):395-400(2008); Hauser S. et al. "B-cell depletion with Rituximab in relapsing-remitting multiple sclerosis."<i>NEJM</i>, 358(7):676-88,(2008); Hawker K et al. "Efficacy and Safety of rituximab in patients with primary progressive multiple sclerosis: results of a randomized, double-blind, placebo-controlled, multicenter trial."<i>Multiple Sclerosis</i> 14(1): S299(2008), abstract; Hawker K et al., "Efficacy and Safety of rituximab in patients with primary progressive multiple sclerosis: results of a randomized, double-blind, placebo-controlled, multicenter trial."<i>Neurology</i> 72(S3): A254(2009), abstract.

The present invention provides a method for treating advanced multiple sclerosis in a patient, which comprises administering to the patient an effective amount of anti-CD20 antibody, wherein the treatment is based on the patient having one or more characteristics selected from the group consisting of: (a) age Less than about 55 years of age, (b) one or more gamma-stained lesions, (c) increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment, and (d) the severity of multiple sclerosis The score (MSSS) is higher than about 5 points.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the patient is less than about 51 years old.

In some embodiments, treatment shortens the time to diagnosed disease progression. In some embodiments, the confirmed disease progression is an increase in EDSS that lasts for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS for twenty-four weeks.

In some embodiments, an effective amount of anti-CD20 antibody is administered to the patient to provide an initial anti-CD20 antibody exposure between about 0.3 grams and about 4.0 grams, followed by a second anti-CD20 antibody exposure between about 0.3 grams and about 4.0 grams. Antibody exposure. In some embodiments, the initial anti-CD20 antibody exposure and/or the second anti-CD20 antibody exposure are between about 0.3 grams and about 1.5 grams. In some embodiments, the second exposure is not provided until about 16 to 60 weeks after the initial exposure. In some embodiments, each anti-CD20 antibody exposure is provided to the patient in the form of one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is ocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumumab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The present invention provides a method for treating patients with progressive multiple sclerosis. The limitation is that the patient has one or more characteristics selected from the group consisting of: (a) younger than about 55 years old, (b) one or more gamma For stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before the start of treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points, and the treatment included An effective amount of anti-CD20 antibody is administered to the patient.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the patient is less than about 51 years old.

In some embodiments, treatment shortens the time to diagnosed disease progression. In some embodiments, the confirmed disease progression is an increase in EDSS that lasts for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS for twenty-four weeks.

In some embodiments, an effective amount of anti-CD20 antibody is administered to the patient to provide an initial anti-CD20 antibody exposure between about 0.3 grams and about 4.0 grams, followed by a second anti-CD20 antibody exposure between about 0.3 grams and about 4.0 grams. Antibody exposure. In some embodiments, the initial anti-CD20 antibody exposure and/or the second anti-CD20 antibody exposure are between about 0.3 grams and about 1.5 grams. In some embodiments, the second exposure is not provided until about 16 to 60 weeks after the initial exposure. In some embodiments, each anti-CD20 antibody exposure is provided to the patient in the form of one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The present invention provides a method for treating progressive multiple sclerosis, which comprises: (a) selecting a patient with progressive multiple sclerosis, wherein the patient has one or more characteristics selected from the group consisting of: (i) age Less than about 55 years of age; (ii) one or more gamma staining lesions, (iii) an increase in the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment, and (iv) the severity of multiple sclerosis The score (MSSS) is higher than about 5 points; and (b) an effective amount of anti-CD20 antibody is administered to the patients thus selected.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the patient is less than about 51 years old.

In some embodiments, treatment shortens the time to diagnosed disease progression. In some embodiments, the confirmed disease progression is an increase in EDSS that lasts for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS for twenty-four weeks.

In some embodiments, an effective amount of anti-CD20 antibody is administered to the patient to provide an initial anti-CD20 antibody exposure between about 0.3 grams and about 4.0 grams, followed by a second anti-CD20 antibody exposure between about 0.3 grams and about 4.0 grams. Antibody exposure. In some embodiments, the initial anti-CD20 antibody exposure and/or the second anti-CD20 antibody exposure are between about 0.3 grams and about 1.5 grams. In some embodiments, the second exposure is not provided until about 16 to 60 weeks after the initial exposure. In some embodiments, each anti-CD20 antibody exposure is provided to the patient in the form of one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The present invention also provides a method for assessing whether a patient with advanced multiple sclerosis responds to treatment with an anti-CD20 antibody, which comprises assessing one or more characteristics selected from the group consisting of: (a) age less than about 55 years old , (B) one or more gamma stained lesions, (c) increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment, and (d) multiple sclerosis severity score (MSSS) Above about 5 points, one or more of these characteristics of the patient indicates that the patient will respond to treatment.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the patient is less than about 51 years old.

In some embodiments, the method further comprises providing advice to the patient.

In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The present invention also provides a method for identifying patients with progressive multiple sclerosis who may be responsive to anti-CD20 antibody therapy, which comprises: (a) evaluating one or more characteristics selected from the group consisting of: (i) younger than Approximately 55 years of age, (ii) one or more gadolinium-stained lesions, (iii) at least about one point increase in the Extended Disability Status Scale (EDSS) within two years before starting treatment, and (iv) multiple sclerosis severity score (MSSS) is higher than about 5 points; and (b) identify patients with one or more characteristics selected from the group consisting of: (i) younger than about 55 years of age, (ii) one or more gamma staining lesions, ( iii) The Extended Disability Status Scale (EDSS) increased by at least about one point within two years before the start of anti-CD20 treatment, and (iv) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the patient is less than about 51 years old.

In some embodiments, the method further comprises providing advice to the patient.

In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The present invention further provides a method of marketing an anti-CD20 antibody or a pharmaceutically acceptable composition thereof for use in a subgroup of patients with progressive multiple sclerosis, the method comprising informing a target audience of the use of the anti-CD20 antibody for the treatment of the subgroup of patients The subgroup of patients is characterized in that the patients of the subgroup have one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) in The Extended Disability Status Scale (EDSS) increased by at least about one point within the first two years of treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was greater than about 5 points.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, a subgroup of patients is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments, samples of the patient subgroup further have signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the EDSS of the patient subgroup is higher than about 5.0 for less than about 15 years. In some embodiments, the EDSS of the patient subgroup is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient subgroup further has two or more relapses within two years prior to starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the patient subgroup is less than about 51 years old.

In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The present invention provides a product comprising a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier packaged together and indicating (that is, indicating) that the anti-CD20 antibody or pharmaceutical composition is designated for the treatment of patients with multiple disease The label of a patient with sclerosis who has one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) two before starting treatment The Extended Disability Status Scale (EDSS) increased by at least about one point during the year, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the patient is less than about 51 years old.

In some embodiments, a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier is in a container. In some embodiments, the container contains between about 0.3 grams and about 4.0 grams of anti-CD20 antibody. In some embodiments the container contains between about 0.3 grams and about 1.5 grams of anti-CD20 antibody.

In some embodiments, the label provides instructions, where the instructions indicate that an effective amount of anti-CD20 antibody is administered to the patient to provide an initial anti-CD20 antibody exposure between about 0.3 grams and about 4.0 grams, followed by about 0.3 grams to about 4.0 grams. Between the second anti-CD20 antibody exposure. In some embodiments, the initial anti-CD20 antibody exposure and/or the second anti-CD20 antibody exposure are between about 0.3 grams and about 1.5 grams. In some embodiments, the second exposure is not provided until about 16 to 60 weeks after the initial exposure. In some embodiments, each anti-CD20 antibody exposure is provided to the patient in the form of one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The present invention also provides a method for predicting whether an individual with advanced multiple sclerosis will respond to a drug treatment used to treat multiple sclerosis, the method comprising evaluating one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma-stained lesions, (c) increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment, and (d) multiple The sclerosis severity score (MSSS) is higher than about 5 points, whereby age, gamma staining lesions, increase in EDDS within two years before starting treatment, MSSS or a combination thereof indicate that the individual will respond to treatment.

In some embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the individual is not diagnosed with relapsing-remitting multiple sclerosis at the beginning of treatment.

In some embodiments, the individual's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments, the individual's EDSS is above about 5.0 for less than about 15 years. In some embodiments, the individual's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, an increase in EDSS of at least about 1.5 points in the two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the individual further has two or more relapses within two years before starting treatment. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments, the age of the individual is less than about 51 years old.

In some embodiments of any of the methods or products described herein, the patient has one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, And (c) Increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment.

The present invention further provides a method for treating multiple sclerosis in a patient, which comprises administering to the patient an effective amount of occlizumab to provide an initial occlizumab exposure between about 0.3 g and about 0.6 g, and then providing A second occlizumab exposure between about 0.3 grams and about 0.6 grams, the second exposure is not provided until about 16 to 60 weeks after the initial exposure, and is in the form of one or two doses of occlizumab Provide patients with individual exposures of okclizumab.

In some embodiments, the initial Okclizumab exposure is about 0.6 grams. In some embodiments, the second okclizumab exposure is about 0.6 grams. In some embodiments, the second exposure is administered about 24 weeks after the initial exposure. In some embodiments, the patient is provided with one or more exposures of occlizumab in the form of one dose of occlizumab. In some embodiments, the patient is provided with one or more occlizumab exposures in the form of two doses of occlizumab. In some embodiments, the initial oxyclizumab exposure includes a first dose and a second dose of occlizumab, wherein the first dose and the second dose of occlizumab are about 0.3 grams. In some embodiments, the second occlizumab exposure comprises a single dose of occlizumab, wherein the single dose of occlizumab is 0.6 grams. In some embodiments, the methods further comprise providing a third okclizumab exposure. In some embodiments, the methods further comprise providing a fourth oklitizumab exposure. In some embodiments, the methods further comprise providing a fifth okclizumab exposure. In some embodiments of any of these methods, the methods further comprise providing between about one to about three subsequent exposures of okclizumab.

The present invention also provides a product comprising: (a) a container containing okclizumab; and (b) a package insert with instructions for treating multiple sclerosis in a patient, wherein the instructions indicate that the administration to the patient is effective Provide an initial exposure of okclizumab between about 0.3 g and about 0.6 g, and then provide okclizumab at a second exposure between about 0.3 g and about 0.6 g. The second exposure was not administered until about 16 to 60 weeks after the initial exposure, and each occlizumab exposure was provided to the patient in the form of one or two doses of occlizumab.

In some embodiments, the initial Okclizumab exposure is about 0.6 grams. In some embodiments, the second okclizumab exposure is about 0.6 grams. In some embodiments, the second exposure is administered about 24 weeks after the initial exposure. In some embodiments, the patient is provided with one or more exposures of occlizumab in the form of one dose of occlizumab. In some embodiments, the patient is provided with one or more occlizumab exposures in the form of two doses of occlizumab. In some embodiments, the initial oxyclizumab exposure includes a first dose and a second dose of occlizumab, wherein the first dose and the second dose of occlizumab are about 0.3 grams. In some embodiments, the instructions further include providing a third exposure of Okclizumab. In some embodiments, the instructions further include providing a fourth okclizumab exposure. In some embodiments, the instructions further include providing a fifth oxyclizumab exposure. In some embodiments of any of these methods, the instructions further include providing between about one to about three subsequent exposures of okclizumab.

<b><i>I. Definition</i></b>

"B cells" are lymphocytes that mature in the bone marrow, and include native B cells, memory B cells, or effector B cells (plasma cells). The B cells herein can be normal B cells or non-malignant B cells.

The "B cell surface marker" or "B cell surface antigen" herein refers to an antigen expressed on the surface of B cells that can be targeted by antibodies bound to it. Exemplary B cell surface markers include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82 , CD83, CDw84, CD85 and CD86 white blood cell surface markers (for description, see The Leukocyte Antigen Facts Book, 2nd Edition 1997, Barclay et al. Edited Academic Press, Harcourt Brace &amp; Co., New York). Other B cell surface markers include RP105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA and 239287. The B cell surface markers of particular interest in this article are preferably expressed on mammalian B cells rather than other non-B cell tissues, and can be expressed on both precursor B cells and mature B cells. The preferred B cell surface marker herein is CD20.

"CD20" antigen or "CD20" is an approximately 35kDa non-glycosylated phosphoprotein found on the surface of more than 90% of B cells in peripheral blood or lymphatic organs. CD20 is present on normal B cells and malignant B cells, but not on stem cells. Other names for CD20 in the literature include "B-lymphocyte restricted antigen" and "Bp35". The CD20 antigen system is described in, for example, Clark et al.<i>Proc. Natl. Acad. Sci</i>. (USA) 82:1766 (1985).

The "antibody antagonist" herein refers to destroying or depleting B cells in mammals when combined with B cell surface markers on B cells and/or interfering with one or the other, for example, by reducing or preventing the humoral response caused by B cells A variety of antibodies for B cell functions. Antibody antagonists are preferably capable of depleting B cells in the mammals treated with them (that is, reducing the content of circulating B cells). This depletion can be achieved by various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or induction of B cell death ( For example, by apoptosis).

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to cell-mediated reactions in which non-specific cytotoxic cells (such as natural killer (NK) cells, neutrophils) that express Fc receptors (FcR) White blood cells and macrophages) recognize the bound antibody on the target cell and then cause the target cell to lyse. The primary cells that mediate ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII, and FcyRIII. The performance of FcR on hematopoietic cells is summarized in Ravetch and Kinet,<i>Annu. Rev. Immunol</i> 9:457-92 (1991) in Table 3 on page 464. To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as the assay described in U.S. Patent Nos. 5,500,362 or 5,821,337. Applicable effector cells for these tests include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Additionally or otherwise, the ADCC activity of the molecule of interest can be in vivo, for example in animal models (such as Clynes et al.<i>PNAS(USA)</i>95:652-656 (1998) in the animal model disclosed in).

"Human effector cells" are white blood cells that express one or more FcRs and perform effector functions. In some embodiments, the cells exhibit at least FcyRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among them, PBMC and NK cells are preferred.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. In addition, the preferred FcR is one that binds IgG antibodies (γ receptors) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allele variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar but mainly different amino acid sequences in the cytoplasmic domain. The activated receptor FcγRIIA contains an activation motif based on immunoreceptor tyrosine (ITAM) in its cytoplasmic domain. The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see<img file="TW201014605A_D0015.tif" /><i>,Annu. Rev. Immunol.</i> 15:203-234 (1997)). FcR reviewed in Ravetch and Kinet,<i>Annu. Rev. Immunol</i> 9:457-92 (1991); Capel et al.,<i>Immunomethods</i> 4:25-34 (1994) and de Haas et al.,<i>J. Lab. Clin. Med.</i> 126:330-41 (1995). Other FcRs (including those to be identified in the future) are covered by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al.,<i>J. Immunol.</i> 117:587 (1976) and Kim et al.,<i>J. Immunol.</i> 24:249(1994))。

"Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to dissolve a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (Clq) of the complement system to a molecule (such as an antibody) complexed with a homologous antigen. To assess complement activation, for example, Gazzano-Santoro et al.,<i>J. Immunol. Methods</i> The CDC test described in 202:163 (1996).

"Growth-inhibiting" antibodies are antibodies that prevent or reduce the proliferation of cells that express antigens bound to the antibody. For example, the antibody can prevent or reduce the proliferation of B cells in vitro and/or in vivo.

Antibodies for "inducing apoptosis" are, for example, as determined by standard apoptosis assays (such as phospholipid binding protein V (annexin V) binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell fragmentation, and/or membrane vesicles). These antibodies are formed (called apoptotic bodies) that are determined to induce progressive cell death (such as B cell death).

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, multi-strain antibodies, multispecific antibodies (such as bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, as long as they exhibit the desired Biological activity is sufficient.

An "antibody fragment" includes a part of a complete antibody, which preferably includes its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab')₂And Fv fragments; bifunctional antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

For the purposes of this document, a "whole antibody" is an antibody comprising a heavy chain variable domain and a light chain variable domain and an Fc region.

"Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons containing two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies with the heavy chains of different immunoglobulin isotypes. Each heavy chain and light chain also have regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V_H), followed by multiple constant domains. Each light chain has a variable domain (V_L) And has a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain.

The term "variable" means that the sequences of certain parts of antibody variable domains are quite different and that these parts are used for the binding and specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in the light chain variable domain and the heavy chain variable domain. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of the natural heavy chain and light chain each contain 4 FRs, most of which are in the β-sheet configuration, connected by three hypervariable regions that form a loop and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of other chains by FR, which promotes the formation of antigen-binding sites of antibodies (see Kabat et al.,<i>Sequences of Proteins of Immunological Interest</i>, The fifth edition of the Public Health Service (Public Health Service), National Institutes of Health (National Institutes of Health), Bethesda, MD. (1991)). Although the constant domain is not directly involved in the binding of an antibody to an antigen, it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments each with a single antigen-binding site and residual "Fc" fragments. The name "Fc" fragment reflects its ability to crystallize easily. Pepsin treatment produces F(ab')₂Fragments that have two antigen binding sites and are still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment containing a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in a tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define V_H-V_LThe antigen binding site on the surface of the dimer. The six hypervariable regions together make the antibody have antigen binding specificity. However, even a single variable domain (or only one half of the Fv containing three hypervariable regions specific to the antigen) still has the ability to recognize and bind to the antigen, but the affinity is lower than that of the complete binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments are different from Fab fragments by adding several residues (including one or more cysteine from the hinge region of an antibody) to the carboxy terminus of the CH1 domain of the heavy chain. Fab'-SH is the name of Fab' with at least one free thiol group for the cysteine residue of the constant domain herein. F(ab')₂Antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteine in the middle. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types (called kappa and lambda) based on the amino acid sequence of their constant domains.

According to the amino acid sequence of the constant domain of the heavy chain, antibodies can be classified into different categories. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the 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" antibody fragments include the V of the antibody_HDomain and V_LDomains, where these domains exist in a single polypeptide chain. In some embodiments, the Fv polypeptide is in V_HDomain and V_LA polypeptide linker is further included between the domains, which enables the scFv to form the structure required for antigen binding. For a review of scFv, see<img file="TW201014605A_D0016.tif" />Of<i>The Pharmacology of Monoclonal </i><i>Antibodies</i>, Volume 13, Rosenburg and Moore eds, Springer-Verlag, New York, pp. 269-315 (1994).

The term "bifunctional antibody" refers to small antibody fragments with two antigen binding sites, these fragments are contained in the same polypeptide chain (V_H-V_L) Medium and light chain variable domain (V_L) Linked heavy chain variable domain (V_H). By using linkers that are too short to allow pairing between two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Bifunctional antibodies are more fully described in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,<i>Proc. Natl. Acad. Sci. USA</i>, 90: 6444-6448 (1993).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homologous antibodies, that is, except for possible variants that may appear during the production of the monoclonal antibody (these variants are generally present in a smaller amount ), the individual antibodies constituting the population are all the same and/or bind to the same epitope. In contrast to multiple antibody preparations that usually include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody system is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies are also suitable because they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a population of substantially homologous antibodies and should not be regarded as requiring the production of antibodies by any specific method. For example, by Kohler et al.,<i>Nature</i>, 256:495 (1975) first described the fusionoma method or recombinant DNA methods (see, for example, US Patent No. 4,816,567) to produce monoclonal antibodies to be used in accordance with the present invention. It is also possible to use such as Clackson et al.,<i>Nature</i>,352:624-628 (1991) and Marks et al.,<i>J. Mol. Biol</i>The technique described in ., 222:581-597 (1991) is used to isolate "monoclonal antibodies" from phage antibody libraries.

The monoclonal antibodies herein especially include "chimeric" antibodies (immunoglobulins), in which a part of the heavy chain and/or light chain is identical or homologous to the corresponding sequence of an antibody derived from a specific species or belonging to a specific antibody class or subclass , And the rest of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass; and fragments of these antibodies as long as they exhibit the required biological activity (US Patent No. 4,816,567; Morrison et al.,<i>Proc. Natl. Acad. Sci. USA</i>, 81:6851-6855 (1984)). The chimeric antibodies of interest herein include "primatized" antibodies, which contain variable domains derived from non-human primates (for example, Old World Monkeys, such as baboons, rhesus monkeys, or rhesus monkeys) Antigen binding sequence and human constant region sequence (US Patent No. 5,693,780).

The "humanized" form of non-human (e.g., murine) antibodies is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. Humanized antibodies are mainly human immunoglobulins (receptor antibodies), in which residues from the hypervariable region of the recipient are derived from non-human species such as mice, rats, rabbits or non-human primates (donor The hypervariable region of the antibody) and the substitution of residues with the required specificity, affinity and ability. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally speaking, humanized antibodies comprise substantially all of at least one and usually two variable domains, wherein all or substantially all hypervariable loops correspond to those of non-human immunoglobulin and all or substantially all hypervariable loops All FRs are those of the human immunoglobulin sequence, but there are FR substitutions as described above. The humanized antibody optionally also contains at least a part of the constant region of an immunoglobulin, usually the constant region of a human immunoglobulin. For details, see Jones et al.,<i>Nature </i>321:522-525 (1986); Riechmann et al.,<i>Nature </i>332:323-329 (1988); and Presta,<i>Curr. Op. Struct. Biol.</i> 2:593-596(1992)。

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region includes the amino acid residues of the "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain , And 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabat et al.,<i>Sequences of Proteins of Immunological Interest</i>, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or residues of the "hypervariable loop" (such as light chain Residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the variable domain, and 26-32 (H1), 53-55 (H2) and 96- of the heavy chain variable domain 101(H3); Chothia and Lesk <i>J. Mol. Biol.</i> 196:901-917 (1987)). "Framework" or "FR" residues are those variable domain residues other than hypervariable region residues as defined herein.

