Materials and methods for treating juvenile idiopathic arthritis

Abstract

This invention provides a pharmaceutical composition for use in the treatment of juvenile idiopathic arthritis (JIA). [Solution] A pharmaceutical composition is provided for use in a method for treating juvenile idiopathic arthritis (JIA) in pediatric patients, wherein the pharmaceutical composition comprises an anti-TNF antibody, and the method comprises administering an intravenous (IV) dose of the anti-TNF antibody to the pediatric patient, wherein the anti-TNF antibody comprises a heavy chain (HC) containing a specific amino acid sequence and a light chain (LC) containing a specific amino acid sequence, and more than 50% of the pediatric patients meet the American College of Rheumatology's JIA ACR30, JIA ACR50, and JIA ACR70 criteria after 52 weeks of treatment.

Description

[Technical Field]

[0001] (Cross-reference of related applications) poly-JIA / integrated / 28 weeks / n=127 This application, "JBI6307USNP1Seq", was created on April 20, 2021. The filename "Listing.txt" is an ASCII format array list for EFS-W Includes a sequence listing of 25kb size submitted electronically via eb. EFS-W The sequence listings submitted via eb are part of this specification and are referred to in their entirety hereby. It will be incorporated into the book.

[0002] (Field of invention) This invention relates to the treatment of juvenile idiopathic arthritis (JIA), particularly polyarticular juvenile idiopathic arthritis ( Anti-TNF antibodies, for example, amino acid formulation of SEQ ID NO: 36, for use in the treatment of pJIA. Anti-T15 has a heavy chain (HC) containing the sequence and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37. This invention relates to compositions and methods using the NF antibody golimumab. [Background technology]

[0003] TNFα is a soluble homotrimer of a 17kD protein subunit. A membrane-bound 26kD precursor form also exists.

[0004] Cells other than monocytes or macrophages also produce TNFα. For example, human nonmonocyte tumor cells The cell line produces TNFα and CD4+ and CD8+ peripheral blood T lymphocytes, and several cultures The T and B cell lines also produce TNFα.

[0005] TNFα promotes cartilage and bone breakdown, induces adhesion molecules, and induces procoagulant activity in vascular endothelial cells. This increases the adhesion of neutrophils and lymphocytes, as well as macrophages, neutrophils and blood vessels. Inflammatory processes that cause tissue damage, such as stimulating the release of platelet-activating factor from endothelial cells. To cause a problem.

[0006] TNFα is associated with infection, immune disorders, neoplastic conditions, autoimmune conditions, and graft-versus-host conditions. The association between TNFα and cancer and infectious diseases is related to the host's catabolic state. This is common. Cancer patients typically suffer from weight loss associated with loss of appetite.

[0007] Significant debilitation associated with cancer and other diseases is known as "cachexia." , progressive weight loss, loss of appetite, and persistent decline in lean body mass in response to the growth of malignant tumors. This includes cachexia, which is a major cause of cancer incidence and mortality. TNFα is a factor in cancer There is evidence that it is involved in cachexia in infectious conditions and other catabolic states.

[0008] TNFα is associated with Gram-negative sepsis and endotoxin, including fever, fatigue, loss of appetite, and cachexia. It is thought to play a central role in primal shock. Endotoxins are monocyte / macrocyte It strongly activates pharmacokinetics and the secretion of TNFα and other cytokines. And other monocyte-derived cytokines mediate the metabolism and neurohormonal responses to endotoxins. Endotoxin administration to human volunteers causes fever, tachycardia, increased metabolic rate, and stress. TNFα causes acute illness accompanied by flu-like symptoms, including the release of steroid hormones. The circulation of this substance increases in patients suffering from Gram-negative sepsis.

[0009] Therefore, TNFα is involved in inflammatory diseases, autoimmune diseases, viral, bacterial, and parasitic infections. It is associated with malignant tumors and / or neurodegenerative diseases, including rheumatoid arthritis and crow's disease. It is a useful target for specific biological therapies in diseases such as nephrosis. It suppresses inflammation and also... The beneficial effects of good retreatment after relapse in rheumatoid arthritis and Crohn's disease are due to TNFα This has been reported in open-label studies using monoclonal antibodies against [the substance]. The effect is due to the suppression of inflammation. Beneficial results in rheumatoid arthritis have also been reported in randomized, double-blind, placebo-controlled trials.

[0010] Neutralizing antiserum or mAbs against TNF are experimental endotoxins in non-human mammals. To suppress adverse physiological changes after a lethal attack in bacteremia and pulmonary embolism, and to prevent death. This has been shown to be the effect of, for example, rodent lethality assays and primate pathology models. This is shown in the system.

[0011] The putative receptor binding sites of hTNF have been disclosed, and amino acids 11-13 and 37 of TNF have been identified. The TNFα receptor binding sites, consisting of ~42, 49~57 and 155~157, are disclosed. Yes, they are.

[0012] Non-human mammals, chimeras, polyclonals (e.g., antiserums), and / or monoclonals Naru antibodies (Mab) and fragments (e.g., proteolytic digestion or fusion protein production) The substance is being investigated in some cases for the purpose of treating certain diseases. It is a powerful therapeutic agent. However, if such antibodies or fragments are administered to humans, they may cause an immune response. This may trigger a response. Such an immune response involves immune complexes of antibodies or fragments from the bloodstream. This can lead to mediation clearance, making repeated administration unsuitable for treatment, thereby affecting the patient. The therapeutic benefit to the patient is reduced, and the re-administration of antibodies or fragments is restricted. For example, non-human parts Repeated administration of antibodies or fragments containing this substance may result in serum sickness and / or anaphylaxis. To avoid these and other problems, as is well known in the art, chimeras Many approaches to reduce the immunogenicity of such antibodies and their portions, including morphogenesis and humanization. —A strategy has been adopted. However, these and other approaches still have some degree of immunity Having virulence, low affinity, low binding activity, or for cell culture, scale-up, production and / or This can result in antibodies or fragments with problems in low yield. Therefore, such antibodies or The fragments may not be ideally suited for manufacture or use as therapeutic proteins. It has a sex.

[0013] There is a need for TNF inhibitors that can resolve one or more of these problems. Currently available anti-TNF antibodies and other TNF inhibitors include, for example, REMICAD E(registered trademark) (infliximab), HUMIRA(registered trademark) (adalimumab), and This led to the development of Simponi® (golimumab), another TNF inhibitor. For example, CIMZIA® (certolizumab pegol), PEGylated antibody fragments, and ENBREL® (etanercept), a soluble TNF receptor fusion protein. For example, see Lis et al., "Arch M See "ed Sci." (December 22, 2014), Vol. 10, No. 6, pp. 1175-1185. Please refer to this. [Overview of the Initiative] [Means for solving the problem]

[0014] For simplicity, the independent and dependent provisions attached to this Specified Reference are incorporated herein by reference. The claims define general and preferred embodiments, respectively. The form, characteristics, and advantages are described in conjunction with the attached drawings, based on the following embodiments for carrying out the invention. It will become clear.

[0015] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Furthermore, the method of meeting the criteria for inactive disease of the Japanese Association of Rheumatology (JIA ACR) provide.

[0016] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 30% of pediatric patients are treated with This method provides a way to meet the JIA ACR criteria for inactive disease after 52 weeks.

[0017] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Furthermore, the criteria for inactive disease set by the Japanese Association of Rheumatology (JIA) (JIA ACR) are met, and the small The method provides for child patients who are between 2 and under 18 years of age.

[0018] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Furthermore, if the criteria for inactive disease are met by the Japanese Association of Rheumatology (JIA) (JIA ACR), the young Juvenile idiopathic arthritis (JIA) is a type of polyarticular juvenile idiopathic arthritis (pJIA), and the method is as follows: To provide.

[0019] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Furthermore, it meets the criteria for inactive disease set by the Japanese Association of Rheumatology (JIA ACR), and is suitable for IV treatment. The dosage is 80 mg / m² of anti-TNF antibody in week 0, week 4, and every 8 weeks thereafter. 2 in I will provide a method.

[0020] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Furthermore, the criteria for inactive disease set by the Japanese Association of Rheumatology (JIA) (JIA ACR) are met, and the method is The present invention provides a method further comprising administering methotrexate (MTX) to a pediatric patient.

[0021] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, the Japanese Association of Rheumatology (JIA) ACR (Japanese Association of Rheumatology) approved the basic criteria for inactive disease. Provide a method that satisfies the criteria.

[0022] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. More than 30% meet the JIA ACR criteria for inactive disease 52 weeks after treatment. To provide.

[0023] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, the Japanese Association of Rheumatology (JIA) ACR (Japanese Association of Rheumatology) approved the basic criteria for inactive disease. The method provides a condition that meets the criteria and the pediatric patient is between 2 and 18 years of age.

[0024] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, the Japanese Association of Rheumatology (JIA) ACR (Japanese Association of Rheumatology) approved the basic criteria for inactive disease. If the criteria are met, the juvenile idiopathic arthritis (JIA) is classified as polyarticular juvenile idiopathic arthritis (pJ). Provides a method that is IA.

[0025] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, the Japanese Association of Rheumatology (JIA) ACR (Japanese Association of Rheumatology) approved the basic criteria for inactive disease. The composition, meeting the criteria, is administered in IV doses at week 0, week 4, and every 8 weeks thereafter, as an anti-T NF antibody 80 mg / m² 2It provides a method to achieve this.

[0026] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, the Japanese Association of Rheumatology (JIA) ACR (Japanese Association of Rheumatology) approved the basic criteria for inactive disease. The method satisfies the requirements and further includes administering methotrexate (MTX) to pediatric patients. , provide a method.

[0027] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Furthermore, we proposed a method that would meet the clinical remission criteria of the Japanese Association of Rheumatology (JIA) (JIA ACR). To provide.

[0028] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 10% of pediatric patients are treated with This method provides a way to meet the JIA ACR clinical remission criteria after 52 weeks.

[0029] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Subsequently, the child met the clinical remission criteria of the Japanese Association of Rheumatology (JIA ACR). The method is provided for patients between 2 and under 18 years of age.

[0030] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Subsequently, the criteria for clinical remission set by the Japanese Association of Rheumatology (JIA ACR) were met, and the young adult Juvenile idiopathic arthritis (JIA) is a type of polyarticular juvenile idiopathic arthritis (pJIA), and the method is as follows: provide.

[0031] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Subsequently, the criteria for clinical remission set by the Japanese Association of Rheumatology (JIA) (JIA ACR) were met, and the IV dose was administered. This involves administering 80 mg / m² of anti-TNF antibody in week 0, week 4, and every 8 weeks thereafter.2 dea To provide a method.

[0032] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. The light chain (LC) contains the amino acid sequence of SEQ ID NO: 37, and pediatric patients should be treated for 52 weeks from the start of treatment. Furthermore, the criteria for clinical remission set by the Japanese Association of Rheumatology (JIA ACR) were met, and the method was as follows: The present invention provides a method further comprising administering methotrexate (MTX) to a pediatric patient.

[0033] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, according to the Japanese Association of American College of Rheumatology (JIA) criteria for clinical remission. To provide a method that satisfies the requirements.

[0034] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. More than 30% meet the JIA ACR criteria for inactive disease 52 weeks after treatment. To provide.

[0035] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, according to the Japanese Association of American College of Rheumatology (JIA) criteria for clinical remission. The present invention provides a method that satisfies the following conditions, and the pediatric patient is between 2 and 18 years of age.

[0036] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, according to the Japanese Association of American College of Rheumatology (JIA) criteria for clinical remission. If the conditions are met, the juvenile idiopathic arthritis (JIA) is classified as polyarticular juvenile idiopathic arthritis (pJI). A) provides a method that is

[0037] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, according to the Japanese Association of American College of Rheumatology (JIA) criteria for clinical remission. The composition satisfies the following conditions, and the IV dose is administered at week 0, week 4, and every 8 weeks thereafter, as an anti-TN 80 mg / m² of F antibody 2 It provides a method to achieve this.

[0038] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, and pediatric patients 52 weeks after treatment, according to the Japanese Association of American College of Rheumatology (JIA) criteria for clinical remission. The method satisfies the requirement and further comprises administering methotrexate (MTX) to a pediatric patient. Provide a method.

[0039] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 50% of pediatric patients are treated with 52 weeks later, JIA American College of Rheumatology (JIA ACR) 30, JIA ACR 50, And provides a method that meets the standards of JIA ACR70.

[0040] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 50% of pediatric patients are treated with 52 weeks later, JIA American College of Rheumatology (JIA ACR) 30, JIA ACR 50, and the criteria of JIA ACR70 are met, and the pediatric patient is between 2 and 18 years of age, method To provide.

[0041] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 50% of pediatric patients are treated with 52 weeks later, JIA American College of Rheumatology (JIA ACR) 30, JIA ACR 50, And meeting the criteria of JIA ACR70, the juvenile idiopathic arthritis (JIA) is polyarticular. This paper provides a method for treating juvenile idiopathic arthritis (pJIA).

[0042] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 50% of pediatric patients are treated with 52 weeks later, JIA American College of Rheumatology (JIA ACR) 30, JIA ACR 50, And meeting the criteria of JIA ACR70, the IV dose is administered in week 0, week 4, and thereafter. Every 8 weeks, 80 mg / m² of anti-TNF antibodies are administered. 2 It provides a method to achieve this.

[0043] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 50% of pediatric patients are treated with 52 weeks later, JIA American College of Rheumatology (JIA ACR) 30, JIA ACR 50, The method meets the criteria of JIA ACR70 and involves administering methotrexate (MTX) to pediatric patients. The present invention provides a method that further includes administering the substance to a person.

[0044] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 20% of pediatric patients are treated with 52 weeks later, regarding low disease activity, the Juvenile Arthritis Disease Activity Score was measured in 71 joints (JA The present invention provides a method having DAS 71).

[0045] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 20% of pediatric patients are treated with 52 weeks later, regarding low disease activity, the Juvenile Arthritis Disease Activity Score was measured in 71 joints (JA The present invention provides a method for pediatric patients who have DAS 71) and are between 2 and 18 years of age.

[0046] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 20% of pediatric patients are treated with 52 weeks later, regarding low disease activity, the Juvenile Arthritis Disease Activity Score was measured in 71 joints (JA DAS 71) is present, and the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis. This provides a method for treating pneumoconiosis (pJIA).

[0047] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 20% of pediatric patients are treated with 52 weeks later, regarding low disease activity, the Juvenile Arthritis Disease Activity Score was measured in 71 joints (JA It has DAS 71), and the IV dose is administered at week 0, week 4, and thereafter every 8 weeks, anti TNF antibody 80 mg / m² 2 It provides a method to achieve this.

[0048] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method of administering an intravenous (IV) dose of anti-TNF antibody to a pediatric patient. The anti-TNF antibody includes the heavy chain (HC) and the amino acid sequence of SEQ ID NO: 36. It contains a light chain (LC) with the amino acid sequence of SEQ ID NO: 37, and more than 20% of pediatric patients are treated with 52 weeks later, regarding low disease activity, the Juvenile Arthritis Disease Activity Score was measured in 71 joints (JA The method involves administering methotrexate (MTX) to pediatric patients, as described in DAS 71). The method further includes the following.

[0049] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 50% of cases occurred 52 weeks after treatment, according to the JIA American College of Rheumatology (JIA ACR) 30. This provides a method that meets the standards of IA ACR 50 and JIA ACR 70.

[0050] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 50% of patients reached the JIA American College of Rheumatology (JIA ACR) 30th Class 52 weeks after treatment. The pediatric patient meets the criteria for JIA ACR 50 and JIA ACR 70, and is 2 Provides a method for those under 18 years of age.

[0051] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 50% of patients reached the JIA American College of Rheumatology (JIA ACR) 30th Class 52 weeks after treatment. The criteria for JIA ACR 50 and JIA ACR 70 are met, and the juvenile idiopathic related This paper provides a method for treating juvenile idiopathic arthritis (JIA) as polyarticular juvenile idiopathic arthritis (pJIA).

[0052] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 50% of patients reached the JIA American College of Rheumatology (JIA ACR) 30th Class 52 weeks after treatment. The composition of the IV dose meets the standards of JIA ACR 50 and JIA ACR 70. In week 0, week 4, and thereafter every 8 weeks, 80 mg / m² of anti-TNF antibody was administered. 2 That is , provide a method.

[0053] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 50% of patients reached the JIA American College of Rheumatology (JIA ACR) 30th Class 52 weeks after treatment. The method meets the criteria of JIA ACR 50 and JIA ACR 70, and involves methotrex The present invention provides a method that further includes administering Sart (MTX) to a pediatric patient.

[0054] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 20% of patients showed low disease activity 52 weeks after treatment, as measured by the Juvenile Arthritis Disease Activity Score. The present invention provides a method having a counting of 71 joints (JADAS 71).

[0055] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 20% of patients showed low disease activity 52 weeks after treatment, as measured by the Juvenile Arthritis Disease Activity Score. The patient has 71 joints (JADAS 71) and is between 2 and 18 years of age. To provide the law.

[0056] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 20% of patients showed low disease activity 52 weeks after treatment, as measured by the Juvenile Arthritis Disease Activity Score. The patient has 71 joints affected (JADAS 71), and this juvenile idiopathic arthritis (JIA) is multifaceted. This paper provides a method for treating juvenile idiopathic arthritis of the rhinoplasty type (pJIA).

[0057] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 20% of patients showed low disease activity 52 weeks after treatment, as measured by the Juvenile Arthritis Disease Activity Score. It has a count of 71 joints (JADAS 71), and the composition of the IV dose is for week 0, week 4, And thereafter, every 8 weeks, 80 mg / m² of anti-TNF antibody. 2 It provides a method to achieve this.

[0058] In certain embodiments, the present invention relates to juvenile idiopathic arthritis (JIA) in pediatric patients. A method for treating the condition, the method comprising a composition comprising an intravenous (IV) dose of anti-TNF antibody. The substance includes administering the substance to pediatric patients, and the anti-TNF antibody contains the amino acid sequence of SEQ ID NO: 36. It contains a heavy chain (HC) and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for pediatric patients. Over 20% of patients showed low disease activity 52 weeks after treatment, as measured by the Juvenile Arthritis Disease Activity Score. The patient had 71 joints (JADAS 71), and the method involved administering methotrexate (MTX) to a child. The present invention provides a method that further includes administering the substance to a patient. [Brief explanation of the drawing]

[0059] [Figure 1] This graph shows an assay of the ability of TNV mAbs in hybridoma cell supernatant to inhibit TNFα binding to recombinant TNF receptors. Various amounts of hybridoma cell supernatant containing known amounts of TNV mAbs were pre-incubated with a fixed concentration (5 ng / mL) of 125I-labeled TNFα. The mixture was transferred to a 96-well Optiplate pre-coated with recombinant TNF receptor / IgG fusion protein p55-sf2. The amount of TNFα bound to the p55 receptor in the presence of the mAb was determined after washing away unbound material and counting using a γ counter. Eight TNV ​​mAb samples were tested in these experiments; however, for simplicity, three mAbs that were shown to be identical to one of the other TNV mAbs by DNA sequencing analysis are not shown here. Each sample was tested in duplicate. The results shown are representative of two independent experiments. [Figure 2A] This shows the DNA sequences of the TNV mAb heavy chain variable regions. The germline genes shown are the DP-46 genes. "TNVs" indicates that the sequences shown are those of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequences define the translation initiation Met codon. The dotted lines in the TNV mAb gene sequences indicate that the nucleotides are the same as those in the germline sequences. The first 19 nucleotides of the TNV sequences (underlined) correspond to the oligonucleotides used to PCR amplify the variable regions. Amino acid translations (single-letter notation) beginning with mature mAbs are shown only for germline genes. The three CDR domains in the germline amino acid translations are shown in bold and underlined. Rows labeled TNV148(B) indicate that the sequences shown relate to both TNV148 and TNV148B. Gaps in the germline DNA sequences (CDR3) are due to sequences that were unknown at the time or not present in the germline genes. The TNV mAb heavy chain uses the J6 binding region. [Figure 2B] This shows the DNA sequences of the TNV mAb heavy chain variable regions. The germline genes shown are the DP-46 genes. "TNVs" indicates that the sequences shown are those of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequences define the translation initiation Met codon. The dotted lines in the TNV mAb gene sequences indicate that the nucleotides are the same as those in the germline sequences. The first 19 nucleotides of the TNV sequences (underlined) correspond to the oligonucleotides used to PCR amplify the variable regions. Amino acid translations (single-letter notation) beginning with mature mAbs are shown only for germline genes. The three CDR domains in the germline amino acid translations are shown in bold and underlined. Rows labeled TNV148(B) indicate that the sequences shown relate to both TNV148 and TNV148B. Gaps in the germline DNA sequences (CDR3) are due to sequences that were unknown at the time or not present in the germline genes. The TNV mAb heavy chain uses the J6 binding region. [Figure 3] This shows the DNA sequence of the TNV mAb light chain variable region. The germline genes shown are representative members of the Vg / 38K family of human κ germline variable region genes. Dotted lines in the TNV mAb gene sequence indicate that the nucleotides are the same as those in the germline sequence. The first 16 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single letter notation) of the mature mAb is shown only for germline genes. The three CDR domains in the germline amino acid translation are shown in bold and underlined. Rows labeled TNV148(B) indicate that the shown sequence relates to both TNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to sequences that are unknown or not present in the germline gene. The TNV mAb light chain uses the J3 binding region. [Figure 4]This shows the putative amino acid sequences of the heavy chain variable regions of TNV mAbs. The amino acid sequences shown (single letter notation) were putative from DNA sequences determined from both non-cloned and cloned PCR products. The amino sequences are shown divided into secretion signal sequence (signal), framework (FW), and complementarity-determining region (CDR) domains. The amino acid sequences of the DP-46 germline gene are shown in the row above each domain. A dotted line indicates that the amino acids in the TNV mAb are identical to those in the germline gene. TNV148(B) indicates that the shown sequence relates to both TNV148 and TNV148B. "TNV" indicates that the shown sequence relates to all TNV mAbs unless a different sequence is shown. A dashed line in the germline sequence (CDR3) indicates that the sequence is unknown or not present in the germline gene. [Figure 5] This shows the putative amino acid sequence of the TNV mAb light chain variable region. The amino acid sequence shown (single letter notation) was putative from DNA sequences determined from both non-cloned and cloned PCR products. The amino sequences are shown divided into secretion signal sequence (signal), framework (FW), and complementarity-determining region (CDR) domains. The amino acid sequences of the Vg / 38K type light chain germline genes are shown in the row above each domain. The dotted line indicates that the amino acids in the TNV mAb are identical to those in the germline gene. TNV148(B) indicates that the shown sequence relates to both TNV148 and TNV148B. "All" indicates that the shown sequence relates to TNV14, TNV15, TNV148, TNV148B, and TNV186. [Figure 6]A schematic diagram of the heavy and light chain expression plasmids used to generate rTNV148B-expressing C466 cells is shown. p1783 is the heavy chain plasmid, and p1776 is the light chain plasmid. The rTNV148B variable and constant region coding domains are shown in black boxes. The immunoglobulin enhancer of the JC intron is shown in gray boxes. The relevant restriction sites are shown. The plasmids are oriented so that transcription of the Ab gene proceeds clockwise. Plasmid p1783 is 19.53 kb long, and plasmid p1776 is 15.06 kb long. The complete nucleotide sequences of both plasmids are known. The variable region coding sequence of p1783 can be readily replaced with another heavy chain variable region sequence by replacing the BsiWI / BstBI restriction fragment. The variable region coding sequence of p1776 can be replaced with another variable region sequence by replacing the SalI / AflII restriction fragment. [Figure 7] The graphs of growth curve analysis for five rTNV148B-producing cell lines are shown. Culturing was initiated on day 0 by seeding cells in I5Q+MHX medium in a T75 flask to achieve a viable cell density of 1.0 × 10⁵ cells / mL in a 30 mL volume. The cell cultures used in these tests were continuous cultures for transfection and subcloning. Subsequently, the cells in the T flask were thoroughly resuspended, and 0.3 mL aliquots of the culture were taken. Growth curve testing was terminated when the cell count fell to less than 1.5 × 10⁵ cells / mL. The number of viable cells in the aliquots was determined by trypan blue exclusion, and the remaining aliquots were stored for later mAb concentration determination. ELISA for human IgG was performed simultaneously on all sample aliquots. [Figure 8]This graph shows a comparison of cell growth rates in the presence of various MHX selector concentrations. Cell subclones C466A and C466B were thawed in MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for a further 2 days. Both cell cultures were then divided into three cultures: one without MHX, one containing 0.2×MHX, and one containing 1×MHX. After 1 day, the cultures were seeded in new T75 flasks at an initial density of 1×10⁵ cells / mL, and cells were counted at 24-hour intervals for 1 week. Doubling times for the first 5 days were calculated using the formula in SOP PD32.025 and are shown above the bars. [Figure 9] The graph shows the time-dependent stability of mAb production from two rTNV148B-producing cell lines. After transfection and subcloning, subclones of cells that had been in continuous culture were used to initiate long-term continuous culture in 24-well culture dishes. Cells were cultured in I5Q medium with or without MHX selector. Cells were continuously subculturified by dividing the culture every 4-6 days to maintain new viable cultures while simultaneously consuming the previous culture. Aliquots of the consumed cell supernatant were collected immediately after the culture was consumed and stored until the mAb concentration could be determined. Human IgG ELISA was performed on all sample aliquots at the same time. [Figure 10] This shows the body weight changes of arthritis mouse model Tg197 in response to the anti-TNF antibody of the present invention, compared to the control in Example 4. Approximately 4-week-old Tg197 test mice were assigned to one of nine treatment groups based on sex and body weight and treated with either Dulbecco's PBS (D-PBS) or a single intraperitoneal bolus of either 1 mg / kg or 10 mg / kg of the anti-TNF antibody of the present invention (TNV14, TNV148, or TNV196). When body weight was analyzed as a change from baseline, animals treated with 10 mg / kg cA2 consistently showed higher body weight gain than animals treated with D-PBS throughout the study. This body weight gain was significant from weeks 3 to 7. Animals treated with 10 mg / kg TNV148 also achieved a significant body weight gain at week 7 of the study. [Figure 11A]This shows the progression of disease severity based on the arthritis index shown in Example 4. The arthritis index in the 10 mg / kg cA2 treatment group was lower than that of the D-PBS control group, which started at week 3 and continued throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and animals treated with 1 mg / kg cA2 did not show a significant reduction in AI from week 3 onward compared to the D-PBS treatment group. When each was compared to other similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there was no significant difference among the 10 mg / kg treatment groups. When compared to the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI at weeks 3, 4, and 7 than 1 mg / kg cA2. 1 mg / kg TNV148 also showed significantly lower AI at weeks 3 and 4 than the group treated with 1 mg / kg TNV14. TNV196 showed a significant reduction in AI up to week 6 of the trial (compared to the group treated with D-PBS), but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the trial. [Figure 11B] This shows the progression of disease severity based on the arthritis index shown in Example 4. The arthritis index in the 10 mg / kg cA2 treatment group was lower than that of the D-PBS control group, which started at week 3 and continued throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and animals treated with 1 mg / kg cA2 did not show a significant reduction in AI from week 3 onward compared to the D-PBS treatment group. When each was compared to other similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there was no significant difference among the 10 mg / kg treatment groups. When compared to the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI at weeks 3, 4, and 7 than 1 mg / kg cA2. 1 mg / kg TNV148 also showed significantly lower AI at weeks 3 and 4 than the group treated with 1 mg / kg TNV14. TNV196 showed a significant reduction in AI up to week 6 of the trial (compared to the group treated with D-PBS), but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the trial. [Figure 11C]This shows the progression of disease severity based on the arthritis index shown in Example 4. The arthritis index in the 10 mg / kg cA2 treatment group was lower than that of the D-PBS control group, which started at week 3 and continued throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and animals treated with 1 mg / kg cA2 did not show a significant reduction in AI from week 3 onward compared to the D-PBS treatment group. When each was compared to other similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there was no significant difference among the 10 mg / kg treatment groups. When compared to the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI at weeks 3, 4, and 7 than 1 mg / kg cA2. 1 mg / kg TNV148 also showed significantly lower AI at weeks 3 and 4 than the group treated with 1 mg / kg TNV14. TNV196 showed a significant reduction in AI up to week 6 of the trial (compared to the group treated with D-PBS), but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the trial. [Figure 12] This shows the body weight change of Tg197 arthritis mouse model mice in response to the anti-TNF antibody of the present invention, compared to the control in Example 5. Approximately 4-week-old Tg197 test mice were assigned to one of eight treatment groups based on body weight and treated with either the control (D-PBS) or an intraperitoneal bolus of 3 mg / kg of antibody (TNV14, TNV148) (week 0). Injections were repeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for the efficacy of the test product. Serum samples obtained from animals in groups 7 and 8 were evaluated for the induction of immune response and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3, and 4. [Figure 13A]This graph shows the progression of disease severity in Example 5 based on the arthritis index. The arthritis index in the group treated with 10 mg / kg cA2 was significantly lower than that of the D-PBS control group, starting from week 2 and continuing throughout the rest of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 did not achieve any significant reduction in AI at any point in the study compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction, starting from week 3 and continuing until week 5, compared to the group treated with d-PBS. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI at weeks 4 and 5 of the study compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg), and was also significantly lower than that of animals treated with TNV14 from weeks 3 to 5. While there appeared to be no significant difference among any of the 3 mg / kg treatment groups, the AI ​​of animals treated with 3 mg / kg TNV14 was significantly higher than that of animals treated with 10 mg / kg at a certain point in time, while animals treated with TNV148 were not significantly different from those treated with 10 mg / kg cA2. [Figure 13B]This graph shows the progression of disease severity in Example 5 based on the arthritis index. The arthritis index in the group treated with 10 mg / kg cA2 was significantly lower than that of the D-PBS control group, starting from week 2 and continuing throughout the rest of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 did not achieve any significant reduction in AI at any point in the study compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction, starting from week 3 and continuing until week 5, compared to the group treated with d-PBS. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI at weeks 4 and 5 of the study compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg), and was also significantly lower than that of animals treated with TNV14 from weeks 3 to 5. While there appeared to be no significant difference among any of the 3 mg / kg treatment groups, the AI ​​of animals treated with 3 mg / kg TNV14 was significantly higher than that of animals treated with 10 mg / kg at a certain point in time, while animals treated with TNV148 were not significantly different from those treated with 10 mg / kg cA2. [Figure 13C]This graph shows the progression of disease severity in Example 5 based on the arthritis index. The arthritis index in the group treated with 10 mg / kg cA2 was significantly lower than that of the D-PBS control group, starting from week 2 and continuing throughout the rest of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 did not achieve any significant reduction in AI at any point in the study compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction, starting from week 3 and continuing until week 5, compared to the group treated with d-PBS. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI at weeks 4 and 5 of the study compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg), and was also significantly lower than that of animals treated with TNV14 from weeks 3 to 5. While there appeared to be no significant difference among any of the 3 mg / kg treatment groups, the AI ​​of animals treated with 3 mg / kg TNV14 was significantly higher than that of animals treated with 10 mg / kg at a certain point in time, while animals treated with TNV148 were not significantly different from those treated with 10 mg / kg cA2. [Figure 14] This shows the body weight change of Tg197 mice, an arthritis mouse model, in response to the anti-TNF antibody of the present invention, compared to the control in Example 6. Approximately 4-week-old Tg197 test mice were assigned to one of six treatment groups based on sex and body weight, and treated with a single intraperitoneal bolus of either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148). This study utilized D-PBS and a 10 mg / kg cA2 control group. [Figure 15]This shows the progression of disease severity based on the arthritis index shown in Example 6. All treatment groups showed some protective effect at the initial stage, with 5 mg / kg cA2 and 5 mg / kg TNV148 showing a significant decrease in AI at weeks 1-3, and all treatment groups showing a significant decrease at week 2. Later in the experiment, animals treated with 5 mg / kg cA2 showed some protective effect and a significant decrease at weeks 4, 6, and 7. Both low doses (3 mg / kg) of cA2 and TNV148 showed a significant decrease at week 6, and all treatment groups showed a significant decrease at week 7. No treatment group was able to maintain a significant decrease at the end of the study (week 8). There were no significant differences between any of the treatment groups (excluding the saline control group) at any given time point. [Figure 16] This shows the body weight change of Tg197 mice, an arthritis mouse model in response to the anti-TNF antibody of the present invention, compared to the control in Example 7. To compare the efficacy of a single intraperitoneal administration of TNV148 (derived from hybridoma cells) and rTNV148B (derived from transfected cells), approximately 4-week-old Tg197 test mice were assigned to one of nine treatment groups based on sex and body weight and treated with a single intraperitoneal bolus of Dulbecco's PBS (D-PBS) or 1 mg / kg of antibody (TNV148, rTNV148B). [Figure 17]The progression of disease severity is shown based on the arthritis index, as presented in Example 7. The arthritis index in the 10 mg / kg cA2-treated group was lower than that in the D-PBS control group, starting at week 4 and continuing throughout the remainder of the trial (week 8). Both the TNV148-treated group and the 1 mg / kg cA2-treated group showed a significant reduction in AI at week 4. While a previous trial (P-099-017) showed that TNV148 was slightly more effective in reducing the arthritis index after a single 1 mg / kg intraperitoneal bolus, this trial showed slightly higher AI from the groups treated with both versions of the TNV antibody. The group treated with 1 mg / kg cA2 (except at week 6) did not show a significant increase compared to the group treated with 10 mg / kg cA2. The group treated with TNV148 showed significantly higher levels at weeks 7 and 8, but there were no significant differences in AI among 1 mg / kg cA2, 1 mg / kg TNV148, and 1 mg / kg TNV148B at any point in the study. [Figure 18] This is a diagram of the clinical trial design for pJIA. DBL = Database Lock, LTE = Long-Term Extension, MSE = Primary Secondary Endpoint, PE = Primary Endpoint. IV infusions of golimumab 80 mg / m2 are marked with arrows at the indicated times. Patients also received commercially available MTX at the same once-weekly BSA-based dose as at the time of study enrollment, at least until week 28. [Figure 19] A diagram showing patient trends throughout the trial is provided. *Since one patient had more than one reason for ineligibility, the maximum number is set to 51. AE: Adverse event; JIA: Juvenile idiopathic arthritis; n: Number of patients. [Figure 20A] The observed steady-state serum trafgolimumab concentration (μg / mL) (A) and the model-predicted AUCss (μg·day / mL) of serum golimumab concentration (B) are shown by age group at 28 weeks in poly-JIA patients and the adult RA reference population. The horizontal line within the box represents the median, the bottom of the box represents the first quartile, and the top of the box represents the third quartile. The whiskers represent the outermost observation within the 1.5 × intraquartile range. AUCss: Area under the steady-state curve; JIA: Juvenile Idiopathic Arthritis; n: Number of patients in the population; RA: Rheumatoid Arthritis; WK: Weeks. [Figure 20B] The observed steady-state serum trafgolimumab concentration (μg / mL) (A) and the model-predicted AUCss (μg·day / mL) of serum golimumab concentration (B) are shown by age group at 28 weeks in poly-JIA patients and the adult RA reference population. The horizontal line within the box represents the median, the bottom of the box represents the first quartile, and the top of the box represents the third quartile. The whiskers represent the outermost observation within the 1.5 × intraquartile range. AUCss: Area under the steady-state curve; JIA: Juvenile Idiopathic Arthritis; n: Number of patients in the population; RA: Rheumatoid Arthritis; WK: Weeks. [Figure 21A]Clinical efficacy up to week 52; percentages of JIA ACR30 / 50 / 70 / 90 responders (A), percentages of patients with JIA ACR inactive disease or clinical remission to the drug (B), mean (standard deviation) parental assessments of CHAQ and pain scores (C), and mean (95% confidence interval) JADAS71 score (D) are shown. For (A), N=127 for all time points of JIA ACR30, 50, 70, and 90; missing data per NRI and LOCF were processed according to the ITT principle. For (B), N=127 for all time points of inactive disease and clinical remission to the drug. Clinical remission to the drug was defined as inactive disease at each visit over a period of 6 months or more during poly-JIA drug administration (all visits including at least 24 weeks prior had to meet the inactive disease criteria). For inactive disease and clinical remission, missing data were processed for each LOCF and NRI according to the ITT principle. For (C), pain and CHAQ scores were based on observed data. For (D), JADAS scores were based on observed data. 95% confidence intervals are based on normal approximations of mean ± 1.96 × SD / √N. ACR, American College of Rheumatology; BSL, baseline; CHAQ, Childhood Health Assessment Questionnaire; HDA, high disease activity; ID, inactive disease; ITT, intention to treat; JADAS, juvenile arthritis disease activity score; JIA, juvenile idiopathic arthritis; LDA, low disease activity; LOCF, last observed value carried over; N, total treated patients; n, number of evaluable patients; NRI, non-responder supplementation; SD, standard deviation. [Figure 21B]Clinical efficacy up to week 52; percentages of JIA ACR30 / 50 / 70 / 90 responders (A), percentages of patients with JIA ACR inactive disease or clinical remission to the drug (B), mean (standard deviation) parental assessments of CHAQ and pain scores (C), and mean (95% confidence interval) JADAS71 score (D) are shown. For (A), N=127 for all time points of JIA ACR30, 50, 70, and 90; missing data per NRI and LOCF were processed according to the ITT principle. For (B), N=127 for all time points of inactive disease and clinical remission to the drug. Clinical remission to the drug was defined as inactive disease at each visit over a period of more than 6 months during poly-JIA drug administration (all visits including at least 24 weeks prior had to meet the inactive disease criteria). For inactive disease and clinical remission, missing data were processed for each LOCF and NRI according to the ITT principle. For (C), pain and CHAQ scores were based on observed data. For (D), JADAS scores were based on observed data. 95% confidence intervals are based on normal approximations of mean ± 1.96 × SD / √N. ACR, American College of Rheumatology; BSL, baseline; CHAQ, Childhood Health Assessment Questionnaire; HDA, high disease activity; ID, inactive disease; ITT, intention to treat; JADAS, juvenile arthritis disease activity score; JIA, juvenile idiopathic arthritis; LDA, low disease activity; LOCF, last observed value carried over; N, total treated patients; n, number of evaluable patients; NRI, non-responder supplementation; SD, standard deviation. [Figure 21C]Clinical efficacy up to week 52; percentages of JIA ACR30 / 50 / 70 / 90 responders (A), percentages of patients with JIA ACR inactive disease or clinical remission to the drug (B), mean (standard deviation) parental assessments of CHAQ and pain scores (C), and mean (95% confidence interval) JADAS71 score (D) are shown. For (A), N=127 for all time points of JIA ACR30, 50, 70, and 90; missing data per NRI and LOCF were processed according to the ITT principle. For (B), N=127 for all time points of inactive disease and clinical remission to the drug. Clinical remission to the drug was defined as inactive disease at each visit over a period of more than 6 months during poly-JIA drug administration (all visits including at least 24 weeks prior had to meet the inactive disease criteria). For inactive disease and clinical remission, missing data were processed for each LOCF and NRI according to the ITT principle. For (C), pain and CHAQ scores were based on observed data. For (D), JADAS scores were based on observed data. 95% confidence intervals are based on normal approximations of mean ± 1.96 × SD / √N. ACR, American College of Rheumatology; BSL, baseline; CHAQ, Childhood Health Assessment Questionnaire; HDA, high disease activity; ID, inactive disease; ITT, intention to treat; JADAS, juvenile arthritis disease activity score; JIA, juvenile idiopathic arthritis; LDA, low disease activity; LOCF, last observed value carried over; N, total treated patients; n, number of evaluable patients; NRI, non-responder supplementation; SD, standard deviation. [Figure 21D]Clinical efficacy up to week 52; percentages of JIA ACR30 / 50 / 70 / 90 responders (A), percentages of patients with JIA ACR inactive disease or clinical remission to the drug (B), mean (standard deviation) parental assessments of CHAQ and pain scores (C), and mean (95% confidence interval) JADAS71 score (D) are shown. For (A), N=127 for all time points of JIA ACR30, 50, 70, and 90; missing data per NRI and LOCF were processed according to the ITT principle. For (B), N=127 for all time points of inactive disease and clinical remission to the drug. Clinical remission to the drug was defined as inactive disease at each visit over a period of more than 6 months during poly-JIA drug administration (all visits including at least 24 weeks prior had to meet the inactive disease criteria). For inactive disease and clinical remission, missing data were processed for each LOCF and NRI according to the ITT principle. For (C), pain and CHAQ scores were based on observed data. For (D), JADAS scores were based on observed data. 95% confidence intervals are based on normal approximations of mean ± 1.96 × SD / √N. ACR, American College of Rheumatology; BSL, baseline; CHAQ, Childhood Health Assessment Questionnaire; HDA, high disease activity; ID, inactive disease; ITT, intention to treat; JADAS, juvenile arthritis disease activity score; JIA, juvenile idiopathic arthritis; LDA, low disease activity; LOCF, last observed value carried over; N, total treated patients; n, number of evaluable patients; NRI, non-responder supplementation; SD, standard deviation. [Modes for carrying out the invention]