A "naked antibody" is an antibody (as defined herein) that is not bound to a heterologous molecule such as a cytotoxic moiety or a radiolabel.

Examples of anti-CD20 antibodies include: "C2B8", now known as "rituximab" ("RITUXAN<img file="TW201014605A_D0017.tif" />/MABTHERA<img file="TW201014605A_D0018.tif" />") (US 5,736,137); called "Y2B8" or "IbritumomabTiuxetan" (ZEVALIN<img file="TW201014605A_D0019.tif" />) 2B8 murine antibody labeled with yttrium [90], which can be purchased from Biogen Idec, Inc. (for example, US 5,736,137; 2B8 deposited with the deposit number HB11388 on June 22, 1993); murine IgG2a "B1", Also known as "Tositumomab", depending on the situation¹³¹I label to produce "131I-B1" or "iodine I131 tositumomab" antibody (BEXXAR^TM), can be purchased from Corixa (see also, for example, US 5,595,721); murine monoclonal antibody "1F5" (for example, Press et al.<i>Blood</i> 69(2):584-591(1987)) and its variants, including "repaired framework" or humanized 1F5 (e.g. WO 2003/002607, Leung, S.; ATCC deposit HB-96450); rodent 2H7 and Chimeric 2H7 antibody (e.g. US 5,677,180); 2H7 antibody (e.g. WO 2004/056312 (Lowman et al.) and as set forth above); HUMAX-CD20^TM(Ofatumomab) a fully human high-affinity antibody that targets CD20 molecules in the cell membrane of B cells (Genmab, Denmark; see, for example, Glennie and van de Winkel,<i>Drug Discovery Today</i> 8:503-510 (2003) and Cragg et al.,<i>Blood</i> 101:1045-1052 (2003)); WO 2004/035607 and WO 2005/103081 (Teeling et al., GenMab/Medarex) described in human monoclonal antibodies; US 2004/0093621 (Shitara et al.) described in Fc region-bound complex N glycoside-linked sugar chain antibody; WO 2006/106959 (Numazaki et al., Biomedics Inc.) described in WO 2006/106959 (Numazaki et al., Biomedics Inc.) described in the chimeric or humanized monomer with high binding affinity to the extracellular epitope of the CD20 antigen Strain antibodies; monoclonal antibodies and antigen-binding fragments that bind to CD20 (for example, WO2005/000901, Tedder et al.), such as HB20-3, HB20-4, HB20-25 and MB20-11; single-chain proteins that bind to CD20, Including (but not limited to) TRU-015 (such as US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and Ledbetter); US 2005/0202028 (Hayden-Ledbetter and Ledbetter); US 2005/ 136049 (Ledbetter et al.); US 2005/0202023 (Hayden-Ledbetter and Ledbetter)-Trubion Pharm Inc.); such as, for example, WO 2004/103404; US 2005/0025764 and US 2006/0251652 (Watkins et al., Applied Molecular Evolution, Inc.) as set forth in CD20 Binding molecules, such as AME series antibodies, such as AME-133^TMAntibodies and anti-CD20 antibodies with Fc mutations as described in, for example, WO 2005/070963 (Allan et al., Applied Molecular Evolution, Inc.); CD20 binding molecules such as those described in WO 2005/016969 and US 2005/0069545 ( Carr et al.); bispecific antibodies as elucidated in WO 2005/014618 (Chang et al.); humanized LL2 monoclonal antibodies and other anti-CD20 antibodies e.g. US 7,151,164 (Hansen et al., Immunomedics; US 2005/0106108 (Leung and Hansen; Immunomedics); as for example WO 2006/130458; Gazit et al., a fully human antibody against CD20 described in Amgen/AstraZeneca); as for example WO 2006/ Antibodies against CD20 described in 126069 (Morawala, Avestha Gengraine Technologies Pvt Ltd.); such as in WO 2005/044859, US 2005/0123546, US 2004/0072290 and US 2003/0175884 (Umana et al.; GlycArt Biotechnology AG) The chimeric or humanized B-Lyl antibody against CD20; A20 antibody or variants thereof, such as the chimeric or humanized A20 antibody (cA20, hA20, respectively) and IMMUN-106 (for example, US 2003/0219433, Immunomedics) ; And can be purchased from International Leukocyte Typing Workshop (for example Valentine et al.,<i>Leukocyte Typing</i> III (McMichael ed., p. 440, Oxford University Press (1987))) monoclonal antibody L27, G28-2, 93-1B3, B-Cl or NU-B2. In some embodiments, the anti-CD20 antibodies herein are chimeric humanized or human anti-CD20 antibodies (more preferably rituximab), 2H7 antibodies, chimeric or humanized A20 antibodies (Immunomedics) and HUMAX-CD20^TMHuman anti-CD20 antibody (Genmab).

The term "rituximab" or "RITUXAN<img file="TW201014605A_D0020.tif" />"Refers to a chimeric murine/human monoclonal antibody that has been genetically engineered to target the CD20 antigen and is named "C2B8" in US Patent No. 5,736,137, including fragments that retain the ability to bind CD20. Rituximab can be purchased from Genentech.

For purely purposes herein and unless otherwise specified, "humanized 2H7" refers to a humanized antibody or antigen-binding fragment thereof that binds to human CD20, wherein the antibody effectively depletes primate B cells in vivo, and the antibody is Its H chain variable region (V_H) Contains at least one CDR H3 sequence from the anti-human CD20 antibody SEQ ID NO: 12 (Figure 1B) and substantially human heavy chain subgroup III (V_HIII) Human Common Framework (FR) residues. In some embodiments, the antibody further includes the H chain CDR H1 sequence of SEQ ID NO: 10 and the CDR H2 sequence of SEQ ID NO: 11, and in some embodiments, further includes the L chain CDR of SEQ ID NO: 4 The L1 sequence, the CDR L2 sequence of SEQ ID NO: 5, the CDR L3 sequence of SEQ ID NO: 6 and the human common framework (FR) residues of substantially human light chain subgroup I (VI), where V_HThe region can be linked to the constant region of the human IgG chain, where the region can be, for example, IgG1 or IgG3. In some embodiments, the antibody comprises the V of SEQ ID NO: 8_HSequence (v16, as shown in Figure 1B), optionally also includes the V of SEQ ID NO: 2_LSequence (v16, as shown in Figure 1A), which can have amino acid substitutions D56A and N100A in the H chain and amino acid substitutions S92A in the L chain (v96). In some embodiments, the antibody is a complete antibody, which includes the light chain and heavy chain amino acid sequences of SEQ ID NOs: 13 and 14 as shown in Figures 2 and 3, respectively. In some embodiments, the antibody is 2H7.v31, which includes the light chain and heavy chain amino acid sequences of SEQ ID NOs: 13 and 15 as shown in FIGS. 2 and 4, respectively. The antibodies herein may further comprise at least one amino acid substitution in the Fc region that improves ADCC and/or CDC activity, such as those in which the amino acid substitution is S298A/E333A/K334A, and in some embodiments has SEQ ID NO 2H7.v31 of the heavy chain amino acid sequence of :15 (as shown in Figure 4). Any of these antibodies may further include at least one amino acid substitution in the Fc region that reduces CDC activity, for example, at least a K322A substitution. See U.S. Patent No. 6,528,624 Bl (Idusogie et al.).

The term "Okclizumab" as used herein refers to a genetically engineered light chain directed against the CD20 antigen and comprising (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and (b) comprising the amine of SEQ ID NO: 14 Humanized monoclonal antibodies of the heavy chain of the base acid sequence include fragments that retain the ability to bind to CD20. Oaklizumab is available from Genentech.

"Isolated" antibodies are antibodies that have been identified and separated and/or recovered from components of their natural environment. The pollution components of the natural environment are substances that interfere with the diagnostic or therapeutic uses of antibodies, and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody will be purified: (1) to greater than 95% by weight of antibody and in some embodiments greater than 99% by weight as determined by the Lowry method; (2) enough to be transformed by using The cup sequencer obtains at least 15 residues of the N-terminal or internal amino acid sequence; or (3) to the use of Coomassie blue under reducing or non-reducing conditions by SDS-PAGE or In some embodiments, it is determined to be homogeneous using silver staining. Since at least one component of the antibody's natural environment will not be present, the isolated antibody includes the antibody in situ in recombinant cells. However, isolated antibodies will usually be prepared by at least one purification step.

The "individual" or "patient" in this article refers to a human individual or patient. Generally speaking, the individual or patient is eligible for treatment of multiple sclerosis. For the purposes of this article, the eligible individual or patient is a patient who has experienced, has experienced, or may experience one or more signs, symptoms or other indicators of multiple sclerosis; has been diagnosed with multiple sclerosis (such as recent Patients who are diagnosed (with "new-onset" MS); previously diagnosed and recently relapsed or worsened; previously diagnosed and in remission, etc.) and/or are at risk of developing multiple sclerosis. Patients with multiple sclerosis or who are at risk of multiple sclerosis can be identified as selected serum, cerebrospinal fluid (CSF), and/or CD20-positive B cells in MS lesions with elevated levels and/or use detection Autoantibodies (qualitative evaluation and preferably quantitative evaluation) screening patients. The exemplary autoantibodies associated with multiple sclerosis include anti-myelin basic protein (MBP), anti-myelin oligodendritic glial cell glycoprotein (MOG), anti-ganglionic glycolipid and/or anti-neural Cilia antibodies. These autoantibodies can be detected in the individual's serum, cerebrospinal fluid (CSF) and/or MS lesions. Herein, the "increased" level of autoantibodies or B cells means that the level of these autoantibodies or B cells significantly exceeds the level of individuals without MS.

"Treatment" as used herein is a means to obtain beneficial or desired results (including clinical results). For the purpose of the present invention, beneficial or desired clinical results include (but are not limited to) one or more of the following results: alleviate one or more symptoms caused by the disease; reduce the severity of the disease; stabilize the disease (for example, prevent or delay the deterioration of the disease) Delay or slow down the progression of the disease; improve the condition of the disease; reduce the dose of one or more other drugs needed to treat the disease and/or improve the quality of life.

As used herein, "delaying" the progression of multiple sclerosis means delaying, hindering, slowing down, delaying, stabilizing, and/or delaying the progression of the disease. Depending on the medical history and/or the individual being treated, this delay can have different lengths of time.

"At the beginning of treatment" as used herein refers to the time or period before the first exposure to multiple sclerosis drugs (such as anti-CD20 antibodies). In some embodiments, "at the time of starting treatment" is roughly any of the first year, nine months, six months, three months, two months, or one month before the use of multiple sclerosis drugs (such as anti-CD20 antibodies) time. In some embodiments, "at the beginning of treatment" is immediately to the same time before the first exposure to multiple sclerosis drugs (such as anti-CD20 antibodies).

"Based on" as used herein includes (1) assessing, measuring or measuring patient characteristics as described herein and preferably selecting patients suitable for treatment, and (2) administering the treatment as described herein.

The "symptoms" of MS are any pathological or abnormal phenomena experienced by an individual and indicative of the structure, function, or sensation of MS.

"Multiple sclerosis" refers to a chronic and usually disabling disease of the central nervous system characterized by progressive myelin sheath damage. There are four internationally recognized forms of MS, namely primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS) and progression Relapsing multiple sclerosis (PRMS).

As used herein, "progressive multiple sclerosis" refers to primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS) . In some embodiments, progressive multiple sclerosis is characterized by a persistent and documented loss of irreversible neurological function that is not attributable to clinical relapse<img file="TW201014605A_D0021.tif" />Months.

"Primary progressive multiple sclerosis" or "PPMS" is characterized by the gradual progression of the disease from the onset, with no superimposed recurrence and remission at all. There may be periods when the disease activity is stable and there may be periods of improvement and deterioration of days or weeks. Because PPMS usually occurs in the early forties or early forties, men and women are equally likely to develop the disease and the initial disease activity is usually in the spinal cord rather than the brain, which is different from RRMS and SPMS. PPMS usually transfers to the brain, but it is less likely to damage the brain area than RRMS or SPMS. For example, people with PPMS are less likely to have cognitive problems than people with RRMS or SPMS. PPMS is the most unlikely MS subtype to show inflammatory (gamma-enhanced) lesions on MRI scans. The number of individuals affected by the primary progressive disease form is between 10% and 15% of all individuals with multiple sclerosis. PPMS can be based on McDonald et al.<i>Ann Neurol</i> 50:121-7 (2001). Individuals with PPMS treated herein are generally individuals with a possible or definite diagnosis of PPMS.

"Relapsing-remitting multiple sclerosis" or "RRMS" is characterized by recurrence (also called exacerbation) in which new symptoms can appear during the period and the old symptoms reappear or worsen. After recurrence is the remission period, during which the individual recovers completely or partially from the defect acquired during the recurrence period. Recurrence can last for days, weeks, or months and the recovery can be slow and gradual or almost instantaneous. The vast majority of people who exhibit MS are first diagnosed with RRMS. This diagnosis is usually in his twenties or thirties, although much earlier or much later diagnoses are known. Females showing this MS subtype are twice as many as males. During relapse, the myelin sheath (the protective barrier surrounding nerve fibers (neurons) in the white matter area of the central nervous system (CNS)) may be damaged in the inflammatory response of the body's autoimmune system. This causes a variety of neurological symptoms, which vary significantly depending on the area of CNS damage. Immediately after recurrence, the inflammatory response gradually disappeared and a specific type of glial cells (called oligodendritic glial cells) in the CNS promote remyelination (a process by which the myelin around axons can be repaired). Perhaps this remyelination is the cause of alleviation. About 50% of patients with RRMS convert to SPMS within 10 years of the onset of the disease. Thirty years later, this figure rose to 90%. At any one time, relapsing-remitting disease accounts for approximately 55% of all individuals with MS.

"Secondary progressive multiple sclerosis" or "SPMS" is characterized by a steady progression of clinical nerve damage, with or without superimposed recurrence, less remission, and a plateau. Individuals who develop SPMS have previously experienced an RRMS period that may have lasted from two to forty years or more. Any superimposed relapses and remissions that exist tend to diminish gradually over time. Starting from the secondary progressive stage of the disease, disability begins to progress at a much faster rate than during RRMS, although in some individuals the process may still be quite slow. After 10 years, 50% of patients with RRMS will develop SPMS. After 25 to 30 years, that number will rise to 90%. SPMS tends to be characterized by a lower degree of inflammatory lesion formation than RRMS, but the total disease burden continues to progress. At any one time, SPMS accounts for approximately 30% of all individuals with multiple sclerosis.

"Progressive relapsing multiple sclerosis" refers to the "PRMS" characterized by the steady progress of clinical nerve damage, accompanied by superimposed recurrence and remission. There is obvious recovery immediately after recurrence, but there is a gradual deterioration of symptoms between recurrences. PRMS affects approximately 5% of all individuals with multiple sclerosis. Some neurologists believe that PRMS is a variant of PPMS.

The expression "effective amount" refers to the amount of the antibody (or other drug) that is effective in improving or treating multiple sclerosis. The effective amount generally improves the signs, symptoms or other indicators of MS, such as reducing the recurrence rate, preventing disability, reducing the number and/or volume of brain MRI lesions, improving the timing of 25 walking, slowing down or delaying the progression of the disease, such as extending to the disease Time to progress (for example, using the Extended Disability Status Scale, EDSS), etc.

"Antibody exposure" refers to contact or exposure to one or more doses of the antibody herein administered within a period of about 1-20 days. These doses can be given at one time or at fixed or random intervals during the exposure period. As described in detail herein, initial and later (e.g., second or third) antibody exposures are separated from each other by a certain amount of time.

The term "immunosuppressive agent" used in adjuvant therapy as used herein refers to a substance used to suppress or mask the immune system of the mammal being treated herein. It will include substances that inhibit cytokine production, down-regulate or inhibit the expression of self-antigens, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); non-steroidal anti-inflammatory drugs (NSAID); ganciclovir, tacrolimus (tacrolimus), glucocorticoids such as cortisol or aldosterone, anti-inflammatory agents such as cyclooxygenase inhibitors, 5-lipoxygenase inhibitors or leukotriene receptor antagonists; purine antagonists, such as sulfur Azoprine or mycophenolate mofetil (mycophenolate mofetil, MMF); alkylating agent, such as cyclophosphamide; bromocryptine (bromocryptine); danazol (danazol); dapsone (dapsone); glutaraldehyde (such as As described in U.S. Patent No. 4,120,649, which shields MHC antigens; MHC antigens and MHC fragments of anti-genetic antibodies; cyclosporin A (cyclosporin A); Steroids, such as corticosteroids or glucocorticoids or glucocorticoid analogs (such as prednisone, methylprednisolone and dexamethasone); dihydrofolate reductase inhibitors, Such as methotrexate (oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists, including anti-interference Anti-tumor necrosis factor alpha antibody (infliximab or adalimumab), anti-TNF alpha immunoadhesin (etanercept), anti-tumor necrosis Factor β antibody, anti-interleukin 2 antibody and anti-IL-2 receptor antibody; anti-LFA-1 antibody, including anti-CD11a and anti-CD18 antibody; anti-L3T4 antibody; heterologous anti-lymphocyte globulin; pan-T antibody (pan -T antibody), preferably an anti-CD3 or anti-CD4/CD4a antibody; a soluble peptide containing the LFA-3 binding domain (7/26/90 published WO 90/08187); streptococcal kinase; TGFβ; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin ( rapamycin); T cell receptor (Cohen et al. U.S. Patent No. 5,114,721); T cell receptor fragment (Offner et al.,<i>Science</i>, 251: 430-432 (1991); WO 90/11294; Ianeway,<i>Nature</i>341:482 (1989); and WO 91/01133); and T cell receptor antibodies (EP 340,109), such as T10B9.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or suppresses cell function and/or causes cell destruction. The term is intended to include radioisotopes (e.g. At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³²And radioisotopes 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 agents" are compounds suitable for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN<img file="TW201014605A_D0022.tif" />Cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa , Carboquone, metredopa and uredopa; ethyleneimine and methylmelamine, including altretamine, triethylenemelamine, three Triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenin (especially bullatacin) and cloth Bullatacinone (bullatacinone); camptothecin (including synthetic analog topotecan (topotecan)); bryostatin; callistatin (callystatin); CC-1065 (including the adrenaline Dozelesin, carzelesin and bizelesin synthetic analogues); cryptophycin (especially Nostoc cyclic peptide 1 and Nostoc cyclic peptide 8); sea Dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1_-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil , Chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosurea, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine ) And ranimnustine (ranimnustine); antibiotics, such as enediyne antibiotics (such as calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, for example, Agnew,<i>Chem Intl. Ed. Engl.</i>, 33:183-186 (1994)); dynemicin (dynemicin), including danemycin A; bisphosphonates, such as clodronate (clodronate); esperamicin (esperamicin) ; And neocarzinostatin chromophore (neocarzinostatin chromophore) and related chromoprotein endiyne antibiotic chromophore), aclacinomysin, actinomycin, authramycin , Azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, color Chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-ortholeucin, ADRIAMYCIN<img file="TW201014605A_D0023.tif" />Cranberries (including morpholinyl-cranberry, cyanomorpholinyl-cranberry, 2-pyrrololinyl-cranberry and deoxycranberry), epirubicin, isol Esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid (mycophenolic acid) acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin , Rodorubicin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin (zorubicin); antimetabolites, such as methotrexate and 5-fluorouracil (5-fluorouracil) (5-FU); folate analogs, such as denoterin, methotrexate, Pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, Such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine , Doxifluridine (doxifluridine), enocitabine (enocitabine), fluridine (floxuridine); androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), ephedrine Alcohol (epitiostanol), mepitiostane (mepitiostane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements, Such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acidacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colchicine (demecolcine); diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; mushroom polysaccharide (lentinan); lonidainine; maytansinoid, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; Mo Mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podoxantrone; podophyllinic acid); 2-ethylhydrazine; procarbazine; PSKacetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoid, such as maytansine maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; methionine Phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; PSKacetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoid, such as maytansine maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; methionine Phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; PSK<img file="TW201014605A_D0024.tif" />Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid ); triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A), myrophyllin A (roridin A) and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol ; Dibromodulcitol (mitolactol); Pipobroman (pipobroman); Gacytosine (gacytosine); Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Paclitaxel ( taxoid), such as TAXOL<img file="TW201014605A_D0025.tif" />Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE^TMPaclitaxel-free cetyl alcohol-free albumin engineered nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXoTERE<img file="TW201014605A_D0026.tif" />Doxetaxel (<img file="TW201014605A_D0027.tif" />-Poulenc Rorer, Antony, France); Chloranbucil; GEMZAR<img file="TW201014605A_D0028.tif" />Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE<img file="TW201014605A_D0029.tif" />Vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda (xeloda); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; catheta Capecitabine; and a pharmaceutically acceptable salt, acid or derivative of any of the above.