[0060] The present invention comprises a heavy chain (HC) containing SEQ ID NO: 36 and a light chain (LC) containing SEQ ID NO: 37. A composition containing an anti-TNF antibody, and a manufacturing process for producing such an anti-TNF antibody. To provide.

[0061] When used in this specification, "antitumor necrosis factor α antibody", "anti-TNF antibody", "anti-TNF The "antibody portion" or "anti-TNF antibody fragment," and / or "anti-TNF antibody variant," etc., At least one complement of heavy chain or light chain that can be incorporated into the antibody of the present invention Determinative region (CDR) or its ligand-binding region, heavy chain or light chain variable region, heavy chain or Alternatively, the light chain steady region, the framework region, or any part thereof, or the TNF receptor Immunotherapy includes, but is not limited to, at least one part of a protein or binding protein. Any protein or peptide containing a molecule that includes at least a portion of a globulin molecule. It contains molecules. Such antibodies, optionally, further affect specific ligands, and these Such antibodies can be used in vitro, in situ, and / or in vivo, in small quantities. At the very least, it modulates or reduces one TNF activity or binding, or TNF receptor activity or binding. To reduce, increase, counteract, activate, mitigate, mitigate, block, inhibit, suppress, and / or interfere. Non-limited. As an example, a preferred anti-TNF antibody of the present invention, a specified part or variant, has at least one It can bind to one TNF or a specific part, variant, or domain thereof. Suitable anti-TNF antibodies, identified parts or variants, can also be optionally used on RNA, DNA, etc. , or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, T TNF activity includes, but is not limited to, NF activity, TNF production, and / or synthesis. Or it may affect at least one of the functions. The term "antibody" is Furthermore, it is intended to include antibodies, their digestive fragments, specific parts, and variants, and this includes antibodies Examples include mimetic drugs, or parts of an antibody that mimic the structure and / or function of an antibody. includes the specific fragment or part thereof, and includes single-chain antibodies and fragments thereof. As a functional fragment , mention may be made of antigen-binding fragments that bind to mammalian TNF. For example, Fab (e.g., obtained by papain digestion), Fab' (e.g., obtained by pepsin digestion and partial reduction) and F(ab')2 (e.g., obtained by pepsin digestion), facb (e.g., obtained by plasmin digestion ), pFc' (e.g., obtained by pepsin or plasmin digestion), Fd (e.g., obtained by pep sin digestion, partial reduction and reaggregation), Fv or scFv (e.g., obtained by molecular biological techni ques), but are not limited thereto; antibody fragments capable of binding TNF or a portion thereof are encompassed by the present invention (see, e.g., Colligan, Imm unology mentioned above).

[0062] Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and / or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons are introduced upstream of the natural stop site. For example, the combination of genes encoding the heavy chain portion of F(ab')2 can be designed to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. Various portions of antibodies can be chemically conjugated by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

[0063] As used herein, the term "human antibody" means that substantially all portions of the protein (e.g., CDRs, framework, C L , C H domain (e.g., C​​H 1, C H 2, and C H3), hinge (V L , V H )) is substantially non-immunogenic in humans and has a sequence This refers to antibodies in which the change or mutation is very minor. Similarly, primates (monkeys, baboons, chins) Pansies, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.), and Antibodies specified for other mammals are antibodies specific to such species, subgenus, genus, subfamily, and family. This refers to... Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations are... Selectively and preferably, compared to unmodified antibodies, antibodies in humans or other species are modified. To maintain or reduce epidemicgenicity. Therefore, human antibodies are chimeric antibodies or humanized antibodies. It is different from the body. Human antibodies are functionally reconstituted human immunoglobulins (e.g., heavy chain and Animals other than humans, or prokaryotes or eukaryotes, that can express the bi / or light chain gene. It has been pointed out that they can be produced by cells. Furthermore, if human antibodies are single-chain antibodies, they can be naturally produced. It may contain linkapeptides not found in human antibodies. For example, Fv is the variable region of the heavy chain. Linkers, such as 2 to about 8 glycine or other amino acid residues, connect the variable region of the light chain to the linker. It may contain peptides. Such linkapeptides are considered to be of human origin.

[0064] Furthermore, monoclonal molecules having binding specificity to at least two different antigens, preferred or human or humanized antibodies, bispecific, for example, DuoBody(registered trademark)( Bispecific antibodies, heterospecific antibodies, heterobinding antibodies, or similar antibodies may be used. In combination, one of the binding specificities is for at least one TNF protein. The other is for any other antigen. The method for producing a bispecific antibody is the said technique. This is known in the field of technology. Conventionally, recombinant generation of bispecific antibodies has been performed using two immunoglobulins. This is based on the co-expression of phosphorus heavy chain-light chain pairs, but here the two heavy chains have different specificities (Mi Ilstein and Cuello, *Nature*, Vol. 305, p. 537 (1983). ). Due to the random combination of immunoglobulin heavy and light chains, these hybrids M(quadroma) produces a possible mixture of 10 different antibody molecules, and of these 1 Only the species possesses the correct bispecific structure. This is usually performed by affinity chromatography. The purification of the correct molecules is time-consuming and results in low product yields, therefore, bispecific antibodies Different methods have been developed to accelerate the generation process.

[0065] Full-length bispecific antibodies can be used, for example, in a cell-free environment in vitro or through co-expression. This is used to facilitate the heterodimer formation of two antibody halves with different specificities. By introducing substitutions at the heavy chain CH3 interface in each half, two monospecific divalents are created. Fab arms can be generated using Fab arm exchange (or half-arm exchange) between antibodies. The exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domain. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. One of the sex antibodies obtained free cysteine ​​is the cysteine ​​of the second parent monospecific antibody molecule. It forms a disulfide bond with the residue and within the heavy chain, and at the same time, the CH3 domain of the parent antibody dissociates. It is released and reformed through association. The CH3 domain of the Fab arm is released through homodimerization. The process may also be manipulated to favor heterodimer formation. The resulting products are, A dual Fab arm or hybrid that can bind to different epitopes. It is a specific antibody.

[0066] As used herein, "homodimerization" refers to having the same CH3 amino acid sequence. This refers to the interaction of two heavy chains. As used herein, "homodimer" means the same This refers to an antibody having two heavy chains, each containing a single CH3 amino acid sequence.

[0067] As used herein, "heterodimer formation" refers to non-identical CH3 amino acid sequences. This refers to the interaction of two heavy chains having a certain characteristic. When used herein, "heterodimer" refers to a heterodimer. This refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0068] The "knob-in-hole" strategy (e.g., International Publication No. 2006 / 02893) (See No. 6) Full-length bispecific antibodies can be generated using this method. In other words, the selected amino acids that form the interface of the CH3 domain in human IgG are In a position that influences CH3 domain interactions to promote heterodimer formation, It can be mutated. An amino acid with a small side chain (hole) specifically binds to the first antigen. An amino acid (nob) with a large side chain is introduced into the heavy chain of the antibody and specifically targets the second antigen. It is introduced into the heavy chain of the antibody that binds heteroto. After co-expression of the two antibodies, a heterodimer is formed. Formed as a result of preferential interaction between heavy chains with "holes" and heavy chains with "knobs". The exemplary CH3 substitution pair forming the knob and hole is T366Y / F405A. , T366W / F405W, F405W / Y407A, T394W / Y407T, T39 4S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (the first CH3 domain of the first heavy chain) As a modified position / modified position in the second CH3 domain of the second heavy chain represent).

[0069] Other strategies include, for example, positively charged residues on one CH3 surface and a second CH3 surface. Heavy chain heterogeneity using electrostatic interactions by substituting negatively charged residues in The promotion of mer formation is described in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. It may be used as described in National Patent Application Publication No. 2011 / 0123532. In this strategy, heterodimer formation is described in U.S. Patent Application Publication No. 2012 / 0149876 or As described in document No. 2013 / 0195849, the following substitution: L351Y_F405 A_Y407V / T394W, T366I_K392M_T394W / F405A_Y4 07V, T366L_K392M_T394W / F405A_Y407V, L351Y_ Y407A / T366A_K409F, L351Y_Y407A / T366V_K409 F, Y407A / T366A_K409F, or T350V_L351Y_F405A_ Y407V / T350V_T366L_K392L_T394W (First heavy chain, first C Modified position in the H3 domain / Modification in the second CH3 domain of the second heavy chain This can be facilitated by (representing the position as it was determined).

[0070] In addition to the methods described above, bispecific antibodies are described in International Publication No. 2011 / 131746. According to the described method, in a cell-free environment in vitro, two monospecific homozygous A reduction is performed by introducing an asymmetric mutation into the CH3 region of a mer antibody, thereby isomerizing the disulfide bond. Under certain conditions, a bispecific heterodimer antibody is obtained from two parent monospecific homodimer antibodies. It can be produced by forming. In this method, the first monospecific bivalent antibody and the second The monospecific bivalent antibody has a CH3 domain that promotes the stability of the heterodimer. These antibodies are modified to have specific substitutions, but these antibodies have cysta in the hinge region. Under sufficient reducing conditions to isomerize the disulfide bond, both are incubated together. This process is performed, and as a result, bispecific antibodies are generated by exchanging the Fab arm. The vating conditions can, in principle, be returned to non-reducing conditions. Examples of reducing agents that can be used are: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioe Lythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine TCEP), L-cysteine, and beta-mercaptoethanol, preferably, 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl A reducing agent selected from the group consisting of phosphines. For example, at a temperature of at least 20°C. In the presence of at least 25 mM 2-MEA or at least 0.5 mM dithio In the presence of slatol, at pH 5-8, for example, at pH 7.0 or pH 7.4, A 90-minute incubation period may be used instead.

[0071] Anti-TNF antibodies (also referred to as TNF antibodies) useful in the methods and compositions of the present invention can optionally be characterized as having high affinity binding to TNF, and optionally and preferably low toxicity . Specifically, individual components such as variable regions, constant regions, and frameworks , individually and / or collectively, optionally and preferably have low immunogenicity, and the antibodies of the invention, identified fragments thereof, or variants thereof are useful in the present invention . Antibodies that can be used in the present invention can optionally be characterized on the basis of measurable alleviation of symptoms and the ability to treat patients for long periods with low and / or acceptable toxicity . Low or acceptable immunogenicity, and / or high affinity, as well as other suitable properties can contribute to the therapeutic results obtained. As used herein, "low immunogenicity" means that a significant increase in HAHA, HACA or HAMA responses occurs in less than about 75%, or preferably less than about 50%, of treated patients, and / or low titers (less than about 300, preferably less than about 100, as measured by double antigen enzyme immunoassay) are increased, which defines low immunogenicity (Elliott et al., Lancet, Vol. 344, pp. 1125-1127 (1994), which is incorporated herein in its entirety by reference) .

[0072] Usefulness: The isolated nucleic acid of the present invention can be measured or act in cells, tissues, organs or animals (including mammals and humans) to diagnose, monitor, modulate, treat, alleviate, prevent the occurrence of at least one TNF-mediated condition, selected from but not limited to at least one of immune disorders or diseases, cardiovascular disorders or diseases, infectious diseases, malignant and / or neurological disorders or diseases ​At least one that can be used to help prevent or reduce the symptoms of It can be used to generate anti-TNF antibodies or identified variants thereof.

[0073] Such methods may regulate, treat, alleviate, prevent, or reduce symptoms, effects, or mechanisms. To cells, tissues, organs, animals, or patients that require it, administer at least one anti-TNF antibody. This may include administering an effective amount of the composition or pharmaceutical composition. The effective amount is as specified herein. To be determined by using known methods, which are published or known in the relevant field, and in a single instance (e.g.) For example, approximately 0.001 to 500 mg / kg per bolus, multiple doses, or continuous infusion. Serum concentration of 0.01 to 5000 μg / mL per dose, or per single, multiple, or continuous infusion. This may include the quantity that achieves the degree, or any effective range or value within it. References. This specification. All publications or patents cited herein are incorporated in their entirety by reference. It represents the highest standards at the time of the invention and / or provides a description and usability of the invention. "Activities" refers to any media format, including all records in electronic or printed form. This refers to any available scientific publication or patent gazette or any other information. (See also: The following references) The entire text is incorporated herein by reference: Ausubel et al., "Current "Protocols in Molecular Biology", John Wi ley&Sons, Inc., NY, NY (1987~2001), Sambrook et al., “Molecular Cloning: A Laboratory Manual ”, 2nd edition, Cold Spring Harbor, NY (1989), Harlo w and Lane, "antibodies", a Laboratory Manual Cold Spring Harbor, NY (1989), edited by Colligan et al. , “Current Protocols in Immunology”, John Wiley & Sons, Inc., NY (1994~2001), Colligan et al. “Current Protocols in Protein Science”, J. ohn Wiley & Sons, NY, NY, (1997~2001).

[0074] The antibody of the present invention: all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3, and / or sequences The present invention includes all of the light chain variable CDR regions numbered 4, 5, and 6, and at least one anti-TN The F antibody is optionally selected from cell lines and mixed cell lines, as is well known in the art. They can be produced by immortalized cells or clonal populations of immortalized cells. For example, Ausube Current Protocols in Molecular Biol, edited by L et al. John Wiley & Sons, Inc., NY, NY (1987~200 1), Sambrook et al., “Molecular Cloning: A Labor atory Manual” 2nd edition, Cold Spring Harbor, NY (1 989), Harlow and Lane, "antibodies", a Laboratories tory manual, Cold Spring Harbor, NY (1989) Colligan et al., eds., "Current Protocols in Immuno John Wiley & Sons, Inc., NY (1994~2001) , Colligan et al., "Current Protocols in Protein Science", John Wiley & Sons, NY, NY, (1997-20 01), the disclosure of which is incorporated herein by reference.

[0075] Human antibodies specific for human TNF protein or fragments thereof can be isolated and / or raised against a suitable immunogenic antigen, such as TNF protein, or portions thereof (including synthetic molecules such as synthetic peptides). Other specific or general mammalian antibodies can also be generated similarly. Preparation of immunogenic antigens and production of monoclonal antibodies can be performed using any suitable technique. can be carried out using art-recognized techniques.

[0076] In one approach, hybridomas are produced by fusing an appropriate immortalized cell line (e.g., Sp2 / 0 , Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3 X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 S A5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT , WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 1 44, NAMAIWA, NEURO 2A, etc., but are not limited to myeloma cell lines , or heteromyelomas, fusion products thereof, or any cell or fused cell derived therefrom, or any other suitable cell line known in the art to produce hybridomas by fusion. See, for example, www.atcc.org, www.life tech.com, etc. Isolated or cloned spleen, peripheral blood, lymph, tonsils, or other immune or B cell-containing cells, but are not limited to Antibody-producing cells, or recombinant or endogenous viruses, bacteria, algae, prokaryotes, Amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep, goats, sheep, spirits Longiform organisms, eukaryotes, genomic DNA, cDNA, rDNA, mitochondrial DNA or R NA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded Or, endogenous or other forms such as triple-stranded, hybridized, or combinations thereof. It is either a heavy chain or a light chain, constant or variable or flame The cell has any other cells that express the Aus or CDR sequence. For example, the Aus mentioned above. See ubel and Chapter 2 of Colligan, Immunology mentioned above. Both documents are incorporated herein by reference in their entirety.

[0077] Antibody-producing cells are obtained from the peripheral blood of a human or other suitable animal immunized with the target antigen, or from a preferred source. Alternatively, it can be obtained from the spleen or lymph nodes. Using any other suitable host cells... Therefore, heterogeneous nucleic acids or endogenous nuclei encoding the antibody of the present invention, a specified fragment or its variant. Acid expression can also be induced. Fusion cells (hybridomas) or recombinant cells are selectively cultured. Isolate using nutrient conditions or other suitable known methods, and perform limiting dilution or cell sorting or other It can be cloned by known methods. It produces antibodies with the desired specificity. The cells can be selected by a suitable assay (e.g., ELISA).

[0078] One method is to select recombinant antibodies from a peptide or protein library. Suitable for producing or isolating antibodies having the required specificity, not limited to those mentioned above. Other methods can be used (e.g., bacteriophages, ribosomes, oligonucleotides) These are display libraries for creotides, RNA, cDNA, etc., but are not limited to these. For example, Cambridge Bold Technologies, Ca mbridgeshire, UK, MorphoSys, Martinsreid / Pl anegg, DE, Biovation, Aberdeen, Scotland, UK, BioInvent, Lund, Sweden, Dyax Corp., Enzon, A From ffymax / Biosite, Xoma, Berkeley, CA, and Ixsys. (Available for purchase). For example, European Patent No. 368,684, International Application No. GB91 / 011 No. 34, International Application No. GB92 / 01755, International Application No. GB92 / 002240, Country International Patent Application No. GB92 / 00883, International Patent Application No. GB93 / 00605, US Patent Application No. Application No. 08 / 350260 (5 / 12 / 94), International Application No. GB94 / 01422, International Application Application No. GB94 / 02662, International Application No. GB97 / 01835, (CAT / MRC) International Publication No. 90 / 14443, International Publication No. 90 / 14424, International Publication No. 90 / 1 Application No. 4430, International Application No. US94 / 1234, International Publication No. 92 / 18619, International Publication Publication No. 96 / 07754 (Scripps), European Patent No. 614 989 (Morph oSys), International Publication No. 95 / 16027 (BioInvent), International Publication No. 88 / Publication No. 06630, International Publication No. 90 / 3809 (Dyax), U.S. Publication No. 4,704,69 Patent No. 2 (Enzon), International Application No. US91 / 02989 (Affymax), International Publication European Patent No. 89 / 06283, European Patent No. 371998, European Patent No. 550400, (Xo (ma) European Patent No. 229046, International Application No. US91 / 07149 (Ixsys) , or probabilistically generated peptides or proteins - U.S. Patent No. 5723323 , No. 5763192, No. 5814476, No. 5817483, No. 5824 Patent No. 514, Patent No. 5976862, International Publication No. 86 / 05803, European Patent No. 5906 Issue 89 (Ixsys, now Applied Molecular Evolution) (AME), each of which is incorporated herein by reference in whole, or A repertoire of human antibodies known in the art and / or described herein It depends on the immunity of transgenic animals that can produce birds (e.g., SCI D mouse, Nguyen et al., "Microbiol. Immunol. Vol. 41, p. 901" ~907 pages (1997), Sandhu et al., Crit. Rev. Biot echnol.16:95-118 (1996);Erenet al.,Immun ol.93:154-161 (1998), each is incorporated into the whole by reference. Such technologies include ribosome displays (Hanes et al., Proc.N). atl.Acad.Sci.USA, Volume 94, pp. 4937-4942 (May 1997) ), Hanes et al., Proc. Natl. Acad. Sci. USA, 95 :14130-14135 (Nov. 1998)); Single-cell antibody production technology (for example, Selective Lymphocyte Antibody Method ("SLAM") (U.S. Patent No. 5,627,052, Wen et al.) "J. Immunol." Vol. 17, pp. 887-892 (1987); Babcook et al.,Proc.Natl.Acad.Sci.USA 93:7843-7 848 (1996)); Gel microdroplets and flow cytometry (Pow ell et al., "Biotechnol." Vol. 8, pp. 333-337 (1990), On e Cell Systems (Cambridge, MA); Gray et al. J.Imm.Meth., Vol. 182, pp. 155-163 (1995); Kenny et al. et al., Bio / Technol. Vol. 13, pp. 787-790 (1995), B cell selection (Steenbakkers et al., Molec. Biol. Re ports 19:125-134 (1994);Jonak et al.,Pro gress Biotech,Vol.5,In Vitro Immunization n in Hybridoma Technology, Borrebaeck, ed. ,Elsevier Science Publishers BV,Amster Examples include dam, Netherlands (1988), but it is not limited to these. stomach.

[0079] Methods for engineering or humanizing non-human antibodies or human antibodies can also be used in the same manner. This is well known in the technical field. Generally, humanized or engineered antibodies are, for example, MAU This includes, but is not limited to, rats, rabbits, non-human primates, or other mammals. These human Amino acid residues are often called "import" residues, and are typically the " "Import" is taken from the variable region, the constant region, or other domains.