This definition also includes anti-hormonal agents, which are used to modulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen (including NOLVADEX<img file="TW201014605A_D0030.tif" />Tamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxy tamoxifen (trioxifene), raloxifene hydrochloride (keoxifene), LY117018, Onapristone and FARESTON-toremifene; aromatase inhibitors, which inhibit aromatase that regulates the production of estrogen in the adrenal glands, such as 4(5)-imidazole, aminogrumi Special (aminoglutethimide), MEGASE<img file="TW201014605A_D0031.tif" />Megestrol acetate, AROMASIN<img file="TW201014605A_D0032.tif" />Exemestane, formestane, fadrozole, RIVISOR<img file="TW201014605A_D0033.tif" />Vorozole, FEMARA<img file="TW201014605A_D0034.tif" />Letrozole and ARIMIDEX<img file="TW201014605A_D0035.tif" />Anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin ; And troxacitabine (troxacitabine) (1,3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, especially for inhibiting gene expression in signal transduction pathways involved in abnormal cell proliferation Their antisense oligonucleotides, such as PKC-α, Ralf and H-Ras; vaccines, such as gene therapy vaccines, such as ALLOVECTIN<img file="TW201014605A_D0036.tif" />Vaccine, LEUVECTIN<img file="TW201014605A_D0037.tif" />Vaccine and VAXID<img file="TW201014605A_D0038.tif" />Vaccine; PROLEUKIN<img file="TW201014605A_D0039.tif" /> rIL-2; LURTOTECAN<img file="TW201014605A_D0040.tif" />Topoisomerase 1 inhibitor; ABARELIX<img file="TW201014605A_D0041.tif" /> rmRH; and a pharmaceutically acceptable salt, acid or derivative of any of the above.

The term "cytokine" is a general term for proteins that are released by a cell population and act as intercellular mediators on another cell. Examples of these cytokines are lymphokines; monocyte hormones; 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 and IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligands (KL). The term cytokine as used herein includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of natural sequence cytokines, including synthetically produced small molecular entities and their pharmaceutically acceptable derivatives and salts .

The term "hormone" refers to polypeptide hormones generally secreted by glands with ducts. Hormones include, for example, growth hormones such as human growth hormone, N-methionine-based human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH); Prolactin, Placental Prolactin, Mouse Gonadotropin Related Peptide; Inhibin; Activin ; Mullerian-inhibiting substance; and thrombopoietin. The term hormone as used herein includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of natural sequence hormones, including synthetically produced small molecule entities and pharmaceutically acceptable derivatives and salts thereof .

The term "growth factor" refers to a protein that promotes growth and includes, for example, hepatocyte growth factor; fibroblast growth factor; vascular endothelial growth factor; nerve growth factor such as NGF-β; platelet-derived growth factor; conversion growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factors I and II; erythropoietin (EPO); osteoinductive factor; interferons, such as interferon α, β, and γ; and community stimulating factor (CSF ), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF). The term growth factor as used herein includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of natural sequence growth factors, including synthetically produced small molecular entities and pharmaceutically acceptable derivatives and Salt.

The term "integrin" refers to a receptor protein that allows cells to bind to and respond to the extracellular matrix, and is involved in a variety of cellular functions, such as wound healing, cell differentiation, tumor cell homing, and apoptosis. It is part of a large family of cell adhesion receptors involved in cell-extracellular matrix and cell-cell interactions. Functional integrins are composed of two non-covalently bound transmembrane glycoprotein subunits (called α and β). The alpha units share some homology with each other, and so does the beta units. The receptor always contains an alpha chain and a beta chain. Examples include α6β1, α3β1, α7β1, LFA-1, α4 integrin, and the like. The term integrin as used herein includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of natural sequence integrins, including synthetically produced small molecule entities and pharmaceutically acceptable derivatives and Salt.

Examples of "integrin antagonists or antibodies" herein include LFA-1 antibodies, such as efalizumab (efalizumab) available from Genentech.<img file="TW201014605A_D0042.tif" />); α4 integrin antibody, such as Natalizumab (TYSABRI<img file="TW201014605A_D0043.tif" />); Diaziridinic acid derivatives (WO 2003/89410); Phenylalanine derivatives (WO 2003/70709, WO 2002/28830, WO 2002/16329 and WO 2003/53926); Phenylpropionic acid derivatives ( WO 2003/10135); enamine derivatives (WO 2001/79173); propionic acid derivatives (WO 2000/37444); alkanoic acid derivatives (WO 2000/32575); substituted phenyl derivatives (US Patent No. 6,677,339 No. and No. 6,348,463); aromatic amine derivatives (US Patent No. 6,369,229); and ADAM disintegrin domain polypeptide (US 2002/0042368), antibody against αvβ3 integrin (EP 633945); aza-bridged bicyclic ring Amino acid derivatives (WO 2002/02556) and so on.

For the purposes of this article, "Tumor Necrosis Factor Alpha (TNF-α)" refers to the human TNF-α molecule, including those described in Pennica et al.,<i>Nature</i>, 312:721 (1984) or Aggarwal et al.,<i>JBC,</i>260: The amino acid sequence in 2345 (1985).

The "TNF-α inhibitor" herein is an agent that generally inhibits the biological function of TNF-α to some extent by binding to TNF-α and neutralizing its activity. An example of a TNF inhibitor specifically covered herein is etanercept (ENBREL<img file="TW201014605A_D0044.tif" />), Infliximab (REMICADE<img file="TW201014605A_D0045.tif" />) And adalimumab (HUMIRA^TM)。

Examples of "disease-modifying antirheumatic drugs" or "DMARDs" include hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (plus oral and subcutaneous methamphetamine) Azathioprine, D-penicillamine, gold preparation (oral), gold preparation (intramuscular), minocycline, cyclosporine, staphylococcal protein A Immunoadsorption, including its salts and derivatives, etc.

Examples of "non-steroidal anti-inflammatory drugs" or "NSAIDs" are acetylsalicylic acid, ibuprofen, naproxen, indomethacin, sulindac, sulindac Tolmetin, including its salts and derivatives.

"Corticosteroid" refers to any one of several synthetic or naturally-occurring substances with the general chemical structure of steroids that mimics or enhances the effects of naturally-occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, glucocorticoids, and betamethasone.

"Pharmaceutical inserts" are used to refer to other therapeutic products that are usually included in the commercial packaging of therapeutic products and contain indications, usage, dosage, administration, contraindications, and other therapeutic products to be combined with the packaged product and/or the use of such therapeutic products Instructions for warnings and other information.

As used herein, "label" refers to information that is usually included with commercial pharmaceutical formulation packaging (including containers such as vials and drug inserts, and other types of packaging).

Mentioning "about" a certain value or a parameter in this article includes (and describes) changes to that value or the parameter itself. For example, the description of "about X" includes the description of "X".

Unless the context clearly dictates otherwise, as used herein and in the scope of the appended application, the singular forms "a", "or" and "the" include plural indicators. It should be understood that the aspects and variants of the present invention described herein include "consisting of aspects and variants" and/or "essentially consisting of aspects and variants."

<b><i>II. Treatment methods</i></b>

The present invention provides a method for treating advanced multiple sclerosis in a patient, which comprises administering an effective amount of anti-CD20 antibody to the patient.

In some embodiments, the present invention provides a method for treating advanced multiple sclerosis in a patient, which comprises administering to the patient an effective amount of anti-CD20 antibody, wherein the treatment is based on the patient having one or more selected from the group consisting of Features: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (d) The multiple sclerosis severity score (MSSS) is higher than about 5 points.

In some embodiments, the present invention provides a method for treating patients with progressive multiple sclerosis. The limitation is that the patient has one or more characteristics selected from the group consisting of: (a) age less than about 55 years old, (b ) One or more gamma stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before the start of treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about At 5 points, treatment involves administering an effective amount of anti-CD20 antibody to the patient.

In some embodiments, the present invention provides a method for treating advanced multiple sclerosis in a patient, which comprises administering to the patient an effective amount of anti-CD20 antibody, wherein the patient has one or more selected from the group consisting of: Characteristics: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (d ) Multiple sclerosis severity score (MSSS) is higher than about 5 points, so that age, gamma staining lesions, increased EDDS within two years before starting treatment, MSSS or a combination of evidence indicates that the individual will be treated with anti-CD20 antibodies Respond.

In some embodiments, the present invention provides a method of treating advanced multiple sclerosis, which comprises: (a) selecting patients with one or more characteristics selected from the group consisting of: (i) age less than about 55 years old; (ii) One or more gamma-stained lesions, (iii) the Extended Disability Status Scale (EDSS) increased by at least about one point within the first two years of treatment, and (iv) the Multiple Sclerosis Severity Score (MSSS) is high At about 5 minutes; and (b) administer an effective amount of anti-CD20 antibody to the thus selected patient.

In some embodiments, the present invention provides a method for treating patients with progressive multiple sclerosis, which comprises administering to the patient an effective amount of an anti-CD20 antibody, and wherein one or more features selected from the group consisting of the following are used as options The basis of the patients receiving treatment: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment , And (d) Multiple Sclerosis Severity Score (MSSS) is higher than about 5 points, and wherein the treatment comprises administering an effective amount of anti-CD20 antibody to the patient.

The present invention further provides a method for treating multiple sclerosis in a patient, which comprises administering to the patient an effective amount of occlizumab to provide an initial occlizumab exposure between about 0.3 g and about 0.6 g, and then providing A second occlizumab exposure between about 0.3 grams and about 0.6 grams, the second exposure is not provided until about 16 to 60 weeks after the initial exposure, and in the form of one or two doses of occlizumab Provide patients with individual exposures of okclizumab. In some embodiments, the initial Okclizumab exposure is about 0.6 grams. In some embodiments, the second okclizumab exposure is about 0.6 grams. In some embodiments, the second exposure is administered about 24 weeks after the initial exposure. In some embodiments, the patient is provided with one or more exposures of occlizumab in the form of one dose of occlizumab. In some embodiments, the patient is provided with one or more occlizumab exposures in the form of two doses of occlizumab. In some embodiments, the two doses of oxyclizumab comprise about 0.3 grams of occlizumab.

The present invention also provides methods for evaluating and/or predicting the responsiveness of patients with progressive multiple sclerosis to anti-CD20 antibody therapy.

In some embodiments, the present invention provides a method for assessing whether a patient with progressive multiple sclerosis is responsive to treatment with an anti-CD20 antibody, which comprises assessing one or more characteristics selected from the group consisting of: (a) Are younger than about 55 years of age, (b) one or more gamma staining lesions, (c) increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment, and (d) severe multiple sclerosis The degree score (MSSS) is higher than about 5 points, where one or more characteristics of the patient indicate that the patient will respond to treatment.

In some embodiments, the present invention provides a method for assessing whether a patient with progressive multiple sclerosis is responsive to treatment with an anti-CD20 antibody, comprising: (a) assessing one or more characteristics selected from the group consisting of : (I) younger than about 55 years of age; (ii) one or more gamma staining lesions, (iii) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (iv) multiple occurrences The Sexual Sclerosis Severity Score (MSSS) is higher than about 5 points; and (b) implement an algorithm to determine whether the patient is responsive to the treatment, and (c) record the specific results of the tested patient.

In some embodiments, the present invention provides a method of treating patients with progressive multiple sclerosis, comprising: (a) evaluating one or more characteristics selected from the group consisting of: (i) age less than about 55 years old, ( ii) One or more gamma-stained lesions, (iii) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before the start of treatment, and (iv) the Multiple Sclerosis Severity Score (MSSS) was higher than Approximately 5 points, among which patients are selected for treatment based on one or more characteristics selected from the group consisting of: (i) younger than about 55 years of age, (ii) one or more gamma staining lesions, (iii) in The Extended Disability Status Scale (EDSS) increased by at least about one point within two years before the start of treatment, and (iv) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points; and (b) by The patient is administered an effective amount of anti-CD20 antibody to treat the selected patient.

In some embodiments, the present invention provides a method for selecting therapies for patients and/or patient populations suffering from progressive multiple sclerosis, comprising: (a) evaluating one or more characteristics selected from the group consisting of: (i) ) Younger than about 55 years of age, (ii) one or more gadolinium-stained lesions, (iii) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (iv) multiple sclerosis The severity score (MSSS) is higher than about 5 points; and (b) anti-CD20 antibodies are selected for treatment when the patient or patient population has one or more characteristics selected from the group consisting of: (i) age less than about 55 Years old, (ii) one or more gadolinium-stained lesions, (iii) at least about one point increase in the Extended Disability Status Scale (EDSS) within two years before starting anti-CD20 treatment, and (iv) multiple sclerosis severity score (MSSS) is higher than about 5 points.

In some embodiments, the present invention provides a method for predicting whether an individual with progressive multiple sclerosis will respond to anti-CD20 antibodies, the method comprising evaluating one or more characteristics selected from the group consisting of: (a) age Less than about 55 years of age, (b) one or more gamma-stained lesions, (c) increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment, and (d) the severity of multiple sclerosis The score (MSSS) is higher than about 5 points, whereby age, gamma-stained lesions, an increase in EDDS within two years before starting treatment, MSSS or a combination thereof indicate that the patient will respond to anti-CD20 antibodies.

In some embodiments, the present invention provides a method for identifying patients with progressive multiple sclerosis who may be responsive to anti-CD20 antibody therapy, which comprises: (a) evaluating one or more characteristics selected from the group consisting of: (i) younger than about 55 years of age, (ii) one or more gamma-stained lesions, (iii) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (iv) multiple The sclerosis severity score (MSSS) is higher than about 5 points; and (b) identify patients with one or more characteristics selected from the group consisting of: (i) younger than about 55 years old, (ii) one or more For gadolinium-stained lesions, (iii) the Extended Disability Status Scale (EDSS) increased by at least about one point within the first two years of treatment, and (iv) the Multiple Sclerosis Severity Score (MSSS) was greater than about 5 points.

In some embodiments of any of the methods for treating, assessing, and/or predicting responsiveness described herein, the patient has one or more characteristics selected from the group consisting of: (a) age less than about 55 years old, (b) One or more gadolinium-stained lesions, and (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment.

In some embodiments of any of the methods for assessing and/or predicting responsiveness described herein, the methods further include providing advice to the patient.

In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, the patient has more than one characteristic selected from the group consisting of: (a) younger than about 55 years of age, (b) one Or multiple gadolinium-stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within the first two years of treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was greater than about 5 points . In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, the patient has two characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one Or multiple gamma-stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within the first two years of treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was greater than about 5 points . In some embodiments, the patient has three characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, and (c) EDSS within two years before starting treatment Increase by at least about one point. In some embodiments, the patient has (a) less than about 55 years of age, (b) one or more gadolinium-stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one within two years before starting treatment Points, and (d) Multiple Sclerosis Severity Score (MSSS) is higher than about 5 points.

In some embodiments of any of the methods of treating and evaluating and/or predicting responsiveness described herein, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient and/or patient population is not diagnosed with relapsing-remitting multiple sclerosis at the beginning of treatment.

In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, the sample of the patient and/or patient population further has signs of inflammation. Signs of inflammation are indicated by evaluating one or more indicators of inflammation. The sample can be any suitable sample used to assess inflammation. In some embodiments, the sample is tissue or fluid. In some embodiments, the fluid sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing. In some embodiments, signs of inflammation are detected by MRI. In some embodiments, signs of inflammation are detected by the presence of Gd enhanced lesions or T2 lesions. Other methods of assessing signs of inflammation are known in the art.

In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, the patient and/or patient population is characterized by changes in EDSS over a period of time. In some embodiments, the patient and/or patient population is characterized by an increase in EDSS of at least about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75 in the two years prior to initiation of anti-CD20 antibody treatment. Any one of points or 3 points. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, the increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the patient and/or patient population is characterized by having an EDSS greater than about 5.0 for less than about 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, Any of 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, or 20 years. In some embodiments, the patient and/or patient population is characterized by having an EDSS greater than about 5.0 for less than about 15 years. In some embodiments, the patient and/or patient population is characterized by having an EDSS less than or equal to about 5.0 for less than about 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years , 13 years, 14 years or 15 years. In some embodiments, the patient and/or patient population is characterized by having an EDSS less than or equal to about 5.0 for less than about 10 years.

In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, the patient and/or patient population is characterized by having a score of about 1.5 to 7, 1.5 to 6.5, and 2 EDSS to 6.5 points or 3 points to 6.5 points. In some embodiments, the patient and/or patient population is characterized by having an EDSS between about 3.0 and about 6.5 at the beginning of treatment.

In some embodiments of any of the methods for treating, evaluating, and/or predicting responsiveness described herein, the patient and/or patient population is characterized by having an MSSS higher than about any of 6, 7, 8, or 9. . In some embodiments of any of the methods of treating and evaluating and/or predicting responsiveness described herein, the patient and/or patient population is characterized by having an MSSS greater than about 9.

In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, the patient and/or patient population is otherwise characterized by having two or more relapses within two years prior to initiation of treatment. In some embodiments, other characteristics of the patient and/or patient population are any of 2 relapses, 3 relapses, 4 relapses, or 5 relapses within two years before starting treatment.

In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, patients and/or patient populations are characterized by age. In some embodiments, the patient and/or patient population is characterized by being less than about any of 55 years old, 54 years old, 53 years old, 52 years old, 51 years old, or 50 years old. In some embodiments, the patient and/or patient population is characterized by being younger than about 51 years old.

In some embodiments of any of the methods of treatment and assessment and/or prediction of responsiveness described herein, treatment shortens the time to diagnosed disease progression. In some embodiments, the confirmed disease progression is an increase in EDSS for about any of 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, or 32 weeks. In some embodiments, the confirmed disease progression is an increase in EDSS that lasts for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS for twenty-four weeks.

In some embodiments of any of the methods of treatment and evaluation and/or prediction of responsiveness described herein, the anti-CD20 antibody comprises: a) comprising SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 The heavy chain variable region, and b) the light chain variable region comprising SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments of any of the methods described herein, the anti-CD20 antibody is okrelizumab. In some embodiments of any of the methods described herein, the anti-CD20 antibody is rituximab. In some embodiments of any of the methods described herein, the anti-CD20 antibody is ofatumomab. In some embodiments of any of the methods described herein, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments of any of the methods described herein, the anti-CD20 antibody is GA101. In some embodiments of any of the methods described herein, the anti-CD20 antibody is hA20.

The methods described herein can encompass any combination of the embodiments described herein. For example, the methods include methods of treatment and evaluation and/or prediction, wherein the patient (a) is less than about 55 years old and (b) has one or more gamma stained lesions.

<b><i>III. Dose</i></b>

According to some embodiments of any of the methods or products described herein, the methods or instructions comprise administering to patients with multiple sclerosis an effective amount of anti-CD20 antibody to provide about 0.3 grams to about 4 grams (preferably about 0.3 grams to about 1.5 grams, such as about 0.6 grams to about 1.0 grams) of initial antibody exposure, followed by about 0.3 grams to about 4 grams (preferably about 0.3 grams to about 1.5 grams, such as about 0.6 grams or about 1.0 grams) of second antibody exposure The second antibody exposure is not provided until about 16 to about 60 weeks after the initial antibody exposure. For the purpose of the present invention, the second antibody exposure is the next time the patient is treated with an anti-CD20 antibody after the initial antibody exposure, and there is no intervening anti-CD20 antibody treatment or exposure between the initial exposure and the second exposure. In some embodiments, the initial antibody exposure and/or the second antibody exposure are approximately any of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams, 0.9 grams, or 1.0 grams.

The time interval between the initial antibody exposure and the second or subsequent antibody exposure can be calculated from the first or second dose of the initial antibody exposure, but in some embodiments, it is calculated from the first dose of the initial antibody exposure.

In some embodiments, antibody exposures are about 24 weeks or 6 months apart or about 48 weeks or 12 months apart.

In one embodiment, the second antibody exposure is not provided until about 20 to about 30 weeks after the initial exposure, and as appropriate, about 0.3 g to about 4 g (preferably about 0.3 g to about 1.5 g) of the third antibody exposure. The third exposure is not administered until about 46 to 60 weeks (preferably about 46 to 54 weeks) after the initial exposure, and then in some embodiments, other antibody exposures are not provided until at least about 70 to 75 weeks after the initial exposure . In some embodiments, the third antibody exposure is approximately any of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams, 0.9 grams, or 1.0 grams.

In an alternative embodiment, the second antibody exposure is not provided until about 46 to 60 weeks after the initial exposure, and the subsequent antibody exposure (if any) is not provided until about 46 to 60 weeks after the previous antibody exposure.

The patient can be provided with any one or more antibody exposures herein in the form of a single dose of the antibody or two divided doses of the antibody (ie, constituting the first dose and the second dose). The specific dose number (one or two) used for each antibody exposure depends on, for example, the type of MS being treated, the type of antibody used, whether and what type of second agent is used, and the method and frequency of administration. When two separate doses are administered, the second dose is preferably administered about 3 to 17 days after the time of administration of the first dose, more preferably about 6 to 16 days, and most preferably about 13 to 16 days. In some embodiments, when two separate doses are administered, the second dose is about 14 days. When two separate doses are administered, the first dose and the second dose of the antibody are preferably about 0.3 g to 1.5 g, more preferably about 0.3 g to about 1.0 g. In some embodiments, when two separate doses are administered, the first dose and the second dose of the antibody are approximately any of 0.3 grams, 0.4 grams, 0.5 grams, or 0.6 grams. In some embodiments, the initial oxyclizumab exposure includes a first dose and a second dose of occlizumab, wherein the first dose and the second dose of occlizumab are about 0.3 grams. In some embodiments, the second occlizumab exposure comprises a single dose of occlizumab, wherein the single dose of occlizumab is 0.6 grams.

In one embodiment, the patient is provided with at least about three, at least about four, or at least about five antibody exposures, such as about 3 to 60 exposures and more specifically about 3 to 40 exposures, most specifically Approximately 3 to 20 exposures. In some embodiments of any of these methods, the methods further comprise providing between about one to about three subsequent exposures of okclizumab. In some embodiments, the exposures are administered at intervals of about 24 weeks or 6 months or 48 weeks or 12 months, respectively. In one embodiment, each antibody exposure is provided as a single dose of antibody. In an alternative embodiment, each antibody exposure is provided in two separate doses of antibody. However, not every antibody exposure needs to be provided in a single dose or in two separate doses.

The antibody can be a naked antibody or can bind to another molecule, such as a cytotoxic agent, such as a radioactive compound. In some embodiments, the antibody is rituximab, humanized 2H7 (e.g., comprising the variable domain sequences of SEQ ID NOs 2 and 8), or humanized 2H7 or humanized 2H7 comprising the variable domain sequences of SEQ ID NOs 23 and 24. huMax-CD20 (Genmab). In some embodiments, the antibody is okrelizumab (e.g., comprising (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 14) .

In one example, the patient has never been previously treated with drugs such as immunosuppressants to treat multiple sclerosis and/or has never been treated with antibodies against B cell surface markers (e.g., have never been treated with CD20 antibodies before. ).

The antibody is administered by any suitable means, including parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Also encompasses intrathecal administration (see, for example, US Patent Application No. 2002/0009444, Grillo-Lopez, A, for intrathecal delivery of CD20 antibodies). In addition, the antibody may be appropriately administered by pulse infusion, for example, in a decreasing antibody dose. In some embodiments, the dosage is administered intravenously, subcutaneously, or intrathecally. In some embodiments, the dosage is administered by intravenous infusion.

When the CD20 antibody may be the only drug to be administered to the patient to treat multiple sclerosis, the second drug, such as a cytotoxic agent, chemotherapy, may be administered together with an antibody that binds to a B cell surface marker (for example, together with a CD20 antibody) as appropriate. Agents, immunosuppressive agents, cytokines, cytokine antagonists or antibodies, growth factors, hormones, integrins, integrin antagonists or antibodies (for example, LFA-1 antibody, such as efalizumab available from Genentech (RAPTIVA<img file="TW201014605A_D0046.tif" />), or α4 integrin antibody, such as natalizumab (TYSABRI<img file="TW201014605A_D0047.tif" />))Wait.

In some embodiments of the combination therapy, the antibody is combined with the following: interferon drugs, such as IFN-β-1a (<img file="TW201014605A_D0048.tif" />and<img file="TW201014605A_D0049.tif" />) Or IFN-β-1b(<img file="TW201014605A_D0050.tif" />); oligopeptides, such as glatiramer acetate (<img file="TW201014605A_D0051.tif" />); Cytotoxic agents, such as mitoxantrone (<img file="TW201014605A_D0052.tif" />), methotrexate, cyclophosphamide, chlorambucil, azathioprine; intravenous immunoglobulin (gamma globulin); lymphocyte depletion therapy (such as mitoxantrone, cyclophosphamide, Campath, anti-CD4, cladribine, whole body irradiation, bone marrow transplantation); corticosteroids (such as methylprednisolone, prednisone, dexamethasone or glucocorticoids), including systemic corticosteroid therapy; Non-lymphocytic depletion immunosuppressive therapy (such as mycophenolate mofetil (MMF) or cyclosporine); "statin" cholesterol-lowering drugs, including cerivastatin (BAYCOL)<img file="TW201014605A_D0053.tif" />), fluvastatin (LESCOL<img file="TW201014605A_D0054.tif" />), atorvastatin (LIPITOR<img file="TW201014605A_D0055.tif" />), lovastatin (MEVACOR<img file="TW201014605A_D0056.tif" />), pravastatin (PRAVACHOL<img file="TW201014605A_D0057.tif" />), Simvastatin (ZOCOR<img file="TW201014605A_D0058.tif" />); estradiol; testosterone (used in high doses as appropriate, Stuve et al.<i>Neurology</i> 8:290-301 (2002)); hormone replacement therapy; treatment of secondary or related symptoms of MS (such as cramps, incontinence, pain, fatigue); TNF inhibitors; disease-modifying antirheumatic drugs (DMARD); Non-steroidal anti-inflammatory drugs (NSAID); plasma depletion; levothyroxine; cyclosporine A; somatostatin analogs; cytokine or cytokine receptor antagonists; antimetabolites; immunosuppressants; rehabilitation surgery; radioactivity Iodine; thyroidectomy; antagonists/antibodies on the surface of another B cell, etc.

The second agent is administered together with the initial exposure and/or later exposure of the CD20 antibody. The combined administration includes co-administration using a single formulation or a single pharmaceutical formulation, and continuous administration in any order, and preferably there are two A period of time during which (or all) active agents exert their biological activities simultaneously.

In addition to administering antibodies to patients, this application covers the administration of antibodies in gene therapy. The expression "effective amount" of antibody administered encompasses the administration of nucleic acid encoding the antibody. See, for example, WO 96/07321 published on March 14, 1996, which relates to the use of gene therapy to generate intracellular antibodies.

There are two main methods for getting nucleic acid (contained in the vector as the case may be) into the patient's cells; in vivo and in vitro. For in vivo delivery, the nucleic acid is usually injected directly into the patient at the site where the antibody is needed. For ex vivo treatment, patient cells are removed, nucleic acids are introduced into these isolated cells and the modified cells are directly administered to the patient, or, for example, encapsulated in a porous membrane implanted in the patient (see, for example, U.S. Patent No. 4,892,538 No. and No. 5,283,187). There are a variety of techniques that can be used to introduce nucleic acids into living cells. These techniques may be different depending on the transfer of nucleic acid into cultured cells in vitro or into cells of a predetermined host in vivo. Techniques suitable for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation methods, and the like. Vectors commonly used to deliver genes in vitro are retroviruses.

In some embodiments, in vivo nucleic acid transfer techniques include viral vectors (such as adenovirus, herpes simplex I virus or adeno-associated virus) and lipid-based systems (the lipids suitable for lipid-mediated gene transfer are, for example, DOTMA, DOPE And DC-Chol) transfection. In some cases, it is necessary to provide nucleic acid sources with drugs that target target cells, such as antibodies specific to cell surface membrane proteins or target cells, and ligands for receptors on target cells. If liposomes are used, proteins that bind to cell surface membrane proteins related to internal drinking can be used to target and/or promote absorption. For example, capsid proteins or fragments thereof that are tropism to specific cell types can undergo internalization in the circulation. Antibodies to the transformed proteins, and proteins that target intracellular localization and enhance intracellular half-life. The technology of receptor-mediated internal drinking is developed by Wu et al.,<i>J</i>.<i> Biol</i>.<i> Chem</i>. 262:4429-4432 (1987); and Wagner et al.,<i>Proc</i>.<i> Natl</i>.<i> Acad</i>.<i> Sci</i>.<i> USA</i> 87:3410-3414 (1990) description. For a review of currently known gene markers and gene therapy protocols, see Anderson et al.,<i>Science</i> 256:808-813 (1992). See also WO 93/25673 and references cited therein.

<b><i>IV. Antibodies and their production</i></b>

The methods and products of the present invention use or incorporate antibodies that bind to B cell surface markers, especially antibodies that bind to CD20. Therefore, methods of producing these antibodies will be described herein.

The B cell surface marker used to produce or screen antibodies can be, for example, a soluble form of a marker containing the desired epitope or a portion thereof. Alternatively or additionally, cells that exhibit markers on the cell surface can be used to produce or screen antibodies. Other forms of B cell surface markers suitable for antibody production are known to those familiar with this technology.

The following describes exemplary techniques for generating antibodies used in accordance with the present invention.

<i>(i) Multiple antibodies</i>

Multiple antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of relevant antigens and adjuvants. It can be suitable for using bifunctional agents or derivatizing agents to bind related antigens to proteins that are immunogenic in the species to be immunized, such as keyhole cyanogenin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor , The bifunctional agent or derivatizing agent is, for example, maleimide benzyl sulfosuccinimide (binding via cysteine residues), N-hydroxysuccinimide (via ionization Amino acid residues), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N=C=NR, where R and R¹Different alkyl groups.

By combining, for example, 100 μg or 5 μg protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intracutaneously at multiple sites with antigens and immunogens The sex conjugate or derivative immunizes the animal. One month later, the animals were strengthened by subcutaneous injection of 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant at multiple sites. After 7 to 14 days, the animals were bled and the antibody value of the serum was checked. Strengthen the animal until the price of power stabilizes. In some embodiments, animals are strengthened with conjugates of the same antigen (but the antigen is bound to different proteins and/or through different cross-linking agents). Conjugates can also be produced as protein fusions in recombinant cell culture. In addition, agglomerating agents such as alum are suitable for enhancing the immune response.

<i>(ii) Monoclonal antibody</i>

The monoclonal antibody system is obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies constituting the population are identical and/or combined except for the possible variants produced during the production of the monoclonal antibody (these variants are generally present in smaller amounts) Same epitope. Therefore, the modifier "monoclonal" indicates that the antibody is not a characteristic of a discrete antibody or a mixture of multiple antibodies.

For example, the monoclonal antibody can be used Kohler et al.,<i>Nature,</i>The fusion tumor method first described in 256:495 (1975) may be prepared by recombinant DNA method (US Patent No. 4,816,567).

In the fusion tumor method, mice or other suitable host animals such as hamsters are immunized as described herein to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Then use a suitable fusion agent such as polyethylene glycol to fuse lymphocytes with myeloma cells to form fusion tumor cells (Goding,<i>Monoclonal Antibodies:Principles and Practice</i>, Pages 59-103 (Academic Press, 1986)).

The fusion tumor cells thus prepared are inoculated and grown in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium used for the fusion tumor will usually include hypoxanthine, aminopterin, and thymidine (HAT medium ), these substances prevent the growth of HGPRT-deficient cells.

In some embodiments, the myeloma cells are those myeloma cells that efficiently fuse, support the selected antibody-producing cells to produce stable and high levels of antibody, and are sensitive to a medium such as HAT medium. Wherein, in some embodiments, the myeloma cell line is a murine myeloma cell line, such as MOPC available from Salk Institute Cell Distribution Center (San Diego, California USA) -21 and MPC-11 mouse tumor cell lines and SP-2 or X63-Ag8-653 cells available from American Type Culture Collection (Rockville, Maryland USA). Also described human myeloma and mouse-human heterologous myeloma cell lines for the preparation of human monoclonal antibodies (Kozbor,<i>J. Immunol</i>.,133:3001 (1984); Brodeur et al.,<i>Monoclonal Antibody Production Techniques and Applications</i>, Pages 51-63 (Marcel Dekker, Inc., New York, 1987)).

To test the production of monoclonal antibodies against the antigen in the growth medium of the fusion tumor cells. In some embodiments, the binding specificity of monoclonal antibodies produced by fusion tumor cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can be obtained, for example, by Munson et al.,<i>Anal. Biochem.</i>, 107:220 (1980) Scatchard analysis (Scatchard analysis) to determine.

After identifying the fusion tumor cells that produce antibodies with the required specificity, affinity and/or activity, pure lines can be subpopulated by a limiting dilution procedure and cultured by standard methods (Goding,<i>Monoclonal Antibodies:Principles and Practice</i>, Pages 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, fusion tumor cells can grow in vivo as ascites tumors in animals.

By conventional immunoglobulin purification procedures such as Protein A-Sepharose, Hydroxyapatite Chromatography, Gel Electrophoresis, Dialysis or Affinity Chromatography, the monoclonal antibody secreted by the sub-pure line is removed from the culture medium, ascites fluid or Properly separated from the serum.

The DNA encoding the monoclonal antibody is easily separated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). In some embodiments, fusion tumor cells are used as the source of the DNA. After isolation, the DNA can be placed in the expression vector, and then the expression vector can be transfected into host cells that do not otherwise produce immunoglobulin (such as Escherichia coli (<i>E. coli</i>) Cells, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells) to obtain monoclonal antibodies synthesized in recombinant host cells. Review articles on the recombination performance of DNA encoding antibodies in bacteria include Skerra et al.,<i>Curr. Opinion in Immunol</i>., 5: 256-262 (1993) and<img file="TW201014605A_D0059.tif" />,<i>Immunol. Revs</i>.,130:151-188(1992)。

In another embodiment, self-use is described in McCafferty et al.,<i>Nature</i>, 348:552-554 (1990) technology in the antibody phage library to isolate antibodies or antibody fragments. Clarkson et al.,<i>Nature</i> 352:624-628 (1991) and Marks et al.,<i>J. Mol. Biol</i>., 222:581-597 (1991) describes the use of phage libraries to separate murine and human antibodies, respectively. The subsequent public case describes the chain reorganization (Marks et al.,<i>Bio/Technology,</i>10:779-783 (1992)) as well as combinatorial infection and in vivo recombination as a strategy to construct extremely large phage libraries (Waterhouse et al.,<i>Nuc. Acids. Res</i>., 21: 2265-2266 (1993)) to generate high-affinity (nM range) human antibodies. Therefore, these techniques are viable alternatives to the traditional monoclonal antibody fusionoma technology used to isolate monoclonal antibodies.

DNA can also be replaced by homologous murine sequences by, for example, human heavy and light chain constant domain coding sequences (US Patent No. 4,816,567; Morrison et al.,<i>Proc. Natl Acad. Sci. USA</i>, 81:6851 (1984)) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

Usually these non-immunoglobulin polypeptides replace the constant domain of an antibody, or replace the variable domain of an antigen combination site of an antibody to produce an antigen combination site that contains one specific for the antigen and another specific for a different antigen The chimeric bivalent antibody of the antigen combination site.

<i>(iii) Humanized antibodies</i>

Methods of humanizing non-human antibodies have been described in this technology. In some embodiments, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from the "import" variable domain. But basically according to Winter and colleagues (Jones et al.,<i>Nature</i>, 321:522-525 (1986); Riechmann et al.,<i>Nature</i>, 332: 323-327 (1988); Verhoeyen et al.,<i>Science</i>, 239:1534-1536 (1988)), humanized by substituting hypervariable region sequences for the corresponding sequences of human antibodies. Therefore, these "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567), in which a portion substantially smaller than a complete human variable domain is replaced with a corresponding sequence from a non-human species. In practice, humanized antibodies are usually human antibodies in which some hypervariable region residues and possibly some FR residues are substituted with residues from similar sites in rodent antibodies.

The selection of human variable domains (light chain and heavy chain) to be used in the preparation of humanized antibodies is extremely important for reducing antigenicity. According to the so-called "best match" method, the variable domain sequences of rodent antibodies are screened against the entire library of known human variable domain sequences. Next, accept the human sequence closest to the rodent sequence as the human framework region (FR) of the humanized antibody (Sims et al.,<i>J. </i><i>Immunol</i>., 151: 2296 (1993); Chothia et al.,<i>J. Mol. Biol.</i>, 196: 901 (1987)). Another method uses specific framework regions derived from the common sequence of all human antibodies of a specific subgroup of light or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al.,<i>Proc. Natl. Acad. Sci. USA</i>, 89: 4285 (1992); Presta et al.,<i>J. Immunol.</i>,151:2623(1993))。

In addition, it is important that the humanized antibody retains high affinity for the antigen and other beneficial biological properties. To achieve this goal, in some embodiments of these methods, humanized antibodies are prepared by using a three-dimensional model of the parental sequence and the humanized sequence to analyze the parental sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly available and are well-known to those familiar with the art. Computer programs that illustrate and present possible three-dimensional configurations of selected candidate immunoglobulin sequences can be used. Examination of these presentations allows the analysis of the possible role of residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the accepted and imported sequences and combined in order to achieve the desired antibody characteristics, such as increased affinity for the target antigen. Generally speaking, the residues in the hypervariable region are directly and most substantially involved in influencing antigen binding.

In some embodiments, the humanized anti-CD20 antibody is a humanized 2H7 antibody. In some embodiments, the humanized 2H7 antibody preferably contains one, two, three, four, five or six of the following CDR sequences: CDR L1 sequence RASSSVSYXH, where X is M or L (SEQ ID NO 18) , Such as SEQ ID NO: 4 (Figure 1A), CDR L2 sequence SEQ ID NO: 5 (Figure 1A), CDR L3 sequence QQWXFNPPT, where X is S or A (SEQ ID NO 19), such as SEQ ID NO: 6 ( Figure 1A), CDR H1 sequence SEQ ID NO: 10 (Figure 1B), CDR H2 sequence AIYPGNGXTSYNQKFKG, where X is D or A (SEQ ID NO 20), such as SEQ ID NO: 11 (Figure 1B), and CDR H3 sequence VVYYSXXYWYFDV, where X at position 6 is N, A, Y, W, or D and X at position 7 is S or R (SEQ ID NO 21), such as SEQ ID NO: 12 (Figure 1B).

The above-mentioned CDR sequences generally exist in human variable light chain and variable heavy chain framework sequences, such as substantially human light chain kappa subgroup I (V_L6I) human common FR residues, and essentially human heavy chain subgroup III (V_HIII) Common human FR residues. See also WO 2004/056312 (Lowman et al.).

In some embodiments, the variable heavy chain region can be linked to the human IgG chain constant region, where the region can be, for example, IgG1 or IgG3, including natural sequences and variant constant regions.

In some embodiments, the antibody includes the variable heavy chain domain sequence SEQ ID NO: 8 (v16, as shown in Figure 1B), and optionally also includes the variable light chain domain sequence SEQ ID NO: 2 (v16, as shown in Figure 1B). 1A), which optionally contains one or more amino acid substitutions at positions 56, 100 and/or 100a in the variable heavy chain domain, such as D56A, N100A or N100Y and/or S100aR, and in the variable light The chain domain contains one or more amino acid substitutions at positions 32 and/or 92, such as M32L and/or S92A. In some embodiments, the antibody is a complete antibody comprising the light chain amino acid sequence of SEQ ID NO 13 or 16 and the heavy chain amino acid sequence of SEQ ID NO 14, 15, 17, 22, or 25. In some embodiments, the humanized 2H7 antibody is okrelizumab (Genentech).

In such embodiments, the humanized 2H7 comprising the variable light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 2); and the variable heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID NO: 8) of full antibody or antibody fragment .

In some embodiments, the humanized 2H7 antibody is an intact antibody, in some embodiments, the light chain comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13); heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) or the heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15).

In some embodiments, the humanized 2H7 antibody comprises 2H7.v511 variable light domain sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPG KAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO: 23) and 2H7.v511 variable heavy domain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQ APGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTL YLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTL VTVSS (SEQ ID NO . twenty four).

In some embodiments, the humanized 2H7.v511 antibody is an intact antibody, it may comprise the light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPG KAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO: 16) and heavy chain amino acid sequence of SEQ ID NO 17 Or: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQ APGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTL YLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAVSSASTKGPSVFPLAPSSKSTSGGTAALVSSTVSLVSSLTVSLVSGSLVSLVSLVSLVSGV TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO. 25).

In some embodiments, the antibodies herein may further include at least one amino acid substitution in the Fc region that improves ADCC activity, such as amino acid substitutions at positions 298, 333 and 334, preferably S298A, E333A and K334A (using Eu numbering of heavy chain residues). See also U.S. Patent No. 6,737,056B1 to Presta. Any of these antibodies may contain at least one substitution in the Fc region that improves FcRn binding or serum half-life, for example a substitution at position 434 of the heavy chain, such as N434W. See also U.S. Patent No. 6,737,056B1 to Presta. Any of these antibodies may further include at least one amino acid substitution in the Fc region that increases CDC activity, for example, at least one substitution at position 326, preferably K326A or K326W. See also U.S. Patent No. 6,528,624 Bl (Idusogie et al.).

In some embodiments, the humanized 2H7 variants are variants comprising the variable light chain domain of SEQ ID NO: 2 and the variable heavy chain domain of SEQ ID NO: 8, including those in the Fc region (if present) Those variants with or without substitutions, and include the variable heavy chain domains of SEQ ID NO: 8 with changes in N100A; or D56A and N100A; or D56A, N100Y, and S100aR, and with changes in M32L; or S92A; Or these variants of the variable light chain domain of SEQ ID NO: 2 of M32L and S92A. M34 in the variable heavy chain domain of 2H7.v16 was identified as a possible source of antibody stability and another possible replacement candidate.

In some embodiments of the present invention, the variable region of the variant based on 2H7.v16 contains the v16 amino acid sequence in addition to the amino acid substitution positions indicated in the table below. Unless otherwise indicated, the 2H7 variant will have the same light chain as v16.

<tables><img file="TW201014605A_D0060.tif" /></tables>

<i>(iv) Human antibodies</i>

As an alternative to humanization, human antibodies can be produced. For example, it is currently possible to produce transgenic animals (such as mice) that can produce a full set of human antibody repertoires without producing endogenous immunoglobulins. For example, it has been described that the homozygous deletion antibody overlaps the junction region in chimeric and germline mutant mice (J_H) Genes lead to complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene array to the germline mutant mice will lead to the production of human antibodies after antigen challenge. See, for example, Jakobovits et al.,<i>Proc. Natl. Acad. Sci USA,</i>90:2551 (1993); Jakobovits et al.,<i>Nature,</i>362:255-258 (1993); Bruggermann et al.,<i>Year in Immuno.,</i>7<i>:</i>33 (1993) and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807.