[0080] Known human Ig sequences have been disclosed in numerous publications and websites, for example, www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / www.abcam.com / www.antibodyresource.com / onlinecomp.htm l; www.public.iastate.edu / ~pedro / research_ tools.html; www.mgen.uni-heidelberg.de / SD / IT / IT.htm l; www.whfreeman.com / immunology / CH05 / kuby0 5.htm; www.library.thinkquest.org / 12429 / Immune / Antibody.html; www.hhmi.org / grants / lectures / 1996 / vlab; www.path.cam.ac.uk / ~mrc7 / mikeimages.htm l; www.antibodyresource.com / www.mcb.harvard.edu / BioLinks / Immunology .html. www.immunologylink.com / www.pathbox.wustl.edu / ~hcenter / index.ht ml; www.biotech.ufl.edu / ~hcl / www.pebio.com / pa / 340913 / 340913.html; www.nal.usda.gov / awic / pubs / antibody / ; www.m.ehime-u.ac.jp / ~yasuhito / Elisa.htm l; www.biodesign.com / table.asp; www.icnet.uk / axp / facs / davies / links.html ; www.biotech.ufl.edu / ~fccl / protocol.html ; www.isac-net.org / sites_geo.html; www.aximt1.imt.uni-marburg.de / ~rek / AEPS tart.html; www.baserv.uci.kun.nl / ~jraats / links1.ht ml; www.recab.uni-hd.de / immuno.bme.nwu.edu / ; www.mrc-cpe.cam.ac.uk / imt-doc / public / IN TRO.html; www.ibt.unam.mx / vir / V_mice.html;imgt.cn usc.fr:8104 / ; www.biochem.ucl.ac.uk / ~martin / abs / index .html;antibody.bath.ac.uk / ; www.abgen.cvm.tamu.edu / lab / www.abgen.html; www.unizh.ch / ~honegger / AHOseminar / Slide 01.html; www.cryst.bbk.ac.uk / ~ubcg07s / ; www.nimr.mrc.ac.uk / CC / ccaewg / ccaewg.htm ; www.path.cam.ac.uk / ~mrc7 / humanisation / T AHHP.html; www.ibt.unam.mx / vir / structure / stat_aim. html; www.biosci.missouri.edu / smithgp / index.h tml; www.cryst.bioc.cam.ac.uk / ~fmolina / Web-p ages / Pept / spottech.html; www.jerini.de / frproducts.html; www.patents.ibm.com / ibm.html, Kabat et al. “Sequences of Proteins of Immunological There is "Interest" and USDept.Health (1983), and each is The entirety is incorporated herein by reference.

[0081] Such imported sequences are used to reduce immunogenicity, or in the field of the relevant technology. As is known, binding, affinity, binding rate constant, dissociation rate constant, binding activity, specificity It can be used to reduce, enhance, or modify the half-life or any other suitable properties. In general, some or all of a non-human or human CDR sequence has variable and constant regions. The human sequence is maintained even while it is being replaced with human or other amino acids. Antibodies also... Selectively, while retaining high affinity for the antigen and other desirable biological properties, It can be humanized. To achieve this objective, a humanized antibody can be optionally used with the parent sequence and Analysis process of parent sequences and various conceptual humanized products using a three-dimensional model of humanized sequences. It can be prepared by [method / tool]. Three-dimensional immunoglobulin models are generally available. This is possible and well known to those skilled in the art. Probability for selected immunoglobulin sequence candidates. Computer programs are available that can illustrate and display highly accurate three-dimensional structures. By examining these indications, we can determine the possible functions that residues may exhibit in candidate immunoglobulin sequences. Analysis of highly effective proteins, specifically analysis of residues that influence the antigen-binding ability of candidate immunoglobulins. This becomes possible. In this way, desirable antibody characteristics, such as increased affinity for the target antigen, can be achieved. To achieve this, FR residues are selected and combined from the consensus sequence and import sequence. It can be made to work. Generally, CDR residues are directly and almost substantially involved in antigen binding. This affects the humanization or engineering of antibodies in the present invention, Winter (Jone et al.) al.,Nature 321:522 (1986);Riechmann et a l.,Nature 332:323 (1988);Verhoeyen et al. .,Science 239:1534 (1988)),Sims et al.,J .Immunol.151:2296 (1993);Chothia and Les k, J. Mol. Biol. 196:901 (1987), Carter et al. .,Proc.Natl.Acad.SCi.USA89:4285 (1992 ); Presta et al., J.Immunol. Vol. 151, p. 2623 (19 (1993), U.S. Patent No. 5723323, No. 5976862, No. 5824514 , No. 5817483, No. 5814476, No. 5763192, No. 5723 323, 5,766886, 5714352, 6204023, 5,766886, 5714352, 6204023, No. 6180370, No. 5693762, No. 5530101, No. 558508 Applications No. 9, No. 5225539, No. 4816567, and International Application No. US98 / 1628 No. 0, No. US96 / 18978, No. US91 / 09630, No. US91 / 05939, U S94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / Publication No. 01755, International Publication No. 90 / 14443, International Publication No. 90 / 14424, International Publication No. 90 / 1 Patent No. 4430, European Patent No. 229246 (each incorporated in its entirety by reference) This includes, but is not limited to, the literature cited within it, This can be done using any known method.

[0082] Anti-TNF antibodies are, optionally, those described herein and / or in the art described herein. Transgenic animals that can generate a known repertoire of human antibodies (e.g.) For example, it can also be produced by the immune response of mice, rats, hamsters, non-human primates, etc. Cells producing human anti-TNF antibodies are isolated from the animal concerned and used according to the method described herein. Immortalization may be achieved using a suitable method such as law.

[0083] Transgenic organisms capable of producing a repertoire of human antibodies that bind to human antigens. Mice can be created by known methods (for example, but not limited to these). U.S. Patent No. 5,770,428 and No. 5,569,8 issued to Lonberg et al. No. 25, No. 5,545,806, No. 5,625,126, No. 5,625,82 No. 5, No. 5,633,425, No. 5,661,016, and No. 5,789,6 Issue 50, International Publication No. 98 / 50433 by Jakobovits et al., Jakobovit International Publication No. 98 / 24893 by s et al., International Publication No. 98 / 24884 by Lonberg et al. Lonberg et al. International Publication No. 97 / 13852, Lonberg et al. International Publication No. Issue 94 / 25585, International Publication No. 96 / 34096 by Kucherlapate et al., K European Patent No. 0463151(B1) by ucherlapate et al., Kucherlap European Patent No. 0710719(A1) by ate et al., U.S. Patent No. 5,54 Issue 5,807, International Publication No. 90 / 04036 by Bruggemann et al., Brugge Mann et al., European Patent No. 0438474(B1), Lonberg et al., European Patent No. 08 Patent No. 14259(A2), British Patent No. 2272440(A) by Lonberg et al., L onberg et al., "Nature" Vol. 368, pp. 856-859 (1994), Ta Ylor et al., "Int. Immunol." Vol. 6, No. 4, pp. 579-591 (1994) (Year), Green et al., "Nature Genetics" Vol. 7, pp. 13-21 (19 1994) Mendez et al., "Nature Genetics" Vol. 15, pp. 146-141 Page 56 (1997), Taylor et al., "Nucleic Acids Research "ch" Vol. 20, No. 23, pp. 6287-6295 (1992), Tuaillon et al. "Proc Natl Acad Sci USA" Vol. 90 No. 8 pp. 3720-372 Page 4 (1993), Lonberg et al., "Int Rev Immunol" Vol. 13. No. 1, pp. 65-93 (1995), and Fishwald et al., "Nat Biote "CHNOL" Vol. 14, No. 7, pp. 845-851 (1996), these are all by reference. The body can be produced by (the body incorporated herein). Generally, these mice at least one that has been functionally reconfigured or is capable of undergoing functional reconfiguration Contains at least one transgene that includes DNA derived from the human immunoglobulin locus. The endogenous immunoglobulin gene locus of such mice is destroyed or deleted, and the endogenous immunoglobulin gene locus of the mouse is destroyed or deleted. The ability to produce antibodies encoded by the causal gene can be eliminated.

[0084] Screening antibodies for specific binding to similar proteins or fragments is done using peptides. This can be successfully achieved using the display library. This method is desired. For each member with a unique function or structure, a large number of peptide samples were collected from the screen. This involves screening. Antibody screening of peptide display libraries is performed using this technology. This is well known in the field of technology. The length of the displayed peptide sequence is 3 to 5000. These are the amino acids, and they frequently have a length of 5 to 100 amino acids, but most have about 8 to 25 amino acids. It may be no acid length. In addition to direct chemical synthesis methods for creating peptide libraries, several Recombinant DNA methods are also described. One type involves bacteriophages or the surface of cells. Includes display of peptide sequences on a surface. Each bacteriophage or cell is specific It contains a nucleotide sequence that codes for the displayed peptide sequence. This refers to International Patent Publication Nos. 91 / 17271, 91 / 18980, and 91 / 1981. This is described in issue 8 and issue 93 / 08278. Other systems include both in vitro chemical synthesis and recombinant methods. International Patent Publication Nos. 92 / 05258, 92 / 14843, and 96 / 19256 See the relevant numbers. See also U.S. Patent Nos. 5,658,754 and 5,643,768. Please be informed. Peptide display libraries, vectors, and screening kits are available. Invitrogen (Carlsbad, CA) and Cambridge Antibacterial Companies like Ody Technologies (Cambridgeshire, UK) It is sold commercially by the supplier. For example, U.S. Patent No. 4704692, which was transferred to Enzon. , No. 4939666, No. 4946778, No. 5260203, No. 5455 No. 030, No. 5518889, No. 5534621, No. 5656730, No. Numbers 5763733, 5767260, and 5856456 were transferred to Dyax. U.S. Patent No. 5,223409, No. 5403484, No. 5571698, No. 5 U.S. Patent No. 837500, U.S. Patent No. 5427908, and U.S. Patent No. 558, which were transferred to Affymax. License No. 0717 transferred to Cambridge Antibody Technologies. U.S. Patent No. 5885793, assigned to Genentech, U.S. Patent No. 575 U.S. Patent No. 0373, U.S. Patent No. 5,618,920 and No. 5,595,898, which were transferred to Xoma. , No. 5576195, No. 5698435, No. 5693493, No. 5698 No. 417, Colligan as described above, Ausubel as described above, or Sambroo as described above See k. Note that each of the above patents and publications is incorporated into this specification by reference. To be absorbed.

[0085] The antibodies of the present invention are also found in the milk of goats, cows, horses, sheep, and other animals that produce such antibodies. To provide a nucleic acid-genic animal or mammal, at least one encoding nucleic acid It can also be prepared using anti-TNF antibodies. Such animals can be prepared using known methods. It can be equipped with. For example, but is not limited to these, U.S. 5,827,690 No. 5,849,992; No. 4,873,316; No. 5,849,992 ; No. 5,994,616; No. 5,565,362; No. 5,304,489 See the references provided (each of which is incorporated into this specification by reference).

[0086] The antibodies of the present invention are used in plant parts or cells cultured therefrom. Transgenic plants that produce modified parts or mutants and cultured plant cells (for example) To provide tobacco and corn (but not limited to these), less Both can be further prepared using a single anti-TNF antibody-coding nucleic acid. Non-limiting examples For example, transgenic proteins express recombinant proteins using an inducible promoter. Large quantities of recombinant proteins have been successfully supplied using tobacco leaves. For example, Cra mer et al.,Curr.Top.Microbol.Immunol.240 See :95-118 (1999) and the references cited therein. Also, Genetically modified maize is a protein or natural product produced in other recombinant systems. Mammalian proteins with biological activity equivalent to proteins purified from the source, commercially It has been used for expression at the generative level. For example, Hood et al., Adv "Exp.Med.Biol.", Vol. 464, pp. 127-147 (1999) and its See the references cited within. The antibodies include single-chain antibodies against tobacco seeds and potato tubers. It is also produced in large quantities from transgenic plant seeds containing antibody fragments such as body (scFv). For example, Conrad et al., "Plant Mol. Biol." Vol. 38, p. 10 See pages 1-109 (1998) and the references cited therein. Therefore, this The antibodies of the invention can also be produced using transgenic plants by known methods. Yes, it is possible. For example, Fischer et al., Biotechnol.Appl.Bi ochem.30:99-108 (Oct.,1999), Ma et al., Tr. ends Biotechnol.13:522-7 (1995);Ma et al .,Plant Physiol.109:341-6 (1995);Whitela m et al.,Biochem.Soc.Trans.22:940-944 (1 See also 994) and the references cited therein. Each of the above references is referenced by The entirety of this is incorporated herein.

[0087] The antibodies of the present invention have a wide range of affinity (K D ) can bind to human TNF. In a preferred embodiment, at least one human mAb of the present invention is optionally human T It can bind to NF with high affinity. For example, human mAbs can bind to human TNF by approximately 10 -7 M or less, for example, 0.1 to 9.9 (or any range or value within that range) x 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11, 10 -12 , 10 -13 or one of the roles within that K is a range of meaning or value (but is not limited to these). D They can be joined together. .

[0088] The affinity or binding activity of an antibody to an antigen can be determined experimentally using any suitable method. It is possible. (For example, Berzofsky et al., "Antibody-Antige n Interactions”, In Fundamental Immunolog Edited by Paul WE, Raven Press: New York, NY (198 4 years), Kuby, Janis, "Immunology", WHFreeman and Company: New York, NY (1992), and as described herein. (See the method for details). The affinity measured for a specific antibody-antigen interaction is different. It may differ when measured under certain conditions (e.g., salt concentration, pH). Therefore, affinity Sex and other antigen-binding parameters (e.g., K D , K a , K d The measurement of ) is preferably done by anti This procedure is performed using standard solutions of the body and antigen, as well as standard buffers such as those described herein. It breaks.

[0089] Nucleic acid molecule. At least one adjacent element from sequence numbers 1, 2, 3, 4, 5, 6, 7, 8. Nucleotide sequences encoding at least 70-100% of the amino acid, identified fragments, variants Allomorphs or their consensus sequences, or containing at least one of these sequences Using the information provided herein, such as the deposited vector, the heavy chain of SEQ ID NOs: 1, 2, and 3 All of the variable CDR area and / or all of the light chain variable CDR areas of sequence numbers 4, 5 and 6 The nucleic acid molecule of the present invention, which encodes at least one anti-TNF antibody, is described herein. It can be obtained by or by using methods known in the art.

[0090] The nucleic acid molecule of the present invention may be mRNA, hnRNA, tRNA, or any other form. RNA in any form, or cDNA obtained by cloning or produced synthetically. and genomic DNA are examples, but are not limited to, any form of DNA, or any of these. Combinations are also acceptable. DNA can be triple-stranded, double-stranded, or single-stranded, or these. Any combination is acceptable. Any part of at least one strand of DNA or RNA. This may be a code chain, also known as a sense chain, or an antisense chain. It may also be a non-coded chain.

[0091] The isolated nucleic acid molecule of the present invention optionally has one or more introns. Open Reading Frames (ORFs), for example, but not limited to these, are at least Another heavy chain (e.g., SEQ ID NOs. 1-3) or light chain (e.g., SEQ ID NOs. 4-6) C At least one CDR such as DR1, CDR2, and / or CDR3 A nucleic acid molecule containing one identified portion, an anti-TNF antibody, or a coding sequence of a variable region (e.g.) For example, nucleic acid molecules including sequence numbers 7 and 8), and nucleic acid molecules that are substantially different from those mentioned above, Due to the degeneracy of the genetic code, the following are described herein and / or known in the art: A nucleic acid molecule containing a nucleotide sequence that still encodes at least one anti-TNF antibody It may include. Naturally, the genetic code is well known in the relevant technical field. Therefore, those skilled in the art will know how to encode such degenerate nucleic acid variants that represent specific anti-TNF antibodies of the present invention. Generating such things is a common occurrence. For example, please refer to Ausubel et al. above. Such nucleic acid variants are included in the present invention. Non-limiting examples of isolated nucleic acid molecules of the present invention include: , respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC Nucleic acids encoding the HC variable region and LC variable region of CDR2, LC CDR3 Sequence numbers 10, 11, 12, 13, 14, and 15 are examples that correspond to typical cases.