Alternatively, phage display technology can be used (McCafferty et al.,<i>Nature</i> 348:552-553 (1990)) in vitro production of human antibodies and antibody fragments from the immunoglobulin variable (V) domain gene lineage of unimmunized donors. According to this technology, antibody V domain genes are cloned into the primary or secondary sheath protein genes of filamentous phage (such as M13 or fd) in frame, and are presented on the surface of phage particles in the form of functional antibody fragments. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also leads to the selection of the gene encoding the antibody exhibiting those properties. Therefore, bacteriophages mimic some of the properties of B cells. Phage presentation can be performed in a variety of formats; for its review, see, for example, Johnson, Kevin S. and Chiswell, DaVid J.,<i>Current Opinion in Structural Biology</i> 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al.,<i>Nature,</i>352:624-628 (1991) Isolation of different arrays of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Can construct the V gene lineage from unimmunized human donors, and can basically follow Marks et al.,<i>J. Mol. Biol. </i>222:581-597 (1991) or Griffith et al.,<i>EMBO J. </i>The technique described in 12:725-734 (1993) separates antibodies against different antigen arrays (including autoantigens). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

Human antibodies can also be produced by activating B cells in vitro (see U.S. Patent Nos. 5,567,610 and 5,229,275).

<i>(v) Antibody fragments</i>

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments are produced by proteolytic digestion of intact antibodies (see, for example, Morimoto et al.,<i>Journal of Biochemical and Biophysical Methods</i> 24:107-117 (1992) and Brennan et al.,<i>Science</i>, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')₂Fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another method, F(ab')₂Fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be understood by skilled practitioners. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894 and U.S. Patent No. 5,587,458. Antibody fragments can also be "wire antibodies", such as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific antibodies.

(vi) Bispecific antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of B cell surface markers. Other such antibodies can bind to B cell surface markers and further bind to a second different B cell surface marker. Alternatively, the anti-B cell surface marker binding arm can bind to trigger molecules on white blood cells (such as T cell receptor molecules (such as CD2 or CD3)) or IgG Fc receptors (FcγR) (such as FcγRI (CD64), FcγRII (CD32). ) And FcγRIII (CD16)) in order to focus the cell defense mechanism on B cells. Bispecific antibodies can also be used to localize cytotoxic agents to B cells. These antibodies have B cell surface marker binding arms and bind to cytotoxic agents (e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or Radioisotope hapten) arm. Bispecific antibodies can be full-length antibodies or antibody fragments (e.g. F(ab')₂Bispecific antibody) format.

Methods for making bispecific antibodies are known in the art. The traditional preparation of full-length bispecific antibodies is based on the common performance of two immunoglobulin heavy chain/light chain pairs, where the two chains have different specificities (Millstein et al.,<i>Nature</i>, 305: 537-539 (1983)). Due to the random collocation of immunoglobulin heavy and light chains, these fusion tumors (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one molecule has the correct bispecific structure. The purification of the correct molecule by affinity chromatography steps is usually quite cumbersome, and the product yield is low. Similar procedures are disclosed in WO 93/08829 and Traunecker et al.,<i>EMBO J.</i>, 10:3655-3659 (1991).

According to different methods, an antibody variable domain with the desired binding specificity (antibody-antigen combination site) is fused with an immunoglobulin constant domain sequence. In some embodiments, it is fused to an immunoglobulin heavy chain constant domain (comprising at least a part of the hinge, CH2, and CH3 regions). In some embodiments, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of these fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain is inserted into an independent expression vector and co-transfected into a suitable host organism. In the embodiment where the unequal ratios of the three polypeptide chains used in the construction provide the best yield, this provides high flexibility to adjust the mutual ratio of the three polypeptide fragments. However, when the performance of at least two polypeptide chains in equal ratios yields high yields or when the ratios are not of particular importance, it is possible to insert two or all three polypeptide chain coding sequences in one expression vector.

In some embodiments of this method, the bispecific antibody comprises a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain light chain pair in the other arm (providing the first Two binding specificity). It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from the undesired combination of immunoglobulin chains. This is because the presence of immunoglobulin light chains in only half of the bispecific molecules provides an easy way of separation. This method is disclosed in WO 94/04690. For other details on the production of bispecific antibodies, see, for example, Suresh et al.,<i>Methods in Enzymology</i>,121:210(1986)。

According to another method described in US Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In some embodiments, the interface includes the C of the antibody constant domain_HAt least part of 3 domains. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (such as tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller side chain (such as alanine or threonine), a compensation "hole" with the same or similar size as the large side chain is generated on the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers over other undesired end products (such as homodimers).

Bispecific antibodies include cross-linked or "heterologous binding" antibodies. For example, one antibody in the form of a heteroconjugate can be coupled to avidin and the other to biotin. These antibodies have been proposed, for example, to target immune system cells to unwanted cells (US Patent No. 4,676,980) and to treat HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Any suitable cross-linking method can be used to prepare heterologously bound antibodies. Suitable crosslinking agents and many crosslinking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980.

Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, chemical linkages can be used to prepare bispecific antibodies. Brennan et al.,<i>Science</i>229:81 (1985) describes a proteolytic cleavage of intact antibodies to produce F(ab')₂Fragment of the program. In the presence of the dithiol complexing agent sodium arsenite, these fragments are reduced to stabilize the o-dithiols and prevent the formation of intermolecular disulfides. The Fab' fragments produced are then converted into thionitrobenzoate (TNB) derivatives. Then, one Fab'-TNB derivative is converted into Fab'-thiol by reduction with mercaptoethylamine, and it is mixed with an equal molar amount of another Fab'-TNB derivative to form a bispecific antibody. The bispecific antibodies produced can be used as reagents for the selective immobilization of enzymes.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al.,<i>J. Immunol.</i>, 148(5): 1547-1553 (1992). The leucine zipper peptides from Fos and Jun proteins were linked to the Fab' parts of two different antibodies by gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer, and then it is reoxidized to form an antibody heterodimer. This method can also be used to produce antibody homodimers. Hollinger et al.,<i>Proc. Natl. Acad. Sci. USA</i>, 90:6444-6448 (1993), the "bifunctional antibody" technology has provided an alternative mechanism for preparing bispecific antibody fragments. These fragments include linkers and light chain variable domains (V_L) Linked heavy chain variable domain (V_H). Therefore, forcing a segment of V_HDomain and V_LComplementarity of domain and another segment V_LDomain and V_HThe domains are paired, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al.,<i>J. Immunol.</i>,152:5368(1994)。

Covers antibodies with more than two valences. For example, trispecific antibodies can be prepared. Tutt et al<i>J. Immunol.</i> 147:60(1991)。

<b><i>V. Binding of antibodies and other modifications</i></b>

The antibodies used in the methods herein or included in the preparations are optionally combined with cytotoxic agents. For example, antibodies can be combined with drugs as described in WO 2004/032828.

The chemotherapeutic agents suitable for the production of these antibody-cytotoxic agent conjugates have been described above.

Conjugates of antibodies and one or more small molecule toxins such as calicheamicir, maytansine (US Patent No. 5,208,020), trichothene and CC1065 are also covered herein. In one embodiment of the present invention, the antibody is combined with one or more maytansine molecules (for example, about 1 to about 10 maytansine molecules per antibody molecule). Maytansine can, for example, be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with modified antibodies (Chari et al.<i>Cancer Research</i> 52:127-131 (1992)) produces maytansine-antibody conjugates.

Alternatively, the antibody is bound to one or more calicheamicin molecules. The calicheamicin family of antibiotics can produce sub-picomolar concentration of double-stranded DNA breaks. Structural analogs of calicheamicin that can be used include (but are not limited to) γ₁^I, Α₂^I, Α₃^I, N-Acetyl-γ₁^I, PSAG and θ^I₁(Hinman et al.<i>Cancer Research</i> 53: 3336-3342 (1993) and Lode et al.<i>Cancer Research</i> 58:2925-2928(1998))。

Enzymatically active toxins and their fragments that can be used include diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa (<i>Pseudomonas aeruginosa</i>)), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, tung protein (<i>Aleurites fordii protein</i>), carnation protein (dianthin protein), pokeweed protein (<i>Phytolaca americana protein</i>) (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, croton, sapaonaria officinalis inhibitor, gelonin ), mitogellin, restrictocin, phenomycin, economycin and tricothecene. See, for example, WO 93/21232 published on October 28, 1993.

The present invention further encompasses antibodies that bind to compounds having nuclear decomposition activity (for example, ribonuclease or DNA endonuclease, such as deoxyribonuclease; DNase).

A variety of radioisotopes can be used to produce radioactively conjugated antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³²And radioactive isotope of Lu.

Combinations of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-butanediimino-3-(2-pyridyldithiol) propionic acid Ester (SPDP), succinimidyl-4-(N-maleiminomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), Difunctional derivatives of imidate esters (such as dimethyl adipimidate HCL), active esters (such as dibutyl imidate suberate), aldehydes (such as glutaraldehyde), diazide Base compounds (such as bis(p-azidobenzyl) hexamethylene diamine), double nitrogen salt derivatives (such as bis(p-diazobenzyl)-ethylene diamine), diisocyanates (such as 2,6 -Toluene diisocyanate) and dual active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the ricin immunotoxin can be as Vitetta et al.,<i>Science</i>, 238:1098 (1987). Carbon 14-labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radionucleotides to antibodies. See WO 94/11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al.<i>Cancer Research</i> 52:127-131(1992))。

Alternatively, a fusion protein containing an antibody and a cytotoxic agent can be prepared, for example, by recombinant technology or peptide synthesis.

In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for pre-targeting tumors, where the antibody-receptor conjugate is administered to the patient, followed by the use of a clearing agent. The bound conjugate is removed from the circulation, and then a "ligand" (e.g., avidin) bound to the cytotoxic agent (e.g., radionucleotide) is administered.

The antibody of the present invention can also be combined with a prodrug activating enzyme that converts a prodrug (for example, a peptide-based chemotherapeutic agent, see WO 81/01145) into an active anticancer drug. See, for example, WO 88/07378 and U.S. Patent No. 4,975,278.

The enzyme components of these conjugates include any enzyme that can act on the prodrug to convert it into a more active cytotoxic form.

Enzymes suitable for the method of the present invention include (but are not limited to): alkaline phosphatase suitable for converting phosphate-containing prodrugs into free drugs; arylsulfuric acid suitable for converting sulfate-containing prodrugs into free drugs Esterase; cytosine deaminase suitable for converting non-toxic 5-fluorocytosine into 5-fluorouracil, an anticancer drug; proteases, such as serratia protease, thermolysin, Bacillus subtilis Protease (subtilisin), carboxypeptidase and cathepsin (such as cathepsin B and L), which are suitable for converting peptide-containing prodrugs into free drugs; suitable for converting D-propylamine containing D-amino acid substituent prodrugs Carbohydrate lyase; carbohydrate lyase, such as β-galactosidase and neuraminidase suitable for converting glycosylated prodrugs into free drugs; suitable for converting β-lactam Β-lactamase that converts the derivative drug into free drug; and penicillin adiminidase, such as penicillin V adiminidase or penicillin G adiminidase, which is suitable for converting phenoxyacetamidase or phenylethyl amidase, respectively The drug derived from the amine group at the amine nitrogen is converted into a free drug. Alternatively, antibodies with enzymatic activity (also referred to in the art as "abzymes") can be used to convert the prodrugs of the present invention into free active drugs (see, for example, Massey,<i>Nature</i> 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein to deliver avidase to tumor cell populations.

The enzyme of the present invention can be covalently bound to the antibody by techniques well known in the art, such as the use of the above-mentioned heterobifunctional cross-linking reagent. Alternatively, recombinant DNA technology well known in the art can be used to construct a fusion protein comprising at least the antigen-binding region of the antibody of the invention and at least the functionally active part of the enzyme of the invention (see, for example, Neuberger et al.,<i>Nature</i>,312:604-608(1984))。

This article covers other antibody modifications. For example, the antibody can be attached to one of a variety of non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or a copolymer of polyethylene glycol and polypropylene glycol. In some embodiments, antibody fragments (such as Fab') are linked to one or more PEG molecules.

The antibodies disclosed herein can also be formulated as liposomes. Antibody-containing liposomes are prepared by methods known in the art, such as Epstein et al.,<i>Proc. Natl. Acad. Sci. USA</i>, 82: 3688 (1985); Hwang et al.,<i>Proc. Natl. Acad. Sci. USA</i>, 77: 4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545, and methods described in WO 97/38731 published on October 23, 1997. Liposomes with enhanced circulation time are disclosed in US Patent No. 5,013,556.

Particularly suitable liposomes can be produced by a reverse phase evaporation method using a lipid composition containing phospholipid choline, cholesterol and PEG-derived phospholipid ethanolamine (PEG-PE). The liposomes are extruded through a filter with a specified pore size to produce liposomes with the desired diameter. But like Martin et al.<i>J. Biol. Chem.</i> As described in 257:286-288 (1982), the Fab' fragment of the antibody of the present invention is bound to liposomes via a disulfide bond exchange reaction. Optionally, the liposome contains a chemotherapeutic agent. See Gabizon et al.,<i>J. National Cancer Inst.</i> 81(19)1484(1989)。

Covers the amino acid sequence modification of antibodies. For example, it may be necessary to improve the binding affinity and/or other biological properties of the antibody. The amino acid sequence variation system of the antibody is prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion and/or insertion and/or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution is performed to obtain the final structure, and the restriction condition is that the final structure has the required characteristics. Amino acid changes can also change the post-translational process of the antibody, such as changing the number or position of glycosylation sites.

The method suitable for identifying certain residues or regions of antibodies as better locations for mutagenesis is called "alanine scanning mutation", such as Cunningham and Wells <i>Science</i>, 244: 1081-1085 (1989). Here, identify residues or target residue groups (for example, charged residues such as arg, asp, his, lys, and glu) and use neutral or negatively charged amino acids (preferably alanine or polyalanine). ) Replacement to achieve the interaction between amino acid and antigen. Then, by introducing additional or other variants at the substitution site or targeting the substitution site, the amino acid positions that exhibit functional sensitivity to substitution are improved. Therefore, although the site at which the amino acid sequence variation is introduced is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the mutation efficiency of a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the displayed antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging from one residue to a polypeptide containing one hundred or more residues, and insertions within a sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionine residues or antibodies fused to cytotoxic polypeptides. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of the antibody with an enzyme or polypeptide, which increases the serum half-life of the antibody.

Another type of variant is the amino acid substitution variant. At least one amino acid residue in the antibody molecule of these variants is replaced by a different residue. The most interesting sites for substitution mutagenesis of antibodies include hypervariable regions, but also include FR changes. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". If these substitutions result in a change in biological activity, more substantial changes named "exemplary substitutions" in Table 2 can be introduced, and the products can be screened.

<tables><img file="TW201014605A_D0061.tif" /></tables>

<tables><img file="TW201014605A_D0062.tif" /></tables>

Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that have significantly different effects on maintaining the following: (a) The structure of the polypeptide backbone in the substitution region, such as a folded sheet or spiral configuration; (b) the target position The charge or hydrophobicity of the molecule at the point; or (c) the side chain volume. Amino acids can be grouped according to the similarity of side chain properties (in AL Lehninger,<i>Biochemistry</i>, Second Edition, pp. 73-75, Worth Publishers, New York (1975)). (1) Non-polarity: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) (2) without Electrode: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q)(3) Acidity: Asp(D), Glu(E) )(4) Basicity: Lys(K), Arg(R), His(H)

Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatics: Trp, Tyr, Phe .

Non-conservative substitutions will require replacing a member of one of these classes with another class.

It can also be substituted (usually with serine) for any cysteine residues that are not involved in maintaining the proper conformation of the antibody to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can be added to the antibody to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitution variant involves the substitution of one or more hypervariable region residues of the parent antibody. Generally speaking, the resulting variants selected for further development usually have improved biological properties relative to the parent antibody from which they were produced. A suitable way for generating these substitution variants is to use the affinity maturation presented by phage. In short, mutate several hypervariable regions (e.g., 6-7 positions) to generate all possible amine substitutions at each position. The antibody variants thus produced are monovalently presented from filamentous phage particles as fusions with the gene III product of M13 encapsulated in each particle. The phage is then screened for biological activity (e.g., binding affinity) of the variant as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. These contact residues and adjacent residues are candidate residues for substitution according to the techniques detailed herein. After the generation of these variants, so the variant of the group is subjected to screening as described herein, and may have excellent properties of anti selected in one or more relevant assays are selected for further development.

Another type of antibody amino acid variants changes the original glycosylation pattern of the antibody. The modification includes the deletion of one or more carbohydrate moieties present in the antibody and/or the addition of one or more glycosylation sites that are not present in the antibody.

Polypeptide glycosylation is usually N-linked or O-linked. The N-linked type refers to the connection of the carbohydrate moiety to the side chain of the asparagine residue. The tripeptide sequence asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) is the carbohydrate moiety and asparagine side chain The recognition sequence for enzymatic ligation. Therefore, the presence of any of these tripeptide sequences in the polypeptide creates potential glycosylation sites. O-linked glycosylation means that one of the sugars N-acetylgalactosamine, galactose, or xylose is attached to a hydroxyl amino acid, most commonly serine or threonine, but 5- Hydroxyproline or 5-hydroxylysine.

The addition of glycosylation sites to the antibody is suitably achieved by changing the amino acid sequence so that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). The change can also be carried out by adding one or more serine or threonine residues or substituting one or more serine or threonine residues in the sequence of the original antibody (for O-linked In terms of glycosylation sites).

When the antibody contains an Fc region, the carbohydrate linked to it can be changed. For example, an antibody having a mature carbohydrate structure lacking trehalose linked to the Fc region of the antibody is described in U.S. Patent Application No. 2003/0157108 A1 (Presta, L.); see also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd), which is about CD20 antibody composition. Antibodies with bisected N-acetylglucosamine (GlcNAc) in carbohydrates linked to the Fc region of antibodies are mentioned in WO 03/011878, Jean-Mairet et al. and Umana et al. U.S. Patent No. 6,602,684 and. Antibodies with at least one galactose residue in the oligosaccharide linked to the Fc region of the antibody are reported in WO 97/30087 (Patel et al.); see also WO 98/58964 (Raju, S.) and WO 99/22764 (Raju ,S.), which is an antibody with altered carbohydrates linked to its Fc region.

In some embodiments, the glycosylation variants herein comprise an Fc region, wherein the carbohydrate structure linked to the Fc region lacks trehalose. These variants have improved ADCC function. Optionally, the Fc region further includes one or more amino acid substitutions in it that further improve ADCC, such as substitutions at positions 298, 333, and/or 334 (Eu numbering of residues) in the Fc region. Examples of publications related to "de-trehaloseylation" or "trehalose deficient" antibodies include: US Patent Application No. US 2003/0157108 A1 of Presta, L; WO 00/61739 A1; WO 01/29246A1; US 2003/0115614A1; US 2002/0164328A1; US 2004/0093621A1; US 2004/0132140A1; US 2004/0110704A1; US 2004/0110282A1; US 2004/0109865A1; WO 03/085119A1; WO 03/084570A1; WO 2005/035778; WO 2005/035586 (Description of RNA Inhibition (RNAi) of Trehalose); Okazaki et al.<i>J. Mol. Biol</i>. 336:1239-1249 (2004); Yamane-Ohnuki et al.<i>Biotech. Bioeng</i>. 87:614 (2004). Examples of cell lines that produce anti-trehaloseylation antibodies include protein trehaloseylation-deficient Lec13 CHO cells (Ripka et al.,<i>Arch. Biochem. Biophys</i>249:533-545 (1986); US Patent Application No. US 2003/0157108 A1 of Presta, L and WO 2004/056312 A1 of Adams et al., especially Example 11) and gene knock-out cell lines, such as α-1 ,6-trehalosyltransferase gene,<i>FUT8</i>, Gene knockout CHO cells (Yamane-Ohnuki et al.,<i>Biotech. Bioeng.</i> 87:614(2004))。

Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods known in the art. These methods include (but are not limited to) isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or mediated by variant or non-variant type oligonucleotides prepared in the early stages of antibody (Or site-specific) mutagenesis, PCR mutagenesis and sequence cassette mutagenesis preparation.

It may be necessary to modify the antibody of the present invention with respect to effector functions in order to, for example, enhance the antibody's antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibody may have improved internalization capabilities and/or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al.,<i>J. Exp Med.</i> 176:1191-1195 (1992) and Shopes, B. <i>J. Immunol.</i> 148:2918-2922 (1992). Such as Wolff et al.<i>Cancer Research</i> The homodimeric antibody with enhanced anti-tumor activity described in 53:2560-2565 (1993) can also be prepared using heterobifunctional crosslinking agents. Alternatively, the antibody can be engineered to have dual Fc regions and can in turn have enhanced complement solubilization and ADCC capabilities. See Stevenson et al.<i>Anti-Cancer Drug </i><i>Design</i> 3:219-230(1989)。

WO 00/42072 (Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies contain amino acid substitutions in their Fc region. In some embodiments, the antibody with improved ADCC contains substitutions at positions 298, 333, and/or 334 of the Fc region. In some embodiments, the altered Fc region is a human IgG1 Fc region comprising one, two, or three substitutions at these positions or consisting of such substitutions.