[0092] As shown herein, the nucleic acid molecule of the present invention comprises a nucleic acid encoding an anti-TNF antibody and As for the above, it includes those that themselves encode the amino acid sequence of the antibody fragment, or the full length of the antibody or This includes sequences that encode a part of an antibody, an antibody, a sequence that encodes a fragment or part of an antibody, and additional encodings. A sequence, for example, with at least one intron, or with the aforementioned additional code sequences. Without accompanying non-coded 5' and 3' sequences, e.g., splicing and polyadenylated sequences Transcription, mRNA processing, including the properties of mRNA (e.g., ribosome binding and stability of mRNA) Additional non-translated sequences, including but not limited to those transcribed, that play a role in the process. Along with the code sequence, at least one coding sequence of a signal reader or fusion peptide, Additional amino acids, for example, additional coding sequences that encode amino acids that provide additional functions. These are some examples, but are not limited to them. Therefore, the sequence encoding the antibody is It can be fused to a marker sequence; for example, the marker sequence can be an antibody fragment to which it is fused. It is a sequence that encodes a peptide that promotes the purification of antibodies containing or part of the antibody.

[0093] Polynucleotides that selectively hybridize to the polynucleotides described herein The present invention provides a selective hybridization of polynucleotides disclosed herein. This invention provides isolated nucleic acids that hybridize under certain conditions. Polynucleotides are used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. It can be used for the purpose of depositing. For example, the polynucleotide of the present invention can be used for deposit. Identifying, isolating, or amplifying partial-length or full-length clones in the library Yes, it is possible. In some embodiments, the polynucleotides are isolated or otherwise If not available, a genome sequence or a genome sequence complementary to the cDNA of a human or mammalian nucleic acid library. This is a cDNA sequence.

[0094] Preferably, the cDNA library contains at least 80% of the complete-length sequence, preferably the complete sequence. At least 85% or 90% of the long sequence, and more preferably at least 95% of the full-length sequence. This includes %. This cDNA library normalizes to increase the expression levels of rare sequences. This is possible. Low or medium stringer stringers use sequences with low sequence identity to complementary sequences. The hybridization conditions of NC are typical, but not limited to them. For sequences with higher unigeneity, use the intermediate and high stringency conditions as an option. This is possible. Low stringency conditions allow for selective extraction of sequences with approximately 70% sequence identity. To enable hybridization and identify orthologous or paralogous sequences It is available.

[0095] Optionally, the polynucleotides of the present invention are the polynucleotides described herein. This will encode at least a portion of the antibodies encoded by the present invention. The polynucleotide is a selective hybrid of the polynucleotide encoding the antibody of the present invention. It contains nucleic acid sequences available for dilation. For example, the Ausubel sequence mentioned above. See Colligan above. Each is incorporated into this specification by reference. Born.

[0096] Construction of nucleic acids. The isolated nucleic acids of the present invention are prepared by (a) a recombinant method, as is well known in the art. (b) synthesis techniques, (c) purification techniques, or a combination thereof can be used to produce them. can.

[0097] In addition to the polynucleotides of the present invention, nucleic acids can conveniently contain sequences. For example, multicloning samples containing one or more endonuclease restriction sites. The thread can be inserted into nucleic acids to aid in the isolation of polynucleotides. It is also translatable. By inserting a suitable sequence, it can be used to help isolate the translated polynucleotide of the present invention. For example, hexahistidine marker sequences are convenient for purifying the proteins of the present invention. The present invention provides a means. The nucleic acid (excluding the coding sequence) of the present invention is optionally selected as the nucleic acid of the present invention. A vector, adapter, or linker for the cloning and / or expression of creotides.

[0098] Additional sequences are added to the cloning sequence and / or expression sequence, and cloning and To optimize their functions in expression, and / or to aid in the isolation of polynucleotides This can improve the introduction of polynucleotides into cells. Cloned vector The use of vectors, expression vectors, adapters, and linkers is well known in the art. For example, see Ausubel or Sambrook mentioned above.

[0099] A recombination method for constructing nucleic acids. RNA, cDNA, genomic DNA, or a combination thereof. The isolated nucleic acid compositions of the present invention, such as combinations of intent, can be any number known to those skilled in the art. It can be obtained from a biological source using a fermentation procedure. In some embodiments, the present invention Ori that selectively hybridizes to polynucleotides under stringent conditions The gonucleotide probe matches the desired sequence within the cDNA or genomic DNA library. It is used for specific purposes. RNA isolation, as well as the construction of cDNA and genomic libraries, are performed by those skilled in the art. This is well known. (See, for example, Ausubel or Sambrook above.) (Regarding the subject).

[0100] Methods for screening and isolating nucleic acids. Using probes based on nucleotide sequences, cDNA or genomic libraries are screened. It can be leaned. Using a probe, the genomic DNA or cDNA sequence can be bled. By breeding, homologous genes from the same or different organisms can be isolated. Therefore, use hybridization stringency of varying degrees in the assay. It is possible to use either a hybridization or washing medium as a stringent. It should be clear that this is possible. The hybridization condition becomes stringent. The degree of complementarity between the probe and target during double-strand formation should increase. The degree of stringency is determined by temperature, ionic strength, pH, and formamide. This can be controlled by the presence of one or more partially denaturing solvents. For example, the stringency of hybridization is, for instance, within the range of 0% to 50%. The polarity of the reaction solution was successfully altered by manipulating the formamide concentration. The degree of complementarity (sequence identity) required for detectable binding is determined by hybridize. It varies depending on the stringency of the cleaning medium and / or washing medium. The degree of complementarity is as follows: Specifically, it is 100%, or 70-100%, or any range or value within that range. However, slight differences in the sequences in the probe and primer can affect hybridization and This can be compensated for by reducing the stringency of the cleaning medium. This should be understood.

[0101] Methods for amplifying RNA or DNA are well known in the art and are not described herein. Based on the instructions and guidelines, the present invention can be used without excessive experimentation.

[0102] Known methods for DNA or RNA amplification include polymerase chain reaction (PCR) and Related amplification processes (for example, Mullis et al., U.S. Patent No. 4,683,195, and the same patent No. No. 4,683,202, No. 4,800,159, No. 4,965,188, Tab or et al. U.S. Patent No. 4,795,699 and No. 4,921,794, Innis U.S. Patent No. 5,142,033, Wilson et al.'s U.S. Patent No. 5,122,464, Innis's U.S. Patent No. 5,091,310, Gyllensten et al.'s U.S. Patent No. 5 U.S. Patent No. 066,584, Gelfand et al.'s U.S. Patent No. 4,889,818, Silve r et al.'s U.S. Patent No. 4,994,370, Biswas's U.S. Patent No. 4,766,067 See U.S. Patent No. 4,656,134 of Ringold, and double-stranded D R uses antisense RNA against a target sequence as a template for NA synthesis. NA-mediated amplification (Malek et al., U.S. Patent No. 5,130,238, trademark name NASBA) (These are examples, but are not limited to these. The full contents of these documents can be found in the references provided.) (To be incorporated into the details). (For example, Ausubel above, or Sambrook above) (Please refer to the following.)

[0103] For example, polymerase chain reaction (PCR) technology can be used to analyze genomic DNA or cDNA. The polynucleotide and related gene sequences of the present invention are amplified directly from Ibrali. PCR and other in vitro amplification methods can also, for example, express the protein to be expressed. Cloning the encoding nucleic acid sequence, detecting the presence of the desired mRNA in the sample. Therefore, nucleic acids are prepared for use as probes for nucleic acid sequencing or other purposes. This may be useful in relation to the following. The in vitro amplification method is sufficient to guide those skilled in the art. Examples of the technology include Berger (mentioned above), Sambrook (mentioned above), and Ausubel (mentioned above). , as well as Mullis et al.'s U.S. Patent No. 4,683,202 (1987), and Innis s et al., “PCR Protocols A Guide to Methods and "Applications", edited by Academic Press Inc., San D. This can be seen in iego, CA (1990). Commercial kits for genome PCR amplification utilize this technology. It is known in the field. For example, Advantage-GC Genomic PCR Please refer to the Kit (Clontech). In addition, for example, T4 gene 32 protein Using a substance (Boehringer Mannheim), the yield of long PCR products was improved. It is possible to do good.

[0104] A synthesis method for constructing nucleic acids. The isolated nucleic acids of the present invention are directly chemically synthesized by known methods. It can also be prepared by (see, for example, Ausubel et al. above). Chemical synthesis is one Generally, this is done through hybridization with complementary sequences, or by using a single strand as a template. By polymerization with the DNA polymerase used, single-stranded DNA can be converted into double-stranded DNA. It produces nucleotides. As those skilled in the art will know, the chemical synthesis of DNA involves more than 100 bases. Although limited to sequences, longer sequences can be obtained through the concatenation reaction of shorter sequences. They will recognize what they are capable of.

[0105] Recombinant expression cassette. The present invention further provides a recombinant expression cassette containing the nucleic acid of the present invention. The nucleic acid sequence of the present invention, for example, the cDNA or genome sequence encoding the antibody of the present invention, is used. Therefore, it is possible to construct a recombinant expression cassette that can be introduced into at least one desired host cell. Yes, it is possible. Recombinant expression cassettes typically produce polynucleo molecules in the intended host cells. The polynucleotide of the present invention, which is functionally linked to a transcription initiation regulatory sequence that guides the transcription of nucleotides. This includes using both heterogeneous and non-heterogeneous (i.e., endogenous) promoters, the core of the present invention. It can induce the expression of acid.

[0106] In some embodiments, an isolated component functions as a promoter, enhancer, or other element. To regulate the expression of the polynucleotide of the present invention, the nucleic acid is used to upregulate or downregulate the polynucleotide of the present invention. Introducing non-heterogeneous nucleotides to the appropriate location (upstream, downstream, or within an intron). This can be done, for example, by mutation, deletion and / or substitution in vivo or in vitro. The sex promoter can be changed.

[0107] Vectors and host cells. The present invention relates to vectors and recombinant vectors comprising the isolated nucleic acid molecules of the present invention. Genetically engineered host cells and recombinant techniques known in the art It also relates to the production of at least one anti-TNF antibody. For example, Sambrook et al. mentioned above, Please refer to Ausubel et al. above, each of which is incorporated herein by reference in whole. It can be done.

[0108] Polynucleotides are vectors that optionally contain a selection marker for host proliferation. It can bind to the phosphate. Generally, plasmid vectors are made of calcium phosphate precipitate. It is introduced into the precipitate or into a complex with charged lipids. If the vector is a virus Then, package this in vitro using an appropriate packaging cell line, and then, It can be transduced into host cells.

[0109] DNA insertions should be functionally linked to the appropriate promoter. The kut is a ribosomal cell that is used for translation within the transcription start site, transcription termination site, and the transcribed region. It further includes a binding site. The coding portion of the mature transcript expressed by the construct is preferably Alternatively, the mRNA to be translated will initially contain a translation initiation site and end with a stop codon (for example, UAA, UGA, or UAG will be appropriately positioned in mammalian or eukaryotic cells. For expression, UAA and UAG are preferred.

[0110] The expression vector preferably contains at least one selection marker, but this is optional. Yes, such markers include, for example, methotrexate (MTX) for eukaryotic cell culture. Hydrofolate reductase (DHFR, U.S. Patent No. 4,399,216, U.S. Patent No. 4,634) , No. 665, No. 4,656,134, No. 4,956,288, No. 5,149, No. 636, No. 5,179,017, Ampicillin, Neomycin (G418), My Cophenolic acid or glutamine synthetase (GS) (U.S. Patent No. 5,122,464) (Patent Nos. 5,770,359 and 5,827,739) Resistance genes, and Escherichia coli Tetracycline or for culture in (E. coli) and other bacteria or prokaryotes This includes, but is not limited to, ampicillin resistance genes (the above patents are fully covered by reference). (The body is incorporated herein). Appropriate culture media and conditions for the above host cells are as follows: This is known in the art. Suitable vectors will be readily apparent to those skilled in the art. The introduction of a vector construct into host cells is performed by calcium phosphate transfects. DEAE-dextran-mediated transfection, cationic lipid-mediated transfection Influence by infection, electroporation, transduction, infection, or other known methods. You may receive this. For details on this method, see Sambrook, Chapters 1-4 and 16- Chapter 18, the above Ausubel, Chapters 1, 9, 13, 15, 16, etc., in the relevant technical field It is written there.

[0111] At least one antibody of the present invention may be expressed in a modified form, such as a fusion protein. This may include not only secretory signals but also additional heterologous functional regions. For example, the region of additional amino acids. By adding charged amino acids, particularly charged amino acids, to the N-terminus of the antibody, during purification or subsequent processing and storage, It can improve stability and persistence in host cells. Furthermore, the peptide portion can be developed in this way. In addition to the antibody, it can also be used to promote purification. (Antibacterial or at least one fragment thereof) Such regions can be removed before the final preparation. Such a method is described above for Sambr ook, Chapters 17.29-17.42 and 18.1-18.74, the above Ausub This is described in many standard laboratory manuals, such as chapters 16, 17, and 18 of EL. .

[0112] Those skilled in the art will know that there are numerous expression techniques available for the expression of nucleic acids encoding the protein of the present invention. He is well-versed in the system.

[0113] Alternatively, the nucleic acid of the present invention may contain endogenous DNA encoding the antibody of the present invention. By switching it on (through manipulation) within the principal cell, it can be expressed in the host cell. Yes, it is possible. Such methods are permitted under U.S. Patent Nos. 5,580,734 and 5,641,670. As described in Articles No. 5,733,746 and No. 5,733,761, The above patents are well known in the art and are incorporated herein by reference in their entirety.

[0114] An example of a cell culture useful for producing antibodies, their specific parts, or variants is a mammalian cell culture. It is a cell. Mammalian cell systems often take the form of a single layer consisting of cells, but mammalian cells Suspensions or bioreactors can also be used. It is possible to express intact glycosylated proteins. Several suitable host cell lines have been developed in the field, including COS -1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL -1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL1610) and BSC-1 (e.g., ATCC CRL-2 6) Cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.65 Examples include 3, SP2 / 0-Ag14, 293 cells, and HeLa cells, which are, for example... , American Type Culture Collection, Manass It is readily available from as,VA. Preferred host cells include CHO cells and bone marrow. Examples include cells derived from the lymphatic system, such as tumor and lymphoma cells. Particularly preferred host cells These are CHO cells, P3X63Ag8.653 cells (ATCC registration number CRL-1580). And SP2 / 0-Ag14 cells (ATCC registration number CRL-1851).

[0115] These cell expression vectors are used as origins of replication, promoters (e.g., late or early SV40) Promoter, CMV Promoter (U.S. Patent No. 5,168,062, and No. 5,385,8) (No. 39), HSV tk promoter, pgk (phosphoglycerate kinase) promoter EF-1α Promotor (U.S. Patent No. 5,266,491), at least one human-powered motor Epidemic globulin promoters, enhancers, and / or ribosome binding sites, RNA splices The sulfate portion, the polyadenylated portion (for example, the SV40 Large T Ag polyaddition portion), and This includes, but is not limited to, processing information sites such as transcription termination sequences, which are involved in expression regulation. It may contain one or more of the elements in the sequence. For example, Ausubel et al. above, S above See ambrook et al. Other useful for the production of nucleic acids or proteins of the present invention The cells are known, and / or, for example, American Type Cultu reCollection's catalog of cell lines and hybridomas or other known Alternatively, it can be obtained from commercial sources.

[0116] When eukaryotic host cells are used, typically polyadenylation or transcription termination occurs within the vector. The sequence is incorporated. An example of a termination sequence is polyadenylation from the bovine growth hormone gene. It is a sequence. Sequences for accurate splicing of transcription can also be included. An example of a pricing sequence is the VP1 intron derived from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983). In addition, the relevant As is well known in the field of technology, gene sequences for controlling replication within host cells are used It can be integrated into the socket.

[0117] Antibody purification. Anti-TNF antibodies are purified by protein A purification, ammonium sulfate or ethanol precipitation. Palatine, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography Raffi, hydrophobic interaction chromatography, affinity chromatography, hydroxy Luapatite chromatography and lectin chromatography are examples, but are not limited to these. It can be recovered and purified from recombinant cell cultures by well-known methods that are not specified. High-performance liquid chromatography (HPLC) It can also be used for purification. For example, Colligan, Current Proto cols in Immunology or Current Protocols in Protein Science,John Wiley & Sons,NY,NY Please refer to chapters 1, 4, 6, 8, 9, and 10 of (1997-2001), for example. These are incorporated in their entirety by reference herein.

[0118] The antibodies of the present invention include naturally purified products, products produced by chemical synthesis procedures, and, for example, Produced by recombinant techniques from eukaryotic hosts, including yeast, higher plants, insects, and mammalian cells. The product contains [a specific product]. Depending on the host used in the recombinant production procedure, the antibody of the present invention is [a specific product]. It is acceptable for it to be sylated or glycosylated, but glycosylation is preferred. The methods described above are Sambrook, Ausubel, and the 10th, 12th, 13th, and 1st. Chapters 6, 18, and 20, Colligan, Protein Science, the above This is described in many standard laboratory manuals, such as in Chapters 12-14, and all of them can be referenced. The entirety is incorporated herein.

[0119] Exemplary anti-TNF antibody All of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3, and / or SEQ ID NOs: 4, 5, and 6 The isolated antibody of the present invention, which includes all of the light chain variable CDR region, can be used in any suitable polynucleotide. The amino acid sequence of the antibody disclosed herein, encoded by Otid, or any single The solution contains a detached or prepared antibody. Preferably, the human antibody or antigen-binding fragment is attached to human TNF. It binds, thereby partially or substantially altering the biological activity of at least one protein. Neutralize. At least one biological activity of at least one TNF protein or fragment. An antibody or a specified part or variant thereof that partially or preferably substantially neutralizes the substance. It binds to proteins or fragments, thereby facilitating the binding of TNF to TNF receptors. Alternatively, it can inhibit activity mediated through other TNF-dependent or mediated mechanisms. When used herein, the term "neutralizing antibody" is used approximately 20-1 depending on the assay. 20%, preferably at least about 10, 20, 30, 40, 50, 55, 60, 65, 7 0, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 This refers to antibodies that can inhibit TNF-dependent activity by 100% or more. The ability of anti-TNF antibodies to inhibit TNF-dependent activity is preferably as described herein and / or at least one suitable TNF protein or receptor known in the art. The antibodies are evaluated by assay. The human antibodies of the present invention belong to any class (IgG, IgA, I (e.g., gM, IgE, IgD) or isotypes, and may contain a κ or λ light chain. It may be seen. In one embodiment, the human antibody is an IgG heavy chain or a defined fragment, for example, I Includes at least one isotype among gG1, IgG2, IgG3, or IgG4 This type of antibody is described herein and / or known in the art, At the very least, one human light chain (e.g., IgG, IgA) and IgM (e.g., γ1, γ2) Transgenic mice or other transgenic mice containing the γ3, γ4) transgenes It can be prepared using non-human mammals. In another embodiment, Human TNF human antibodies contain IgG1 heavy chains and IgG1 light chains.

[0120] As used herein, the terms “antibody” or “multiple antibodies” are defined in accordance with the 2009 standards. Iologics Price Competition and Innovation Act (BPCI Act) and similar global laws Contains biosimilar antibody molecules approved under laws and regulations. Under the BPCI Act, antibodies are Despite slight differences in clinically inactive components, it is "very similar" to the reference product. Therefore, it is "expected" that clinical results equivalent to those of the standard product will be obtained in terms of safety, purity, and efficacy. If the data indicates that it is a biosimilar (Endocrine Practice: February 2018, Vol. 24, No. 2, pp. 195-204). These The Iosimira antibody molecule provides a shortened approval pathway, thereby allowing the applicant to obtain legal approval. To ensure approval, we rely on clinical data from the innovator's standard product. Clinical trial success Compared to the original Innovator Criteria antibody approved by the FDA based on biosimilarities, The antibody molecule is referred to herein as a "biosimilar." Based on the success of clinical trials, SIMPONI® (golimumab) has been approved by the FDA. It is an approved original innovator-reference anti-TNF antibody. Golimumab was developed in 2009. It is sold in the United States.

[0121] Example sequence In various embodiments, the TNF inhibitor is the anti-TNF antibody SIMPONI®. (Golimumab), or its antigen-binding fragment containing the sequence shown below. Anti-TNF antibody SI For further information regarding MPONI® (golimumab) and other anti-TNF antibodies, For example, U.S. Patent No. 7,250,165, No. 7,691,378, No. 7, No. 521,206, No. 7,815,909, No. 7,820,169, No. 8,2 Nos. 41,899, 8,603,778, 9,321,836, and 9, Please refer to issues 828 and 424.

[0122] Example sequence of an exemplary anti-TNF antibody - SIMPONI® (golimumab) Heavy-chain CDRs (HCDRs) and light-chain CDRs (LCDRs) are defined by Kabat. .

[0123] Amino acid sequence of the underlined golimumab heavy chain (HC) in CDR: (SEQ ID NO: 36)

[0124] JPEG2026153027000001.jpg43136

[0125] Amino acid sequence of the golimumab light chain (LC) underlined in CDR: (SEQ ID NO: 37)

[0126] JPEG2026153027000002.jpg22136

[0127] Amino acid sequence of golimumab variable heavy chain (VH) underlined in CDR: (SEQ ID NO: 38) )

[0128] JPEG2026153027000003.jpg19136

[0129] Amino acid sequence of golimumab variable light chain (VL) underlined in CDR: (SEQ ID NO: 39) )

[0130] JPEG2026153027000004.jpg13134

[0131] Amino acid sequence of complementarity-determining region 1 (HCDR1) of the golimumab heavy chain: (SEQ ID NO: 40) SYAMH

[0132] Amino acid sequence of complementarity-determining region 2 (HCDR2) of the golimumab antibody heavy chain: (SEQ ID NO: 4) 1) FMSYDGSNKKYADSVKG

[0133] Amino acid sequence of complementarity-determining region 3 (HCDR3) of the golimumab heavy chain: (SEQ ID NO: 42) DRGIAAGGNYYYYGMDV

[0134] Amino acid sequence of complementarity-determining region 1 (LCDR1) of the golimumab light chain: (SEQ ID NO: 43) RASQSVYSYLA

[0135] Amino acid sequence of complementarity-determining region 2 (LCDR2) of the golimumab light chain: (SEQ ID NO: 44) DASNRAT

[0136] Amino acid sequence of complementarity-determining region 3 (LCDRL) of the golimumab light chain: (SEQ ID NO: 45) QQRSNWPPFT

[0137] The present invention comprises at least one antibody, at least one TNF protein, subunit , fragment, part, or any combination thereof, at least one specific epitome It binds to the epitope. This at least one epitope is at least part of this protein. It may include at least one antibody-binding region, and this epitope is preferably , at least one extracellular portion of this protein, soluble portion, hydrophilic portion, external portion, Alternatively, it is composed of cytoplasmic granular portions. At least one identified epitope is distributed At least 1 to 3 amino acids relative to the entire identified portion of the adjacent amino acid in column number 9 It can contain any combination of at least one amino acid sequence.

[0138] Generally, the human antibody or antigen-binding fragment of the present invention has at least one human complementarity-determining region. (CDR1, CDR2 and CDR3) or variants of at least one heavy chain variable region, and At least one human complementarity-determining region (CDR1, CDR2, and CDR3) or at least It also includes an antigen-binding region containing a variant of the light chain variable region. As a non-limiting example, an antibody or The antigen-binding portion or variant is a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 and / or may contain at least one of the light chain CDR3s having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment corresponds to CDR1, 2 and / or 3. At least one heavy chain having the amino acid sequence (e.g., SEQ ID NOs: 1, 2 and / or 3) Antivirus containing at least a portion of CDR (i.e., CDR1, CDR2 and / or CDR3) It may have a protobinding region. In another specific embodiment, the antibody or antigen binding portion Alternatively, the mutants may have the corresponding CDR1, 2, and / or 3 amino acid sequences (e.g., sequence number At least one light chain CDR having numbers 4, 5, and / or 6 (i.e., CDR1, It can have an antigen-binding region that includes at least a portion of CDR2 and / or CDR3. In a preferred embodiment, three heavy chain CDRs and three light chains of the antibody or antigen-binding fragment are used. The chain CDRs are described herein as mAb TNV148, TNV14, TNV15, At least one pair of TNV196, TNV118, TNV32, and TNV86 It has the amino acid sequence of the corresponding CDR. Such antibodies are based on conventional techniques related to recombinant DNA technology. Using this technique, nucleic acid molecules encoding antibodies (i.e., one or more) are prepared and developed. By making it apparent, or by using any other suitable method, the prior art can be made apparent. Using this method, various parts of the antibody (e.g., CDR, framework) are chemically bound together. It can be prepared by adding it.

[0139] Anti-TNF antibodies are derived from a small portion of the heavy chain or light chain variable region that has a defined amino acid sequence. It may contain at least one. For example, in a preferred embodiment, the anti-TNF antibody is A heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 optionally and / or a sequence as desired. At least one of the light chain variable regions having amino acid sequence number 8 Includes. Antibodies that bind to human TNF and contain a defined heavy chain or light chain variable region are preferred methods. For example, phages known in the art and / or described herein Display (Katsube, Y. et al., "Int J Mol.Med" Vol. 1, No. 5) 863-868 (1998)) or a suitable method such as employing transgenic animals. It can be prepared using the following methods. For example, functionally rearranged human immunoglobulins Human immunoglobulin light chain genes that can undergo heavy chain introduction and functional rearrangement. Transgenic mice containing an introduced gene with DNA from a translocus, and human TNF Alternatively, antibody production can be induced by immunization with the fragment. If desired, antibody production fragments Cells can be isolated as described herein and / or in the art. As is already known, prepare hybridomas or other immortalized antibody-producing cells. This can be done. Alternatively, the antibody, identified portion, or variant can encode in a suitable host cell. It can be expressed using nucleic acids or a portion thereof.

[0140] The present invention also provides an amino acid sequence that is substantially the same as the amino acid sequence described herein. This relates to antibodies containing ano acids, antigen-binding fragments, immunoglobulin chains, and CDRs. Preferably, Antibodies or antigen-binding fragments and antibodies containing such chains or CDRs have high affinity (for example) Ba, K D about 10 -9 It can bind to human TNF (M or less). (As described herein) Amino acid sequences that are substantially the same as the sequence being performed include conservative amino acid substitutions and Examples include sequences containing amino acid deletions and / or insertions. Conservative amino acid substitutions are the first A The amino acid possesses chemical and / or physical properties similar to those of the first amino acid (e.g., charge). This refers to substitution with a second amino acid that has the same properties (structure, polarity, hydrophobic / hydrophilic). Conservative Substitutions include replacing one amino acid with another amino acid from the following group: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate Sodium (E); asparagine (N), glutamine (Q), serine (S), threonine (T) ), tyrosine (Y), K, R, H, D, and E; alanine (A), valine (V), leucine ¹(L), isoleucine(I), proline(P), phenylalanine(F), tryptophosphate ammonium compounds (W), methionine (M), cysteine ​​(C), and glycine (G); F, W, and Y;C, S, and T.

[0141] Amino acid code. The amino acids constituting the anti-TNF antibody of this invention are often abbreviated. Amino acid notation consists of its single-letter code, its three-letter code, its name, or three nucleotides. This can be shown by representing amino acids using codons, and in the art, It is well understood (Alberts, B. et al., "Molecular Biology") "of The Cell," 3rd edition, Garland Publishing, Inc. See New York (1994).

[0142] [Table 1]

[0143] The anti-TNF antibodies of the present invention are, as specified herein, naturally occurring or manipulated by humans. This may include the substitution, deletion, or addition of one or more amino acids by any of the following:

[0144] Naturally, the number of amino acid substitutions that a person skilled in the art can perform is numerous, including those mentioned above. It depends on the factor. Generally, amino acids for any given anti-TNF antibody, fragment, or variant. The number of acid substitutions, insertions, or deletions is 40, 30, 20, or 19, as specified herein. , 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, for example, not exceeding 1 to 30, or any range or value within this range.

[0145] The amino acids in the anti-TNF antibody of the present invention, which are functionally essential, are site-directed mutagenesis or This can be identified by methods known in the relevant art, such as lanin scanning mutagenesis. This is possible (for example, Ausubel, Chapters 8 and 15 above; Cunningham and Wells, Science 244:1081-1085 (1989). The latter hand Next, a single alanine mutation is introduced into each residue within the molecule. Then, the result obtained The mutant molecules include, for example, at least one TNF neutralizing activity, but are not limited to this. The biological activity that is not detected is tested. Sites that are extremely important for antibody binding are also tested. It can be identified by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (S mith et al., “J.Mol.Biol.” Vol. 224, pp. 899-904 (1992) And de Vos et al., "Science," Vol. 255, pp. 306-312 (1992) ).

[0146] The anti-TNF antibody of the present invention is at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 6 At least one part, sequence, or combination selected from one to all of the adjacent amino acids. This may include, but is not limited to, combinations.

[0147] The anti-TNF antibody is further optionally adjacent to at least one of SEQ ID NOs. 7 and 8. It may contain at least one polypeptide comprising 70-100% of its amino acids.

[0148] In one embodiment, an immunoglobulin chain or a portion thereof (e.g., variable region, CDR) The amino acid sequence is the amino acid sequence of at least one corresponding chain from sequence numbers 7 and 8, Approximately 70-100% identity (for example, 70, 71, 72, 73, 74, 75, 76, 77) , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any of these. It has a range or value. For example, the amino acid sequence of the light chain variable region is the sequence of SEQ ID NO: 8. It can be compared with, or the amino acid sequence of heavy chain CDR3 can be compared with SEQ ID NO: 7. Yes, it is possible. Preferably, 70-100% amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range within these numbers The value) is obtained using a suitable computer algorithm, as is known in the art. It is determined by using it.

[0149] Exemplary arrangements of heavy and light chain variable regions are shown in Sequence IDs 7 and 8. The body or its specified variant may contain any number of adjacent amino acid residues from the antibody of the present invention. The number is selected from an integer group consisting of 10-100% of the number of adjacent residues in the anti-TNF antibody. Selected. Optionally, this subsequence of adjacent amino acids is at least approximately 10, 20, 3 0, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, An amino acid length of 250 or more, or any range or value within that range. Furthermore, such portion The number of sequences is selected from a group consisting of 1 to 20, such as at least 2, 3, 4, or 5. It can be any integer.

[0150] As will be apparent to those skilled in the art, the present invention includes at least one biological activity of the present invention It contains antibodies. Biologically active antibodies are natural (non-synthetic), endogenous, or related and pre-existing. The antibody of known substance shall be at least 20%, 30%, or 40%, and preferably at least 5 0%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%~1 It has a specific activity of 00%. The assay and quantitative measurement methods for enzyme activity and substrate specificity are as follows: This is well known to those skilled in the art.

[0151] In another aspect, the present invention is modified by covalent bonding of organic parts as described herein. This relates to human antibodies and antigen-binding fragments. Such modifications result in improved pharmacokinetic properties (e.g., It is possible to produce antibodies or antigen-binding fragments that have an increased serum half-life in vivo. The organic portion consists of linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups. It is possible. In certain embodiments, the hydrophilic polymer group has a molecular weight of about 800 to about 1 20,000 Daltons, and polyalkane glycol (e.g., polyethylene glycol) Polypropylene glycol (PEG), polypropylene glycol (PPG), carbohydrate polymer, amino acids It may be a polymer or polyvinylpyrrolidone, and the fatty acid group or fatty acid ester group is about 8~ It may contain approximately 40 carbon atoms.