Antibodies with altered Clq binding and/or complement dependent cytotoxicity (CDC) are described in WO 99/51642, U.S. Patent No. 6,194,551B1, U.S. Patent No. 6,242,195B1, U.S. Patent No. 6,528,624B1, and U.S. Patent No. 6,538,124 Number (Idusogie et al.). The antibodies contain amino acid substitutions at one or more of the amino acid positions 270, 322, 326, 327, 329, 313, 333, and/or 334 of the Fc region.

In order to increase the serum half-life of antibodies, salvage receptor binding epitopes can be incorporated into antibodies (especially antibody fragments), for example, as described in U.S. Patent No. 5,739,277. As used herein, the term "rescue receptor binding epitope" refers to an IgG molecule (e.g., IgG₁, IgG₂, IgG₃Or IgG₄The epitope in the Fc region of IgG is responsible for increasing the serum half-life of IgG molecules in vivo. Antibodies with substitutions in the Fc region and increased serum half-life are also described in WO 00/42072 (Presta, L.).

It also covers engineered antibodies with three or more (preferably four) functional antigen binding sites (US Patent No. US 2002/0004587 A1, Miller et al.).

<b><i>VI. Pharmaceutical formulations</i></b>

The therapeutic formulation of the antibody used according to the present invention is prepared by mixing the antibody with the desired purity and optionally pharmaceutically acceptable carriers, excipients or stabilizers in the form of a lyophilized formulation or an aqueous solution For storage (<i>Remington's Pharmaceutical Sciences</i>16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration used and include: buffers, such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine ; Preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroxy quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoate , Such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) Polypeptides; proteins, such as serum albumin, gelatin or immunoglobulin; 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; Salt relative ions, such as sodium; metal complexes (such as Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN^TM, PLURONICS^TMOr polyethylene glycol (PEG).

Exemplary anti-CD20 antibody formulations are described in WO 98/56418. This publication describes a liquid multiple-dose formulation containing 40 mg/mL Rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, and 0.02% polysorbate 20 with a pH of 5.0. , Its minimum storage period is two years under 2-8BC. Another anti-CD20 formulation of interest contains 10 mg/mL in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and sterile water for injection (pH 6.5). mL Rituximab (Rituximab).

Lyophilized formulations suitable for subcutaneous administration are described in US Patent No. 6,267,958 (Andya et al.). The lyophilized formulations can be reconstituted to a high protein concentration with a suitable diluent, and the reconstituted formulations can be administered subcutaneously to the mammal to be treated herein.

It also covers antibodies in crystalline form. See, for example, US 2002/0136719A1 (Shenoy et al.).

The formulation herein may also contain more than one active compound necessary for the specific indication being treated, and in some embodiments active compounds with complementary activities that do not adversely affect each other. For example, it may be necessary to further provide a cytotoxic agent; chemotherapeutic agent; immunosuppressant; cytokine; cytokine antagonist or antibody; growth factor; hormone; integrin; integrin antagonist or antibody (For example, LFA-1 antibody, such as efalizumab/RAPTIVA available from Genentech, or α4 integrin antibody, such as natalizumab/TYSABRI available from Biogen Idec/Elan Pharmaceuticals, Inc.<img file="TW201014605A_D0063.tif" />); Interferon drugs, such as IFN-β-1a (<img file="TW201014605A_D0064.tif" />and<img file="TW201014605A_D0065.tif" />) Or IFN-β-1b(<img file="TW201014605A_D0066.tif" />); oligopeptides, such as glatiramer acetate (<img file="TW201014605A_D0067.tif" />); Cytotoxic agents, such as mitoxantrone (<img file="TW201014605A_D0068.tif" />), methotrexate, cyclophosphamide, chlorambucil or azathioprine; intravenous immunoglobulin (gamma globulin); lymphocyte depleting drugs (such as mitoxantrone, cyclophosphamide, Perth, anti-CD4 or Cladribine); non-lymphocyte depleting immunosuppressive drugs (such as mycophenolate mofetil (MMF) or cyclosporine); "statin" cholesterol-lowering drugs; estradiol; testosterone; hormone replacement Therapies; drugs for the treatment of secondary or related symptoms of MS (such as cramps, incontinence, pain, fatigue); TNF inhibitors; disease-modifying antirheumatic drugs (DMARD); non-steroidal anti-inflammatory drugs (NSAID); corticosteroids ( For example, methylprednisolone, prednisone, dexamethasone or glucocorticoids); levothyroxine; cyclosporine A; somatostatin analogs; cytokine antagonists; antimetabolites; immunosuppressants; integrins Anti-agent or antibody (for example, LFA-1 antibody, such as efalizumab or α4 integrin antibody, such as natalizumab); or another B cell surface antagonist/antibody, etc. The types and effective amounts of these other agents depend, for example, on the amount of antibodies present in the formulation, the type of multiple sclerosis being treated, and the clinical parameters of the patient. These equivalent amounts are generally used at the same dosage and route of administration as used above, or about 1% to 99% of the dosage used so far.

The active ingredient can also be enclosed in microcapsules prepared by coacervation technology or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), In colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. These technologies are disclosed in<i>Remington's Pharmaceutical Sciences</i>16th edition, Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g. poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L-bran Amino acid and γ-ethyl-L-glutamate copolymer, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (such as LUPRON DEPOT^TM(Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

The formulation to be used for in vivo administration must be sterile. This can be easily achieved by filtering through a sterile filter membrane.

In some embodiments, the formulation includes one or more of the group consisting of histidine buffer, trehalose, sucrose, and polysorbate 20. In some embodiments, the histidine buffer is histidine-acetate buffer (pH 6.0). Examples of formulations suitable for the administration of anti-CD20 antibodies are found in US 2006/0088523 of Andya et al., which is incorporated by reference in its entirety on formulations.

Exemplary anti-CD20 antibody formulations are described in US 2006/0088523 and WO 98/56418 of Andya et al., which are incorporated herein by reference in their entirety. In some embodiments, the formulation is a liquid multiple dose formulation comprising the following: 40 mg/mL anti-CD20 antibody, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 (pH 5.0 ), its minimum storage period is two years at 2-8°C. In some embodiments, the anti-CD20 formulation of interest is contained in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and sterile water for injection (pH 6.5) Among the 10mg/mL antibody. In some embodiments, the anti-CD20 antibody contains 10-30 mM sodium acetate with a pH value of about 4.8 to about 5.5, preferably a pH value of 5.5; a polymer as a surfactant in an amount of about 0.01-0.1% v/v Sorbitol ester; an aqueous pharmaceutical formulation of trehalose with an amount of about 2-10% w/v and benzyl alcohol used as a preservative (US 6,171,586, which is incorporated herein by reference in its entirety). Lyophilized formulations suitable for subcutaneous administration are described in WO 97/04801, which is incorporated herein by reference in its entirety. The lyophilized formulations can be reconstituted to a high protein concentration with a suitable diluent, and the reconstituted formulations can be administered subcutaneously to the mammal to be treated herein.

In some embodiments, the humanized 2H7 variant formulation is 12-14 mg/mL antibody in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20 (pH 5.8). In a specific embodiment, 2H7 variants and especially 2H7.v16 are formulated in 10mM histidine sulfate, 60mg/ml sucrose, 0.2mg/ml polysorbate 20 and sterile water for injection (pH 5.8). 20mg/mL antibody. In a specific embodiment, a humanized 2H7 v16 IV formulation is: 20mM sodium acetate, 4% trehalose dihydrate, 0.02% polysorbate 20 (Tween 20^TM) (in pH 5.3) 30mg/ml antibody. In some embodiments, the humanized 2H7.v511 variant formulation is based on 10mM histidine sulfate, 60mg/ml sucrose (6%), 0.2mg/ml polysorbate 20 (0.02%) and sterile water for injection (pH 5.8) 15-30mg/ml antibody, preferably 20mg/ml antibody. In another embodiment, the 2H7 variant and especially the formulation of 2H7.v511 is 20mg/ml 2H7, 20mM sodium acetate, 4% trehalose dihydrate, 0.02% polysorbate 20 (pH 5.5). In some embodiments, the 2H7.v 114 formulation is 15-25 mg/ml in 20 mM sodium acetate, 240 mM (8%) trehalose dihydrate, 0.02% polysorbate 20 (pH 5.3), preferably 20 mg /ml antibody.

<b><i>VII. Products and manufacturing methods</i></b>

The present invention provides a product comprising: (a) a container containing okclizumab; and (b) a package insert with instructions for treating multiple sclerosis in a patient, wherein the instructions (for example, instructions) are provided to the patient Administer an amount of okclizumab that is effective to provide an initial exposure of okclizumab between about 0.3 g and about 0.6 g, and then provide a second okclizumab exposure between about 0.3 g and about 0.6 g The second exposure is not administered until about 16 to 60 weeks after the initial exposure, and each occlizumab exposure is provided to the patient in the form of one or two doses of occlizumab. In some embodiments, the initial Okclizumab exposure is about 0.6 grams. In some embodiments, the second okclizumab exposure is about 0.6 grams. In some embodiments, the second exposure is administered about 24 weeks after the initial exposure. In some embodiments, the patient is provided with one or more exposures of occlizumab in the form of one dose of occlizumab. In some embodiments, the patient is provided with one or more occlizumab exposures in the form of two doses of occlizumab. In some embodiments, the two doses of oxyclizumab comprise about 0.3 grams of occlizumab.

The present invention further provides products containing substances suitable for the treatment of progressive multiple sclerosis described herein. In some embodiments, there is provided an article of manufacture comprising a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier packaged together and indicating that the anti-CD20 antibody or pharmaceutical composition is designated for treatment with one or more The label of a patient suffering from multiple sclerosis with characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma-stained lesions, (c) expansion within two years before starting treatment The disability status scale (EDSS) is increased by at least about one point, and (d) the multiple sclerosis severity score (MSSS) is higher than about 5 points.

In some embodiments, there is provided an article of manufacture comprising a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier packaged together and an instruction to administer the anti-CD20 antibody or the pharmaceutical composition is based on the patient having one or A variety of labels selected from the characteristics of the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) extended disability status scale (EDSS) within two years before starting treatment Increase by at least about one point, and (d) Multiple Sclerosis Severity Score (MSSS) is higher than about 5 points.

In some embodiments, there is provided an article of manufacture comprising a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier packaged together and a label indicating the administration of the pharmaceutical composition to a selected patient, wherein the selected The patient has one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) expanding the disability status scale within two years before starting treatment ( EDSS) increased by at least about one point, and (d) the multiple sclerosis severity score (MSSS) was higher than about 5 points.

In some embodiments of any of the articles described herein, the patient has one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, and ( c) Increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment.

In some embodiments of any of the articles described herein, the patient has more than one characteristic selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) The Extended Disability Status Scale (EDSS) increased by at least about one point within two years before the start of treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was greater than about 5 points. In some embodiments of any of the articles described herein, the patient has two characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, and (c ) Increase in EDSS by at least about one point within two years before starting treatment. In some embodiments, the patient has three characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, (c) extended disability within two years before starting treatment The status scale (EDSS) is increased by at least about one point, and (d) the multiple sclerosis severity score (MSSS) is higher than about 5 points. In some embodiments, the patient has (a) less than about 55 years of age, (b) one or more gadolinium-stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one within two years before starting treatment Points, and (d) Multiple Sclerosis Severity Score (MSSS) is higher than about 5 points.

In some embodiments of any product, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments of any product, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments of any product, the increase in EDSS within two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, the 1.5 point increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment.

In some embodiments of any of the articles described herein, the patient and/or patient population is characterized by having an MSSS higher than about any of 6, 7, 8, or 9. In some embodiments of any article, the patient and/or patient population is characterized by having an MSSS higher than about 9.

In some embodiments of any article, the patient is less than about 51 years old.

In some embodiments of any product, the treatment shortens the time to diagnosed disease progression. In some embodiments, the confirmed disease progression is an increase in EDSS that lasts for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS for twenty-four weeks.

In some embodiments of any product, the anti-CD20 antibody comprises: a) comprises the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprises SEQ ID NO: 4 , SEQ ID NO: 5 and SEQ ID NO: 6 light chain variable regions. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The product includes a container and a label or drug insert on or combined with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed of various materials such as glass or plastic. The container contains or contains a composition effective for treating multiple sclerosis, and may have a sterile access hole (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody. In some embodiments, the container contains between about 0.3 grams and about 4.0 grams of anti-CD20 antibody. In some embodiments, the container contains between about 0.3 grams and about 1.5 grams of anti-CD20 antibody.

The label or package insert indicates that the composition is used to treat multiple sclerosis in patients suffering from multiple sclerosis, and provides specific guidelines on the dosage and time interval of the antibody and any other drugs provided. The article may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrorotatory Sugar solution. The product may further include other substances that are desirable from the perspective of business and users, including other buffers, diluents, filters, needles and syringes.

Optionally, the product herein further includes a container containing an agent other than the antibody used for treatment and further containing instructions on using the agent to treat patients. The agent is preferably a chemotherapeutic agent or an immunosuppressant; an interferon drug, Such as IFN-β-1a(<img file="TW201014605A_D0069.tif" />and<img file="TW201014605A_D0070.tif" />) Or IFN-β-1b(<img file="TW201014605A_D0071.tif" />); oligopeptides, such as glatiramer acetate (COPAXONE<img file="TW201014605A_D0072.tif" />); Cytotoxic agents, such as mitoxantrone (NOVANTRONE<img file="TW201014605A_D0073.tif" />)), methotrexate, cyclophosphamide, chlorambucil or azathioprine; intravenous immunoglobulin (gamma globulin); lymphocyte depleting drugs (such as mitoxantrone, cyclophosphamide, Campas, anti-CD4 or Cladribine); non-lymphocyte depleting immunosuppressive drugs (such as mycophenolate mofetil (MMF) or cyclosporine); "statin" cholesterol-lowering drugs; estradiol; hormone replacement therapy ; Drugs for the treatment of secondary or related symptoms of MS (such as cramps, incontinence, pain, fatigue); TNF inhibitors; disease-modifying antirheumatic drugs (DMARD); non-steroidal anti-inflammatory drugs (NSAID); corticosteroids (such as Methylprednisolone, prednisone, dexamethasone, or glucocorticoid); levothyroxine; cyclosporine A; somatostatin analogs; cytokine or cytokine receptor antagonists; antimetabolites; immunosuppressive agents ; Integrin antagonist or antibody (for example, LFA-1 antibody, such as falibizumab, or α4 integrin antibody, such as natalizumab); or another B cell surface marker antibody, etc.

In some embodiments, the label may further indicate any of the embodiments described herein. For example, the label may indicate that the patient has (a) an age less than about 55 years of age, and (b) one or more gamma-stained lesions.

In another embodiment of the present invention, there is provided a method of manufacturing a substance suitable for treating the progressive multiple sclerosis described herein. In some embodiments, there is provided a method of manufacturing an anti-CD20 antibody or a pharmaceutical composition thereof, which comprises combining an anti-CD20 antibody or pharmaceutical composition in a package and indicating that the anti-CD20 antibody or pharmaceutical composition is designated for the treatment of patients with progressive polymorphism A label for patients with sclerosis, where the patients have one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) at the beginning of treatment The Extended Disability Status Scale (EDSS) increased by at least about one point in the first two years, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points.

In some embodiments of any of the manufacturing methods described herein, the patient has one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, and (c) Increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment.

<b><i>VIII. Methods of promotion and sales</i></b>

The present invention also provides a method of promoting anti-CD20 antibody or a pharmaceutically acceptable composition thereof, the method comprising promoting the anti-CD20 antibody or a pharmaceutical composition thereof to target audiences for the treatment of patients or patients suffering from progressive multiple sclerosis Use of a population, wherein the patient or patient population has one or more characteristics selected from the group consisting of: (a) younger than about 55 years old, (b) one or more gamma staining lesions, (c) before starting treatment The Extended Disability Status Scale (EDSS) increased by at least about one point within two years, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points.

Also provided herein is a method of marketing an anti-CD20 antibody or a pharmaceutically acceptable composition thereof for use in a subgroup of patients with progressive multiple sclerosis, the method comprising informing a target audience of the use of the anti-CD20 antibody for the treatment of the subgroup of patients , The characteristics of the patient subgroup are that the patients of the subgroup have one or more characteristics selected from the group consisting of: (a) younger than about 55 years old, (b) one or more gamma staining lesions, (c) in The Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points.

In addition, the present invention provides a method for specifying the use of anti-CD20 antibodies in a subgroup of patients with progressive multiple sclerosis, the method comprising providing instructions for administering the anti-CD20 antibody or a pharmaceutically acceptable composition thereof to the subgroup of patients, The characteristics of the patient subgroup are that the subgroup has one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma staining lesions, (c) before starting treatment The Extended Disability Status Scale (EDSS) increased by at least about one point within two years, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points.

The present invention further provides a method of providing treatment options to patients suffering from advanced multiple sclerosis, the method comprising packaging an anti-CD20 antibody in a vial together with a package insert containing instructions for treating patients suffering from advanced multiple sclerosis, These patients have one or more characteristics selected from the group consisting of: (a) younger than about 55 years old, (b) one or more gamma staining lesions, (c) extended disability status within two years before starting treatment The EDSS is increased by at least about one point, and (d) the multiple sclerosis severity score (MSSS) is higher than about 5 points.

In some embodiments of any of these methods, the patient has one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, and (c) at The Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment.

In some embodiments of any of these methods, the patient has two characteristics selected from the group consisting of (a) younger than about 55 years of age, (b) one or more gamma stained lesions, (c) at the beginning of treatment The Extended Disability Status Scale (EDSS) increased by at least about one point in the first two years, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points. In some embodiments, the patient has three characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, (c) extended disability within two years before starting treatment The status scale (EDSS) is increased by at least about one point, and (d) the multiple sclerosis severity score (MSSS) is higher than about 5 points. In some embodiments, the patient has (a) less than about 55 years of age, (b) one or more gadolinium-stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one within two years before starting treatment Points, and (d) Multiple Sclerosis Severity Score (MSSS) is higher than about 5 points.

In some embodiments of any of these methods, the progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments of any of these methods, the patient's sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are indicated by an increase in the IgG index. In some embodiments, signs of inflammation are indicated by IgG oligoclonal bands detected by isoelectric focusing.

In some embodiments of any of these methods, the increase in EDSS within two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, the 1.5 point increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment.

In some embodiments of any of these methods, the patient is less than about 51 years old.

In some embodiments of any of these methods, treatment shortens the time to diagnosed disease progression. In some embodiments, the confirmed disease progression is an increase in EDSS that lasts for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS for twenty-four weeks.

In some embodiments of any of these methods, the anti-CD20 antibody is okrelizumab. In some embodiments of any of these methods, the anti-CD20 antibody is rituximab. In some embodiments of any of these methods, the anti-CD20 antibody is ofatumomab. In some embodiments of any of these methods, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments of any of these methods, the anti-CD20 antibody is GA101. In some embodiments of any of these methods, the anti-CD20 antibody is hA20.

The methods described herein can encompass any combination of the embodiments described herein. For example, these methods include methods where the patient has (a) an age less than about 55 years of age and (b) one or more gamma-stained lesions.

<b><i>IX. System and method for predicting responsiveness to multiple sclerosis treatment</i></b>

The present invention also provides a system and method for analyzing whether an individual and/or patient suffering from advanced multiple sclerosis is responsive to the use of drugs for the treatment of multiple sclerosis. The present invention provides a system for analyzing the responsiveness of patients suffering from advanced multiple sclerosis to the use of medications for the treatment of multiple sclerosis, which comprises: (a) evaluating one or more selected from the group consisting of Features: (i) younger than about 55 years of age; (ii) one or more gamma stained lesions, (iii) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (iv) The Multiple Sclerosis Severity Score (MSSS) is higher than about 5 points; (b) the hardware that performs the evaluation of (a); and (c) the algorithm is executed to determine whether the patient is susceptible to the treatment or is affected by the treatment The computational component of the reaction.

The present invention further provides a method for predicting whether an individual with progressive multiple sclerosis will respond to the use of a drug therapy for the treatment of multiple sclerosis, the method comprising evaluating one or more characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma-stained lesions, (c) increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment, and (d) multiple The sclerosis severity score (MSSS) is higher than about 5 points, based on the patients age, gamma staining lesions, increase in EDSS within two years before the start of treatment, MSSS or a combination thereof will indicate that the individual will respond to treatment.

In some embodiments of any of these systems and/or methods, the patient has more than one characteristic selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, and ( c) Increase the Extended Disability Status Scale (EDSS) by at least about one point within two years before starting treatment.

In some embodiments of any of the systems and/or methods described herein, the patient has more than one characteristic selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma For stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one point within two years before starting treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was greater than about 5 points. In some embodiments of any of these systems and/or methods, the patient has two characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, (c ) The Extended Disability Status Scale (EDSS) increased by at least about one point within two years before the start of treatment, and (d) the Multiple Sclerosis Severity Score (MSSS) was higher than about 5 points. In some embodiments, the patient has three characteristics selected from the group consisting of: (a) younger than about 55 years of age, (b) one or more gamma stained lesions, (c) extended disability within two years before starting treatment The status scale (EDSS) is increased by at least about one point, and (d) the multiple sclerosis severity score (MSSS) is higher than about 5 points. In some embodiments, the patient has (a) less than about 55 years of age, (b) one or more gadolinium-stained lesions, (c) the Extended Disability Status Scale (EDSS) increased by at least about one within two years before starting treatment Points, and (d) Multiple Sclerosis Severity Score (MSSS) is higher than about 5 points.

In some embodiments of any of these systems and/or methods, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, progressive multiple sclerosis is progressive relapsing multiple sclerosis. In some embodiments, the patient is not diagnosed with relapsing-remitting multiple sclerosis at the start of treatment.

In some embodiments of any of these systems or methods, the patient sample further has signs of inflammation. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, signs of inflammation are due to an increase in the IgG index. In some embodiments, signs of inflammation are shown by IgG oligocolonization fringes detected by isoelectric focusing.

In some embodiments of any of these systems and/or methods, the increase in EDSS within two years prior to initiation of treatment is not attributable to relapse. In some embodiments, the patient's EDSS is higher than about 5.0 for less than about 15 years. In some embodiments, the patient's EDSS is less than or equal to about 5.0 for less than about 10 years. In some embodiments, the EDSS is between about 3.0 and about 6.5 at the beginning of treatment. In some embodiments, the increase in EDSS is an increase in EDSS by at least about 1.5 points in the two years prior to initiation of treatment. In some embodiments, the 1.5 point increase in EDSS within the first two years of starting treatment is not attributable to relapse. In some embodiments, the patient further has two or more relapses within two years before starting treatment.

In some embodiments of any of these systems and/or methods, the patient is less than about 51 years old.

In some embodiments of any of these systems and/or methods, the system or method further includes providing advice to the patient.

In some embodiments of any of these systems and/or methods, treatment shortens the time to diagnosed disease progression. In some embodiments, the confirmed disease progression is an increase in EDSS that lasts for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS for twenty-four weeks.

In some embodiments of any of these systems and/or methods, the drug is interferon beta-1b (e.g. Betaseron<img file="TW201014605A_D0074.tif" />), interferon β-1a (e.g. AVonex<img file="TW201014605A_D0075.tif" />Or Rebif<img file="TW201014605A_D0076.tif" />), Glatiramer (e.g. Copaxone<img file="TW201014605A_D0077.tif" />), mitoxantrone (e.g. Novantrone), corticosteroids (e.g. ethylprednisolone, prednisone, dexamethasone), 3-4 diaminopyridine, ABT-874, alemtuzumab (Alemtuzumab), Albuterol (Proventil<img file="TW201014605A_D0078.tif" />), ATL1102, Atorvastatin (Lipitor<img file="TW201014605A_D0079.tif" />), azathioprine, BG00012 (dimethyl fumarate), BHT-3009, Botulinum toxin A (Botulinum toxin A) (Botox<img file="TW201014605A_D0080.tif" />), C-105, cannador, dronabinol, tetrahydrocannabinol, cannabidiol, CDP323, cladribine, CNTO 1275, cyclophosphamide, Dali Daclizumab, Dextromethorphan/quinidine (AVP-923, ZenviaTM), Donepezil (Aricept<img file="TW201014605A_D0081.tif" />), Doxycycline, estradiol, estriol, estroprogestins, Fampridine-SR (4-aminopyridine, sustained release), fingolimod ( Fingolimod) (FTY720), interferon σ, Lamotrigine (Lamictal<img file="TW201014605A_D0082.tif" />), Laquinimod, Lidocaine + Prilocaine (EMLA), MBP8298 (synthetic myelin basic protein peptide), Memantine (Namenda<img file="TW201014605A_D0083.tif" />), methylprednisolone, MN-166, Modafinil (Provigil<img file="TW201014605A_D0084.tif" />), mycophenolate mofetil (Cellcept<img file="TW201014605A_D0085.tif" />), naltrexone, natalizumab (Tysabri<img file="TW201014605A_D0086.tif" />), Paroxetine (Paxil<img file="TW201014605A_D0087.tif" />), PI-2301 (copolymer), Pioglitazone (Actos<img file="TW201014605A_D0088.tif" />), Pixantrone (BBR 2778), Pravastatin (Pravachol<img file="TW201014605A_D0089.tif" />), Pregabalin (Lyrica<img file="TW201014605A_D0090.tif" />), Progesterone, RG2077, Riluzole (Rilutek<img file="TW201014605A_D0091.tif" />), Rolipram (phosphodiesterase-4 inhibitor), RTL1000, SB-683699, Simvastatin (Zocor<img file="TW201014605A_D0092.tif" />), T cell receptor peptide vaccine (NeuroVaxTM), Teriflunomide, testosterone gel (Androgel<img file="TW201014605A_D0093.tif" />) Or Trimethoprim (Trimethoprim).

In some embodiments of any of these systems and/or methods, the drug used to treat multiple sclerosis is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody comprises: a) comprising the heavy chain variable region of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and b) comprising SEQ ID NO: 4, SEQ ID The light chain variable region of NO:5 and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody is okrelizumab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is ofatumomab. In some embodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibody is hA20.

The systems and/or methods described herein can encompass any combination of the embodiments described herein. For example, these methods include methods in which patients have (a) an age less than about 55 years of age and (b) one or more gamma-stained lesions.

Other details of the invention are illustrated by the following non-limiting examples. The disclosures of all cited documents in this specification are expressly incorporated herein by reference.

Instance

The examples intended to be used only to illustrate the invention and therefore should not be regarded as limiting the invention in any way also describe and detail the aspects and embodiments of the invention discussed above. The above examples and detailed description are provided by way of illustration rather than limitation.

<b><i>Example 1: Phase II study of okclizumab in relapsing-remitting multiple sclerosis (RRMS)</i></b>

A phase II multicenter randomized parallel group partially blinded placebo and Avonex controlled dose discovery study was conducted to evaluate the efficacy of two oclizumab dose regimens in patients with relapsing-remitting multiple sclerosis (RRMS) ( As measured by brain magnetic resonance imaging (MRI) lesions) and safety.

The two oclizumab dose regimens studied are as follows: 1) oclizumab 1000 mg dose regimen: consists of two infusions of 1000 mg in the first treatment cycle followed by a single infusion of 1000 mg in the subsequent treatment cycles, and 2) oclizumab Clibizumab 600 mg dosage regimen: consists of two infusions of 300 mg in the first treatment cycle followed by a single infusion of 600 mg in the subsequent treatment cycles.

As shown in Figure 7, eligible patients were randomly (1:1:1:1) assigned to one of four treatment groups A, B, C or D. An overview of the study design is illustrated in Figure 7.

Group A (Oclizumab 1000 mg): In the first treatment cycle, two intravenous (iv) infusions of occlizumab, 1000 mg each time, 14 days apart. The patient is then subjected to a maintenance dose regimen, that is, a single infusion of 1000 mg is subsequently given every 24 weeks in the treatment cycle. Subsequently, in order to maintain the double-blind study during the second treatment cycle, the patients received two infusions 14 days apart, the first infusion was oclitizumab 1000 mg and the second infusion was placebo. In the third and fourth treatment cycles, patients were treated with a single 1000 mg infusion without a second placebo infusion in a double-blind manner until the best dose was selected based on the initial analysis.

Group B (Okclizumab 600 mg): In the first treatment cycle, oklizumab was injected twice intravenously, 300 mg each time, with an interval of 14 days. The patient was then subjected to a maintenance dose regimen, which was followed by a single infusion of 600 mg every 24 weeks in the treatment cycle. Subsequently, in order to maintain the double-blind study during the second treatment cycle, the patients received two infusions 14 days apart, the first infusion was oclizumab 600 mg and the second infusion was placebo. In the third and fourth treatment cycles, patients were treated with a single 600 mg infusion without a second placebo infusion in a double-blind manner until a better dose was selected based on the initial analysis.

Group C (placebo): Placebo was injected twice intravenously in the first treatment cycle with an interval of 14 days. Thereafter, the patient received a 600 mg occlizumab dose regimen, which started with two double-blind intravenous infusions of occlizumab 300 mg at an interval of 14 days at the beginning of the second treatment cycle. The patient was then subjected to a maintenance dose regimen, that is, a single infusion of 600 mg was administered in a double-blind manner in the third and fourth treatment cycles until a better dose was selected based on the initial analysis.

Group D (Avonex): 30 μg Avonex was administered intramuscularly (im) every week in the first treatment cycle. Thereafter, based on a voluntary and open label, patients were provided with a 600 mg occlizumab dosage regimen, which started with two intravenous infusions of occlizumab 300 mg at an interval of 14 days at the beginning of the second treatment cycle. A single 600 mg infusion was used to treat patients in the third and fourth treatment cycles until the best dose was selected based on the initial analysis.

For all groups, after the researchers and the ethics committee were informed of the preferred dose, the patients received the preferred dose (600 mg or 1000 mg) as a single infusion during their next continuous treatment cycle.

The first administration of the study drug (intravenous infusion (Oclizumab or placebo) or the first intramuscular injection of Avonex) defines the start of the treatment period (day 1). All patients also received intravenous infusion of 100 mg methylprednisolone on the first day of the study and then each received occlizumab or placebo infusion or the patients received Avonex according to the time point required for occlizumab infusion (group D) .

There are four treatment cycles, namely cycle 1 = baseline to week 24; cycle 2 = week 24 to week 48; cycle 3 = week 48 to week 72; cycle 4 = week 72 to week 96. After the first cycle of the infusion visit (visits 2 and 3 on day 1 and week 2, respectively), the visit was conducted in the 4th week and every 4 weeks thereafter for the first 24 weeks. After the second cycle of the infusion visit (visits 9 and 10 of the 24th week and the 26th week, respectively), the visit was carried out in the 36th week and every 12 weeks thereafter until the end of the treatment and follow-up period. Efforts should be made to schedule the visit within the provided period. Additional unscheduled visits to assess potential recurrences, new neurological symptoms, or safety events can be performed at any time.

<i>Research groups and selection criteria</i>

According to the revised McDonald criteria (2005), men and women who were diagnosed with relapsing-remitting multiple sclerosis (RRMS) and met the inclusion/exclusion criteria provided below are 18 to 55 years old (inclusive end point) males and females are eligible to participate in the study .

<i>Inclusion criteria:</i>

Patients must meet the following criteria to be eligible to participate in the study:

1. Diagnosed as RRMS according to the revised McDonald criteria (2005);

2. Age 18-55 (including endpoints);

3. At least two recorded recurrences in the last 3 years before screening, of which at least one occurred within one year before screening;

4. The baseline extended disability status scale (EDSS) is 1.0 to 6.0;

5. Signs of burden of multiple sclerosis (MS) disease as defined below:

a. Based on local readings, at least six T2 lesions in the MRI scan performed one year before screening. If the MRI scan has not been used in the previous year or the MRI scan shows less than six T2 lesions, the screening MRI scan of eligible patients needs to have at least six T2 lesions, or

b. The patient had 2 recorded relapses within one year before screening.

<i>Exclusion criteria:</i>

Exclude patients who meet the following criteria from participating in the study:

1. Having secondary or primary progressive multiple sclerosis at the time of screening (visit 1);

2.<img file="TW201014605A_D0094.tif" />The patients disease lasted more than 15 years.

<i>Power analysis</i>

The main goal of this study is to give occlizumab (see Figure 7) to two doses of intravenous 600mg or 1000mg in the 12th, 16th, 20th and 24th week compared with placebo. Compare the effect of gamma enhancement on the total number of T1 lesions observed on brain MRI scans.

The secondary objective of this study is to evaluate the efficacy and safety of okclizumab compared to placebo as reflected in the following: the recurrence rate as defined by the annual agreement by the 24th week; patients who remain relapse-free by the 24th week (Recurrence defined in the agreement); the total number of gamma-enhanced T1 lesions observed in the brain MRI scan at the 4th, 8th, 12th, 16th, 20th and 24th weeks; the 4th week, The total number of new and/or continuous gamma-enhanced T1 lesions in brain MRI scans at 8, 12, 16, 20, and 24 weeks; the total volume of T2 lesions in brain MRI scans from baseline to week 24 Changes to evaluate the safety and tolerability of the two dose regimens of occlizumab compared with placebo and Avonex in patients with RRMS at week 24, and to administer occlizumab for up to 96 weeks Overall safety, and study the pharmacokinetics and other pharmacodynamics end points of Okclizumab.

In this example, recurrence is defined as the occurrence of new neurological symptoms or worsening neurological symptoms attributable to MS, followed by a relatively stable or improved neurological state for at least 30 days. Symptoms must last more than 24 hours and should not be attributed to confounding clinical factors (such as fever, infection, injury, adverse reactions to concomitant medications). New neurological symptoms or worsening neurological symptoms must be accompanied by an objective neurological deterioration consistent with an increase in EDSS by at least half a grade or an appropriate functional system score (FSS) increase by 2 points or two or more appropriate FSS increases by 1 point. The change must affect the selected FSS (ie, cones, gait, cerebellum, brainstem, sensation, or vision). Sensory changes, intermittent cramps, fatigue, mood changes, or bladder or bowel urgency or incontinence are not sufficient to establish a recurrence. Check the investigator to confirm that they meet the above criteria for recurrence.

The exploration objectives of this study include (but are not limited to): the brain volume changes from the baseline scan to the 12th week of brain MRI scan; the brain volume of the 12th to 96th week brain MRI scan in the subgroup of patients receiving oclibizumab The total number of new T2 lesions and/or enlarged T2 lesions observed by brain MRI scans at 4th week, 8th week, 12th week, 16th week, 20th week and 24th week; remained unchanged until 24th week Proportion of patients with T1 lesions enhanced by neogluium; time to the first batch of neogluium-enhancing T1 lesions within 24 weeks; with the aid of receiving up to 4 treatment cycles of oklitizumab in a patient subgroup, gamma-enhancement at 48 weeks The total number of T1 lesions evaluates the effect of stopping treatment; the proportion of patients who maintain no recurrence (clinical and agreement-defined recurrence) during each treatment cycle and at the 48th and 96th weeks; during each treatment cycle and at the 48th and 96th weeks Proportion of MS relapsed patients requiring systemic treatment with methylprednisolone at 96 weeks; the annual clinical and protocol-defined recurrence rate during each treatment cycle and the 48th and 96th weeks; the relapse within 24 weeks to the first agreed-upon definition The time from 96 weeks to the first recurrence defined in the agreement; the time to the onset of continuous disability progression, which is defined as the continuous deterioration of EDSS with a score of 1.0 or higher until the 96th week for 12 weeks; The time to the onset of continuous disability progression, which is defined as the continuous deterioration of EDSS with a score of 1.0 or higher until the 96th week for 24 weeks; the study of okclizumab compared with Avonex is the primary and secondary endpoint of the study Role; to study the correlation between genetic polymorphisms and okclizumab activity related to RRMS susceptibility and the responsiveness of RRMS patients to okclizumab treatment; to study the cyclic biomarkers and oxclizumab related to RRMS susceptibility The relationship between the activity of crilizumab and the response of RRMS patients to treatment with oclizumab; the changes in the Modified Fatigue Intensity Scale (MFIS) from the baseline to the 24th and 48th weeks; from the baseline to the first Changes in the Fatigue Scale for Exercise and Cognitive Function (FSMC) at Week 24 and Week 48; Comparing the baseline with Week 24 and Week 48, from "severe" fatigue to "moderate" fatigue and from "moderate" fatigue "Changes in the proportion of patients whose fatigue turns into "mild" fatigue; changes in the Epidemiology Research Center-Depression Scale (CES-D) from baseline to week 24 and week 48; and changes from baseline to week 24 Compared with the 48th week, the change in the proportion of patients with CES-D from a state with more depression symptoms to a state with fewer depression symptoms.

<i>Brain MRI</i>

MRI is a tool suitable for monitoring central nervous system (CNS) lesions in MS. Only some patients' brain MRI scans at screening (see secondary endpoints) and all patients' brain MRI scans at baseline and at four-week intervals between baseline and week 24 were obtained. In addition, in the patient subgroups (group A and group B), brain MRI scans were performed at the 96th week (visit 16) and 48 weeks thereafter (that is, the 144th week).

MRI includes the following scans obtained at each time point: T2-weighted MRI scan, T1-weighted MRI scan (with no gamma enhancement), and T1-weighted MRI scan (with gamma enhancement).

<i>Disability assessment</i>

The independent inspection investigator evaluated the disability progression as measured by EDSS in all patients at the time of screening and every 12 weeks throughout the study until the observation period (when the disability progression was assessed after 24 weeks).

Disability progression is defined as an increase from the baseline EDSS score that is not attributable to other causes (such as fever, concurrent disease, or concomitant medication) when the baseline score is 5.0 or below 5.0<img file="TW201014605A_D0095.tif" />Score and when the baseline score is 5.5 or higher<img file="TW201014605A_D0096.tif" />. When the increase in EDSS is confirmed by regularly scheduled visits at least 12 weeks after the initial recording of the progress, the disease progression is considered to be continuous. The alternative definition of persistent disability progression requires confirmation of an increase in EDSS at least 24 weeks after the initial recording of the progression.

EDSS is based on standard neurological examinations; seven EDSS grades and scores (collectively called Score for the functional system or FSS). Each FSS score is a sequential clinical grading scale ranging from 0 to 5 or 6. These ratings are then used in conjunction with observations and information about walking and assistive use to determine the EDSS score. The EDSS is a disability scale with 0.5 points and a first level ranging from 0 (normal) to 10 (death).

<b><i>Example 2: Phase II/III study of rituximab in primary progressive multiple sclerosis (PPMS)</i></b>

A randomized, double-blind, parallel group, placebo-controlled, multicenter Phase II/III study (U2786g) was conducted to evaluate the risk of rituximab as defined by McDonald et al. (Ann Neurol 50:121-7 (2001)). Safety and efficacy in adults with progressive multiple sclerosis (PPMS).

Individuals are randomized at a 2:1 ratio to receive rituximab or placebo. Rituximab available from Genentech is formulated into 9.0mg/ml sodium chloride, 0.7mg/ml polysorbate 80, 7.35mg/ml sodium citrate dihydrate and sterile water for injection (pH 6.5) The sterile product for intravenous administration. Each time course of the study drug consisted of two intravenous infusions (14 days apart) of 1000 mg rituximab or placebo. Individuals receive the first treatment schedule on Day 1 and Day 15 and additional schedules on Week 24, Week 48, and Week 72. Individuals received oral acetaminophen (1 g) and diphenhydramine HCl (50 mg) or equivalent 30 minutes to 60 minutes before the start of each infusion. Glucocorticoids are not administered before infusion. During the 96-week test duration, individuals were inspected for physical examination, neurological and MRI assessments during regularly scheduled visits to collect adverse reactions and vital signs and complete routine hematology, serum chemistry, and urinalysis laboratory tests.

The baseline demographics of the intention to treat (ITT) individuals are presented in Table 3.

<tables><img file="TW201014605A_D0097.tif" /></tables>

The duration of MS disease was similar in the two treatment groups. The baseline MRI results are summarized in Table 4. The baseline MRI characteristics of the placebo group and the rituximab group were similar.

<tables><img file="TW201014605A_D0098.tif" /></tables>

According to the research site, by EDSS (<img file="TW201014605A_D0099.tif" />) To classify the randomization by the baseline disease severity defined by ). The baseline EDSS is summarized in Table 5. Due to dynamic randomization, all grading factors for the percentage of individuals in each treatment group are at similar levels.

<tables><img file="TW201014605A_D0100.tif" /></tables>

In addition, the other baseline disease severity indicators of the two treatment groups were similar: EDSS, Kurtzke functional system score, multiple sclerosis function comprehensive scale (MSFCS) score and MSFCS components (timed 25 walking, nine-hole cylindrical test and PASAT -3).

<i>Efficacy results</i>

The initial efficacy analysis of this trial compared rituximab and placebo to the time to disease progression diagnosed during the 96-week treatment period. Disease progression is defined as an increase from baseline EDSS if the baseline EDSS is between 2.0 and 5.5 points (inclusive of endpoints)<img file="TW201014605A_D0101.tif" />Points (Kurtzke <i>J. Neurology</i> 33(11):1444-52(1983)), or increase if baseline EDSS&gt;5.5 points<img file="TW201014605A_D0102.tif" />The change cannot be attributed to other causes (such as fever, concurrent disease, recurrence or worsening of MS, or concomitant medication).

Grading analysis showed that rituximab did not significantly delay the confirmed disease progression compared with placebo (p=0.1442, graded logarithmic scale). It is estimated that the percentages of patients in the placebo group and rituximab group that progressed within 96 weeks were 38.5% and 30.2%, respectively (Table 6). The Kaplan-Meier plot of the time to confirmed disease progression is shown in FIG. 8.

<tables><img file="TW201014605A_D0103.tif" /></tables>

Secondary efficacy endpoints include the change in total T2 lesion volume from baseline to week 96 and the change in brain volume from baseline to week 96. The Hochberg-Bonferroni program was used to control the Type I error rate in testing these two secondary endpoints. There was no significant difference in brain volume changes from baseline to 96th week in the two treatment groups (p=0.6237). See Table 7.

<tables><img file="TW201014605A_D0104.tif" /></tables>

A significant difference was observed in the changes in T2 lesion volume from baseline to week 96 between the two treatments (p=0.0008). The median increase in T2 lesion volume in the placebo group and rituximab group was 809.50 mm, respectively³And 301.95mm³. (See Table 8 and Figure 9).

<tables><img file="TW201014605A_D0105.tif" /></tables>

Analysis of all study endpoints (except T2 lesion volume change, enlarged T2 lesion and new T2 lesion) showed that the difference between the placebo group and the rituximab group was not statistically significant. Compared with placebo, the rituximab group experienced a significantly smaller increase in T2 lesion volume at weeks 48 and 122 (p=0.0051 and 0.0222, respectively); it had less at weeks 48 and 96 New T2 lesions (p&lt;0.001); the number of T2 lesions with less expansion at the 48th and 96th weeks (p=0.008 and 0.072, respectively).

<i>Subgroup analysis</i>

The subgroup analysis of the primary endpoint included the time to diagnosed disease progression based on the following demographics and baseline disease characteristics: location, age, gender, race, previous MS therapy, baseline EDSS, since the onset of MS symptoms and baseline gamma (Gd) lesions The duration and baseline Multiple Sclerosis Severity Score (MSSS) (an indicator of the rapid progress of the patient, see Roxburgh et al.<i>Neurology</i> 64;1144-1151(2005))。

The results of the subgroup analysis showed a potential therapeutic effect for patients who are younger, progressing faster (higher MSSS), or have baseline Gd lesions (Figure 10). In addition, the additive predictive effect of age, baseline Gd lesions and MSSS of the treatment effect has been verified by multivariate analysis (Figure 11 and Figure 12). See also Table 9. Based on the results of these MSSS, the modified inclusion/exclusion criteria were used to select the subgroup (age<img file="TW201014605A_D0106.tif" />，<img file="TW201014605A_D0107.tif" />, If the patients baseline EDSS is &lt;5, exclude patients with disease duration&gt; 10, or if the patients baseline<img file="TW201014605A_D0108.tif" />, Then exclude patients with disease duration&gt; 15). The significant therapeutic effect of this subgroup is also shown (P value of hierarchical log-level test = 0.01; Figure 13).

<tables><img file="TW201014605A_D0109.tif" /></tables>

Subgroup analysis showed that PPMS patients with signs of active disease showed significant clinical signs such as treatment-related benefits as measured by the time to diagnosed disease progression, changes from baseline in EDSS, MSFC, and T2 lesions in brain MRI (data not shown) exhibit). Independent factors that show the prognosis of disease progression in the placebo group and may predict treatment response in the rituximab group include the following: younger, especially younger than 51 years old; baseline contrast-enhancing lesions in brain MRI; and higher MS Severity score. These observations are used to generate hypotheses supporting the potential therapeutic benefit of B cell depletion for the confirmed disease progression of appropriately selected patients with progressive MS.

Although this study failed to show the main efficacy in the overall PPMS population, the subgroup efficacy analysis indicated that patients with baseline contrast-enhanced brain MRI lesions may respond to rituximab treatment, where the treatment group was compared with the placebo group The risk of disease progression (1-HR) with a diagnosis of 57% was relatively reduced, which was mainly but not entirely due to the extremely high rate of placebo-diagnosed disease progression (CDP) of 52.8% in the 96th week (Figure 10). PPMS patients younger than 51 years of age can also benefit, with a 43% relative reduction in the risk of confirmed disease progression and a placebo progression rate of 44.9%. Although it is associated with the presence of contrast-enhancing lesions and age &lt;51 years, the post-hoc analysis of 72 patients exhibiting two characteristics revealed a more significant effect, with the confirmed risk of disease progression being relatively reduced by 77% (Table 9). In this subgroup, the placebo progression rate of 51.6% is not higher than the rate of all patients with baseline enhanced MRI lesions, while the lower progression rate (24.6%) of the rituximab group indicates that treatment may be greatly reduced danger. These OLYMPUS placebo data confirm the natural history observation of clinical and MRI heterogeneity in PPMS patients (Sastre-Garriga et al.<i>Neurology</i> 65(4):633-5 (2005), Ingle et al.<i>Brain</i> 126 (Part 11): 2528-36 (2003), Tremlett et al.<i>Mult Scler</i>. 14(3):314-24 (2008), Tremlett et al.<i>Neurology</i> 65(12): 1919-23 (2005), Kremenchutzky et al.<i>Brain</i> 129 (Part 3): 584-94. (2006)). In addition, MAGNIMS clinical and MRI clustering studies describe a subset of PPMS patients who have more inflammatory MRI activity early in the course of the disease and have a poorer prognosis for disability progression (Ingle et al.<i>J. Neurol Neurosurg Psychiatry</i> 76(9):1255-8(2005)); OLYMPUS placebo data seems to confirm these observations for the first time.

<b><i>Example 3: Phase III study of okclizumab in progressive multiple sclerosis</i></b>

A phase III randomized double-blind parallel group multi-center study was conducted to evaluate the safety and efficacy of 600 mg occlizumab compared with placebo in adults with progressive MS.

A total of 630 patients with progressive MS (315 with progressive MS and 315 with relapsing MS) participated and assigned (2:1 randomized) to Oakley beads classified by the site and type of multiple sclerosis Monoclonal antibody group or placebo group. This study consists of the following three periods applied to all patients: the screening period, the treatment period, and the treatment-free follow-up period. In the first study schedule, drug therapy (300 mg oclizumab or placebo infusion × 2) was administered on day 1 and day 15. In the follow-up treatment schedule, the patient was administered every 24 weeks (a single infusion of 600 mg oclitizumab) until the last patient who participated received the administration at the 96th week for its final treatment schedule.

Before the infusion of each study drug, patients received analgesics/antipyretics (such as acetaminophen/paracetamol (1 g)) and intravenous or oral antihistamines (such as diphenhydramine 50 mg) and 100 mg Intravenous methylprednisolone or equivalent treatment to reduce the incidence of possible infusion reactions. In patients with CTCAE Grade 3 or higher (severe) infusion reactions associated with respiratory symptoms (wheezing, asthma, or bronchospasm), additional treatment with bronchodilators can be specified.

Routine laboratory studies were obtained throughout the study, in which other tests were performed after the study drug treatment schedule. Immune group, serum human anti-human antibody (HAHA) and thyroid test are also performed. Serum samples of all patients were collected for pharmacokinetic analysis and blood samples were collected for B cell count determination. Monitoring the number of B cells is used as a pharmacokinetic marker of Okelizumab.

<i>Patient groups and selection criteria</i>

The target population of this study includes patients with progressive MS with or without a history of superimposed recurrence. The characteristics of patients with progressive MS who meet the conditions of this study are diagnosed according to the revised McDonald criteria (2005) and documented irreversible neurological loss lasting 6 months or longer in the absence of recurrence. Using the criteria identified as potential risk factors in previous clinical trials conducted on patients with progressive MS, patients with higher risk signs of active disease and faster disability progression are selected. These factors include younger age, signs of inflammation in the cerebrospinal fluid (CSF) (oligoselective zone or elevated IgG index), contrast-enhanced lesions in brain MRI, high recurrence activity superimposed with non-recurring related progress, and relatively Accumulated history of rapid disability.

Screen all patients who meet the following inclusion and exclusion criteria who are willing and eligible to participate in the study.

<i>Inclusion criteria include:</i>

1. Diagnosed multiple sclerosis according to the revised McDonald criteria (2005);

2. Progressive MS, characterized by documented evidence, not attributable to clinical recurrence, persistent<img file="TW201014605A_D0110.tif" />Months of irreversible neurological loss;

3. Age 18-55 (including endpoints);

4. EDSS is 3.0 to 6.5 points during screening.

5. Due to the results of the lower limbs, the pyramidal system or the gait functional system (FS) scale score<img file="TW201014605A_D0111.tif" />。

6. There are at least one of the following laboratory findings in the CSF samples obtained during the screening period or recorded in the previous six months, such as (for example) increased IgG index and/or IgG oligosity detected by isoelectric aggregation Instructed by the selection belt.

7. There is at least one of the following criteria:

Age &lt;50

Gd+ lesions in brain MRI during screening or within 6 months of screening

In the past 2 years, EDSS that is not attributable to relapse has increased by at least 1.5 points

Two relapses in the past two years

<i>Exclusion criteria include:</i>

1. With relapsing-remitting multiple sclerosis at the time of screening (visit 1)

2. The duration of the disease since the onset of MS symptoms: more than 15 years for patients with EDSS&gt;5.0 at the time of screening, or for patients at the time of screening<img file="TW201014605A_D0112.tif" />For patients, it is more than 10 years.

<i>Power analysis</i>

The primary efficacy endpoint is the time to progression of the confirmed disease. Disease progression is defined as an increase from baseline EDSS if the baseline EDSS is between 2.0 and 5.5 points (inclusive of endpoints)<img file="TW201014605A_D0113.tif" />Points, or increase if baseline EDSS&gt;5.5 points<img file="TW201014605A_D0114.tif" />The change cannot be attributed to other causes (such as fever, concurrent disease, recurrence or worsening of MS, or concomitant medication).

EDSS is based on standard neurological examinations; seven EDSS grades and scores (collectively called Functional system score or FSS). Each FSS score is a sequential clinical grading scale ranging from 0 to 5 or 6. These ratings are then used in conjunction with observations and information about walking and assistive use to determine the EDSS score. EDSS is a disability scale with 0.5 points and a first level ranging from 0 (normal) to 10 (death).

Secondary efficacy endpoints that support the primary efficacy endpoints include: the change in total T2 lesion volume in brain MRI scans from baseline to week 120; the change in 25-step timed walking from baseline to week 120; the time to confirmed disease progression, where Confirm that at least 24 weeks after initial disease progression (<img file="TW201014605A_D0115.tif" />Days).

<i>Assess for recurrence</i>

At each visit in the entire study, the treatment investigator will assess the patient's recurrence and, if necessary, will use occasional visits to confirm the recurrence between visits. In order to meet the recurrence criteria defined in the agreement, recurrence is defined as the occurrence of new or worsening neurological symptoms attributable to MS followed by a relatively stable or improved neurological state for at least 30 days. Symptoms must last more than 24 hours and should not be attributed to confounding clinical factors (such as fever, infection, injury, adverse reactions to concomitant medications). New neurological symptoms or worsening neurological symptoms must be accompanied by objective neurological deterioration consistent with an increase in EDSS by at least half a grade or an appropriate FSS increase of 2 points or two or more appropriate FSS increases of 1 point. The change must affect the selected FSS (ie, cones, gait, cerebellum, brainstem, sensation, or vision). Intermittent cramps, sexual dysfunction, fatigue, mood changes, or bladder or bowel urgency or incontinence are not enough to establish a recurrence.

<i>Brain MRI imaging</i>

Magnetic resonance imaging of the brain and cervical spinal cord was obtained at various time points (including baseline) during this study. Brain MRI includes the following scans obtained at each time point: T2-weighted MRI scan and T1-weighted MRI scan (without gamma enhancement).

The variance classification analysis was used to compare the total volume of T2 lesions between okclizumab and placebo and the changes from baseline to the 120th week of regular 25-walking. The model includes the two grading factors recorded in the initial analysis.

<b><i>Example 4: Phase III study of okclizumab in primary progressive multiple sclerosis</i></b>

A phase III randomized double-blind parallel group multicenter study was conducted to evaluate the safety and efficacy of one of the two oclizumab dosage regimens compared with placebo in adults with primary progressive MS.

The two dose regimens of oxyclizumab studied are as follows: 1) oxyclizumab 1000 mg dose regimen: consisting of two infusions of 1000 mg in the first treatment cycle followed by a single infusion of 1000 mg in the subsequent treatment cycles, and 2) Oakley The 600mg dosage regimen of virizumab: consists of two infusions of 300mg in the first treatment cycle followed by a single infusion of 600mg in the subsequent treatment cycles.

A total of 630 patients with primary progressive MS participated and were assigned (2:1 randomized) to the oxyclizumab group or the placebo group classified by the location and type of multiple sclerosis. This study consists of the following three periods applied to all patients: the screening period, the treatment period, and the safety follow-up period. In the first study schedule, drug treatment (Oclizumab or placebo infusion × 2) was administered on Day 1 and Day 15. In the follow-up treatment schedule, patients were administered (single infusion of occlizumab or placebo) every 24 weeks until the last patient who participated received the administration at the 96th week for their final treatment schedule.

Prior to the infusion of each study drug, patients received analgesics/antipyretics (such as acetaminophen/paracetamol (1 g)) and intravenous or oral antihistamines (such as diphenhydramine 50 mg) and 100 mg intravenous Prednisone or equivalent treatment to reduce the incidence of possible infusion reactions. In patients with CTCAE Grade 3 or higher (severe) infusion reactions associated with respiratory symptoms (wheezing, asthma, or bronchospasm), additional treatment with bronchodilators can be specified.

Routine laboratory studies were obtained throughout the study, in which other tests were performed after the study drug treatment schedule. Immune group, serum human anti-human antibody (HAHA) and thyroid test are also performed. Serum samples of all patients were collected for pharmacokinetic analysis and blood samples were collected for B cell count determination. Monitoring the number of B cells is used as a pharmacokinetic marker of Okelizumab.

<i>Patient groups and selection criteria</i>

The target group of this study includes patients with primary progressive MS. The characteristics of patients with primary progressive MS who meet the conditions of this study are diagnosed according to the revised McDonald criteria (2005). Using the criteria identified as potential risk factors in previous clinical trials on patients with progressive MS, patients with higher risk signs of active disease and faster disability progression are selected. These factors include younger age, signs of inflammation in the cerebrospinal fluid (CSF) (increased oligoselective zone or IgG index), and a history of faster accumulation of disability.

Screen all patients who meet the following inclusion and exclusion criteria who are willing and eligible to participate in the study:

<i>Inclusion criteria include:</i>

1. Diagnosed primary progressive multiple sclerosis according to the revised McDonald criteria (2005);

2. Age 18-55 (including endpoints);

3. When screening, EDSS is 3.0 to 6.5 points;

4. Due to the results of the lower limbs, the function system (FS) scale of the pyramidal system was scored<img file="TW201014605A_D0116.tif" />；

5. At the time of screening, the CSF sample has a record of at least one of the following laboratory findings or the presence of at least one of the following laboratory findings in the CSF sample, such as detected by an increase in IgG index and/or by isoelectric focusing As indicated by the IgG oligo-selective bands tested;

6. The duration of the disease since the onset of MS symptoms: less than 15 years for patients with EDSS&gt;5.0 at the time of screening, or for patients at the time of screening<img file="TW201014605A_D0117.tif" />For patients, it is less than 10 years.

<i>Exclusion criteria include:</i>

1. Have a history of relapsing-remitting, secondary progressive, or progressive relapsing multiple sclerosis at the time of screening (visit 1).

<i>Power analysis</i>

The primary efficacy endpoint is the time to progression of the confirmed disease. Disease progression is defined as an increase from baseline EDSS if the baseline EDSS is between 2.0 and 5.5 points (inclusive of endpoints)<img file="TW201014605A_D0118.tif" />Points, or increase if baseline EDSS&gt;5.5 points<img file="TW201014605A_D0119.tif" />The change cannot be attributed to other causes (such as fever, concurrent disease, recurrence or worsening of MS, or concomitant medication).

EDSS is based on standard neurological examinations; seven EDSS grades and scores (collectively called Functional system score or FSS). Each FSS score is a sequential clinical grading scale ranging from 0 to 5 or 6. These ratings are then used in conjunction with observations and information about walking and assistive use to determine the EDSS score. EDSS is a disability scale with a scale of 0.5 points and a range of 0 (normal) to 10 (death).

Secondary efficacy endpoints that support the primary efficacy endpoints include: the change in total T2 lesion volume in brain MRI scans from baseline to week 120; the change in 25-step timed walking from baseline to week 120; the time to confirmed disease progression, where Confirm that at least 24 weeks after initial disease progression (<img file="TW201014605A_D0120.tif" />Days).

<i>Assess for recurrence</i>

At each visit in the entire study, the treatment investigator will assess the patient's recurrence and, if necessary, will use occasional visits to confirm the recurrence between visits. In order to meet the recurrence criteria confirmed by the agreement, recurrence is defined as the occurrence of new or worsening neurological symptoms attributable to MS followed by a relatively stable or improved neurological state for at least 30 days. Symptoms must last more than 24 hours and should not be attributed to confounding clinical factors (such as fever, infection, injury, adverse reactions to concomitant medications). New neurological symptoms or worsening neurological symptoms must be accompanied by objective neurological deterioration consistent with an increase in EDSS by at least half a grade or an appropriate FSS increase of 2 points or two or more appropriate FSS increases of 1 point. The change must affect the selected FSS (ie, cones, gait, cerebellum, brainstem, sensation, or vision). Intermittent cramps, sexual dysfunction, fatigue, mood changes, or bladder or bowel urgency or incontinence are insufficient to establish a recurrence.

<i>Brain MRI imaging</i>

Magnetic resonance imaging of the brain and cervical spinal cord was obtained at various time points (including baseline) during this study. Brain MRI includes the following scans obtained at each time point: T2-weighted MRI scan and T1-weighted MRI scan (without gamma enhancement).

The variance classification analysis was used to compare the total volume of T2 lesions between okclizumab and placebo and the changes from baseline to the 120th week of regular 25-walking. The model includes the two grading factors recorded in the initial analysis.

<img file="TW201014605A_D0121.tif" />

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Figure 1A is a comparison of the light chain variable domains of various murine 2H7 (SEQ ID NO: 1), humanized 2H7.v16 variants (SEQ ID NO: 2) and human kappa light chain subgroup I (SEQ ID NO: 3) (V_L) Sequence alignment of amino acid sequences. V of 2H7 and hu2H7.v16_LThe CDRs are as follows: CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5) and CDR3 (SEQ ID NO: 6);

Figure 1B is a comparison between the murine 2H7 (SEQ ID NO: 7), the humanized 2H7.v16 variant (SEQ ID NO: 8) and the human common sequence of the heavy chain subgroup III (SEQ ID NO: 9). Variable domain (V_H) Sequence alignment of amino acid sequences. V of 2H7 and hu2H7.v16_HThe CDRs are as follows: CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12);

In Figure 1A and Figure 1B, as shown, the CDR1, CDR2, and CDR3 of each chain are framed in brackets, and the framework regions FR1-FR4 are on both sides. 2H7 refers to the murine 2H7 antibody. The asterisk between the two sequences indicates the different positions between the two sequences. Residue numbering is based on Kabat et al.<i>Sequences of Immunological Interest</i>, The 5th edition of the Public Health Service (Public Health Service), National Institutes of Health (National Institutes of Health), Bethesda, Md. (1991), where the inserts are shown as a, b, c, d and e;

Figure 2 shows the amino acid sequence of the mature 2H7.v16 light chain (SEQ ID NO: 13);

Figure 3 shows the amino acid sequence of the mature 2H7.v16 heavy chain (SEQ ID NO: 14);

Figure 4 shows the amino acid sequence of the mature 2H7.v31 heavy chain (SEQ ID NO: 15). The L chain of 2H7.v31 is the same as the L chain of 2H7.v16;

Figure 5 shows the comparison of mature 2H7.v16 and 2H7.v511 light chains (SEQ ID NOs 13 and 16, respectively), using Kabat variable domain residue numbering and Eu constant domain residue numbering;

Figure 6 shows the comparison of mature 2H7.v16 and 2H7.v511 heavy chains (SEQ ID NOs 14 and 17, respectively), using Kabat variable domain residue numbering and Eu constant domain residue numbering;

Figure 7 shows an overview of the study design for the treatment of relapsing-remitting multiple sclerosis using oclizumab;

Figure 8 shows the Kaplan Meier curve of the time to diagnosed disease progression of individuals in the placebo group and the rituximab group;

Figure 9 shows the median change in T2 lesion volume from baseline to week 96. Y axis shows T2 lesion volume mm³；

Figure 10 shows a summary of baseline characteristics and risk ratios of individuals in the placebo group and rituximab group;

Figure 11 shows a multivariate analysis of the additive predictive effect of age of treatment effect and baseline gamma (Gd) lesions in the placebo group and rituximab group. Figure 11A shows a multivariate analysis of age younger than 51 years and baseline Gd lesion equal to zero. Figure 11B shows age<img file="TW201014605A_D0140.tif" />Multivariate analysis of age and baseline Gd lesion equal to 0. Figure 11C shows age less than 51 years old and baseline Gd lesions<img file="TW201014605A_D0141.tif" />Multivariate analysis. Figure 11D shows age<img file="TW201014605A_D0142.tif" />Years old with baseline Gd lesions<img file="TW201014605A_D0143.tif" />Multivariate analysis;

Figure 12 shows a multivariate analysis of the additive predictive effect of age and multiple sclerosis severity score (MSSS) of the treatment effect in the placebo group and rituximab group. Figure 12A shows age<img file="TW201014605A_D0144.tif" />Multivariate analysis of age and MSSS less than 5. Figure 12B shows a multivariate analysis of age greater than 55 years and MSSS less than 5. Figure 12C shows age<img file="TW201014605A_D0145.tif" />Years old<img file="TW201014605A_D0146.tif" />Multivariate analysis. Figure 12D shows that the age is greater than 55 years old and<img file="TW201014605A_D0147.tif" />Multivariate analysis; and

Figure 13 shows the Carben-Meier curve of the time to diagnosed disease progression for individuals with the following characteristics in the placebo group and rituximab group: age<img file="TW201014605A_D0148.tif" />；<img file="TW201014605A_D0149.tif" />; Exclude the duration of the disease more than 10 years and the baseline EDSS less than 5 or the duration of the disease more than 15 years and the baseline<img file="TW201014605A_D0150.tif" />The patient.