[0152] The modified antibodies and antigen-binding fragments of the present invention are covalently bound to the antibody, either directly or indirectly. It may contain one or more organic moieties. It is bound to the antibody or antigen-binding fragment of the present invention. Each organic part is independently a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. Obtain. As used herein, the term "fatty acid" refers to monocarboxylic acids and dicarbic acid. It contains octa acids. When used herein, the term "hydrophilic polymer group" is used with respect to octa acids. This refers to organic polymers that have a higher solubility in water than ions. For example, polylysine is an organic polymer that has a higher solubility in water than ions. It has higher solubility in water than tan. Therefore, the antibody modified by the covalent bond of polylysine The body is included in the present invention. A hydrophilic polymer suitable for modifying the antibody of the present invention is linear. It may be in a symmetric or branched form, for example, polyalkane glycol (e.g., PEG, monomethyl (e.g., C-polyethylene glycol (mPEG), PPG), carbohydrates (e.g., dextrose) (e.g., orchids, cellulose, oligosaccharides, polysaccharides), hydrophilic amino acid polymers (e.g., polypropyl alcohol) Polyarginine, polyaspartic acid, etc.), polyalkane oxides (for example, poly Includes ethylene oxide, polypropylene oxide, etc., and polyvinylpyrrolidone. Preferably, the hydrophilic polymer that modifies the antibody of the present invention is as individual molecules, about 80 It has a molecular weight of 0 to approximately 150,000 daltons. For example, PEG. 5000 and PEG2 0,000 You can use the subscript, where the subscript represents the average molecular weight (Daltons) of the polymer. Yes, there are. The hydrophilic polymer group consists of 1 to about 6 alkyl groups, fatty acid groups, or fatty acid ester groups. Substitution is possible. Hydrophilic polymers substituted with fatty acids or fatty acid ester groups are It can be prepared by using a suitable method. For example, a poly containing an amine group Mer can be linked to a carboxylate salt of a fatty acid or fatty acid ester, and fatty acid or Activated carboxylate salts on fatty acid esters (for example, N,N-carbonyldiimidazole) The activated group can be linked to the hydroxyl group on the polymer.

[0153] Fatty acids and fatty acid esters suitable for modifying the antibodies of the present invention may be saturated. or may contain one or more unsaturated units. Suitable fatty acids include, for example, n-dodecanoate (C 12 (laurate), n-teto Radecanoate (C 14 (Myristic acid), n-octadecanoate (C 18 , stearin Salts), n-eicosane salts (C 20 , arachidinate), n-docosanate (C 22 , Behenic acid, n-triacontane (C 30 ), n-tetracontane (C 40 ), cis-Δ9-octadecanoate (C 18 (Oleate), all cis-Δ5,8,11 ,14-eicosatetraenoic acid (C 20 (Arachidone), octanedionic acid, tetraphosphate Examples include ladecanedionic acid, octadecanedionic acid, and docosanedionic acid. Fatty acid esters are monoesters of dicarboxylic acids containing a linear or branched lower alkyl group. It contains a lower alkyl group. The lower alkyl group may contain 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms. ru.

[0154] Modified human antibodies and antigen-binding fragments are preferably reacted with one or more modifying agents. It can be prepared using the following methods. When used herein, the term "modifier" is used. The term refers to a suitable organic group containing an activating group (e.g., hydrophilic polymers, fatty acids, fatty acid esters). This means "activating group". An "activating group" is a group that reacts with a second chemical group under appropriate conditions, thereby A chemical moiety or functional group capable of forming a covalent bond between the modifying agent and the second chemical group. Yes, there are. For example, amine-reactive activating groups include tosylates, mesylates, and halo(chloro, bro). Electrophilic groups such as mo, fluoro, and iodine, and N-hydroxysuccinimidyl esters (N Includes HS, etc. Examples of activating groups that can react with thiols include maleimide, yo Acryloyl acetyl, pyridyl disulfide, 5-thiol-2-nitrobenzoate Examples include acid thiols (TNB-thiols). The aldehyde functional group is amine- or It can be linked to hydrazide-containing molecules, and the azide group reacts with the trivalent phosphorus group to form a phosphorus group. It can form luamidate or phosphorimide bonds. Activating groups can be introduced into the molecule. A preferred method for doing so is known in the art (for example, Hermanso n,GT, “Bioconjugate Techniques”, Academy See Press: San Diego, CA (1996). Activating group This is directly applied to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters), or to phosphorus. The C portion, for example, divalent C1~C 12 A group (where one or more carbon atoms are oxygen, They can be bonded via (which may be substituted with heteroatoms such as nitrogen or sulfur). Suitable linker portions include, for example, tetraethylene glycol, -(CH2)3-, -NH-( CH2)6-NH-, -(CH2)2-NH- and -CH2-O-CH2-CH2-O- It contains CH2-CH2-O-CH-NH-. Modifiers containing the linker moiety include, for example, 1-eth In the presence of 3-(3-dimethylaminopropyl)carbodiimide (EDC), mono- Boc-alkyldiamines (e.g., mono-Boc-ethylenediamine, mono-Boc- By reacting diaminohexane with fatty acids, free amines and fatty acid carboxylates are produced. It can be produced by forming an amide bond between the Boc protecting group and the tort. Removed from the product by fluoroacetic acid (TFA) treatment, and separated into another carboxylate as described. A primary amine that can be coupled to the salt can be exposed, or this can be done with malean anhydride. The product is reacted with an acid, and the resulting product is cyclized to produce an activated maleimide derivative of a fatty acid. (For example, the entire teaching is incorporated herein by reference, Thomp See International Publication No. 92 / 16221 by Son et al.

[0155] The modified antibody of the present invention is obtained by reacting a human antibody or antigen-binding fragment with a modifying agent. It can be produced in this way. For example, the organic part is an amine-reactive modifier, such as PEG. By utilizing NHS esters, it is possible to bind antibodies in a non-site-specific manner. Yes, it is possible. The disulfide bonds of the antibody or antigen-binding fragment (e.g., intrachain disulfide bonds) Modified human antibodies or antigen-binding fragments can also be prepared by reduction. At this time, the reduced antibody or antigen-binding fragment is reacted with a thiol-reactive modifier to produce the present product. It is possible to produce antibodies with the specified modification. The antibody of the present invention binds to a specific site. Modified human antibodies and antigen-binding fragments containing organic moieties undergo reverse proteolysis (Fisc h et al., "Bioconjugate Chem." Vol. 3, pp. 147-153 (1992) (Year), Werlen et al., "Bioconjugate Chem." Vol. 5, pp. 411-44 Page 17 (1994), Kumaran et al., "Protein Sci." Vol. 6, No. 10 Issues 2233-2241 (1997); Itoh et al., "Bioorg.Chem." Vol. 2 Volume 4, Number 1, pp. 59-68 (1996), Capellas et al., "Biotechno "l.Bioeng." Vol. 56, No. 4, pp. 456-463 (1997) and Herm anson, GT, "Bioconjugate Techniques", Aca The method described in Demic Press: San Diego, CA (1996) It can be prepared using any suitable method.

[0156] Anti-idiotype antibodies against anti-TNF antibody compositions. Monoclonal or chimeric anti-TN In addition to the F antibody, the present invention provides an anti-idiotype (anti-Id) specific to such antibody. This also relates to antibodies. Anti-Id antibodies generally have unique determinants related to the antigen-binding region of other antibodies. It is an antibody that recognizes Id. Anti-Id is an antibody that recognizes animals of the same species and genotype as the Id antibody source (e.g., maw). The strain can be prepared by immunizing it with an antibody or its CDR-containing region. The immunized animal recognizes and responds to the idiotype determinant of the immunizing antibody, and the anti-Id It produces antibodies. Anti-Id antibodies can also be used as "immunogens" to induce an immune response in other animals. It can be used to produce so-called anti-anti-Id antibodies.

[0157] Anti-TNF antibody composition. The present invention is also described herein and / or in the art. As is known, at least a non-naturally occurring composition, mixture, or form provided 1, at least 2, at least 3, at least 4, at least 5, at least A composition containing at least one anti-TNF antibody, comprising six or more of its anti-TNF antibodies. Provided. Such compositions are the adjacent amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8. Selected from a group consisting of 70-100% or the identified fragment, domain, or variant thereof. The amino acid sequence of the anti-TNF antibody to be used has at least one or two full lengths, C and / or This is a non-naturally occurring composition including N-terminal deletion mutants, domains, fragments, or identified mutants thereof. Contains substances. Preferred anti-TNF antibody compositions are 70-1 of SEQ ID NOs: 1, 2, 3, 4, 5, 6. 00% anti-TNF antibody, or a specified fragment, domain or variant thereof, Also, as one CDR or LBR containing portion, at least one or two full-length, fragment, or dormant portions The composition includes the in or variants of the 7 SEQ ID NOs: 1, 2, 3, 4, 5, 6. 0-100%, or at least one of the identified fragments, domains, or variants thereof. It contains 40-99% of one of these. The percentages of such compositions are known in the art. As such, or as described herein, weight, volume, concentration, volume molar concentration, or Moles by weight as a liquid or dry solution, mixture, suspension, emulsion, or colloid. It depends on the concentration.

[0158] The anti-TNF antibody composition of the present invention further applies to cells requiring such regulation, treatment, or therapy. , comprising at least one anti-TNF antibody against tissue, organ, animal or patient, and optionally in addition, at least one TNF antagonist (e.g., TNF antibody or fragment, soluble TNF receptor) This includes the substance or fragments thereof, fusion proteins thereof, or small molecule TNF antagonists, etc. (and not limited to these), anti-rheumatic drugs (e.g., methotrexate, auranofin, etc.) Urothioglucose, azathiopurine, etanercept, sodium aurothiomalate, sulfur Hydroxychloroquine acid, leflunomide, sulfasalazine), muscle relaxants, anesthetics (narc otic, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedation Agents, local anesthetics, neuromuscular blockers, antibacterial agents (e.g., aminoglycosides, antifungal agents, anthelmintics) Drugs, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides (penicillin, sulfonamide, tetracycline, and other antibacterial agents), antipsoriatic agents, corticosteroids Thycosteroids, anabolic steroids, diabetes-related medications, minerals, nutritional supplements, thyroid medications Vitamins, calcium-related hormones, antidiarrheals, cough suppressants, antiemetics, anti-ulcer agents, laxatives, anti- Anticoagulants, erythropoietin (e.g., epoetin α), filgrastim (e.g., G- CSF (Neupogen), Salglamostim (GM-CSF, Leukine) prevention Inoculation, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, dacri). Zumab, growth hormone, hormone replacement therapy, estrogen receptor modulators, mydriatics, ciliary muscle Regulators, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanic drugs Antipsychotics, anxiolytics, hypnotics, sympathomimetic drugs, stimulants, donepezil, tacrine, asthma Breath medications, β-agonists, inhaled corticosteroids, leukotriene inhibitors, methylxanthines, chromoly N, epinephrine or similar drugs, dorunase α (Pulmozyme), cytokines or Any preferred and effective amount further comprising at least one selected from cytokine antagonists It may contain at least one of the composition or pharmaceutical composition. Such cytokine non A limited example would be any of IL-1 to IL-23, but it is not limited to these. It is not possible. Suitable dosages are well known in the art. For example, Wells et al., "Pharmacotherapy Handbook" 2nd Edition, Appleton nd Lange, Stamford, CT (2000), “PDR Pharmac. opoeia, Tarascon Pocket Pharmacopoeia 200 0” Special Edition, Tarascon Publishing, Loma Linda, CA ( See (2000), each of these is incorporated herein in whole by reference. ru.

[0159] Such anticancer agents or anti-infective agents are associated with, bind to, and the same at least one antibody of the present invention. This may include toxic molecules used in combination with or at other times. The toxins selectively kill diseased cells or tissues. It can be selectively controlled to cause a particular condition. Pathogenic cells are cancer cells or other cells. It can be a cell. Such toxins are not limited to these, but for example, ricin, A small amount of toxin selected from at least one of diphtheria toxin, snake toxin, or bacterial toxin A purified or recombinant toxin or toxin fragment containing at least one functional cytotoxic domain It is possible. The term toxin also refers to a toxin that can be fatal in humans and other mammals. Any naturally occurring, mutated, or recombinant bacteria that may cause any pathological condition, including shock. This includes both endotoxins and exotoxins produced by viruses. Such toxins include intestinal toxins. Tuberototoxigenic Escherichia coli (E. coli) heat-unstable enterotoxin (LT), heat-stable enterotoxin Kixin (ST), Shigella cytotoxin, Aeromonas enterotoxin, toxic shock Candidate toxin-1 (TSST-1), Staphylococcus enterotoxin A (SEA), B (SEB) Examples include ), or C(SEC), streptococcal enterotoxin, etc. This is not limited to enterotoxigenic Escherichia coli (ETEC). ), enterohemorrhagic Escherichia coli (E. coli) (e.g., strain serotype 0157: H7), Staphylococcus aureus (St aphylococcus) species (e.g., Staphylococcus aureus, Staphylococcus pyogenes) (e.g., *Lococcus aureus*), Shigella species (e.g., *Shigella dysentery*) IAE), Shigella flexneri, Shigella boydii and Shigella sonnei), Salmonella species (for example, intestinal bacteria) Bacillus subtilis, Salmonella cholera (Switzerland), Salmonella enteritidis, Clostridium erythrorhizon species (for example, Clostridium perfringens) Clostridium dificile, Clostridium botulism (Clostridium botulinum), Campylobacter species (e.g., Campylobacter) Campholobacter jejuni, Campylobacter phytus Helicobacter fetus), Helicobacter species (e.g., Helicobacter pyro Heliocbacter pylori, Aeromonas species (for example, Aeromonas- Aeromonas sobria, Aeromonas hydrophila ), Aeromonas caviae), Plesiomonas sigeroides ( Pleisomonas shigelloides, Yersinia enterocolitica, Vibrio (Vibrio) species (e.g., Vibrio cholerae, Vibrio parahemorrhagicus) (Vibrio parahemolyticus), Klebsiella species, Pseudomonas Examples include strains of *Streptococcus aeruginosa* and *Streptococcus* species, but are not limited to these. It's not possible. For example, Stein, ed., "INTERNAL MEDICINE" 3rd edition, chapter 1- 13 pages, Little, Brown and Co., Boston (1990), E Vans et al., eds., "Bacterial Infections of Humans: E "pidemiology and Control" 2nd edition, pp. 239-254, Ple num Medical Book Co., New York (1991), Man dell et al., “Principles and Practice of Infect "European Diseases," 3rd edition, Churchill Livingstone, New York (1990), edited by Berkow et al., "The Merck Manuscript" al” 16th edition, Merck and Co., Rahway, NJ (1992). Wood et al., "FEMS Microbiology Immunology" No. 76 Volume 121-134 (1991), Marrack et al., "Science" No. 248. See pages 705-711 of Volume 1990 (The contents of these documents are as follows: (The entirety is incorporated herein.)

[0160] The anti-TNF antibody compounds, compositions, or mixtures of the present invention may further include diluents, binders, stabilizers, and stimulants. These include, but are not limited to, amphetants, salts, lipophilic solvents, preservatives, and adjuvants; they are optional. It may contain at least one of the preferred excipients. Pharmaceutically acceptable excipients are preferred. Non-limiting examples and methods for preparing such sterilization solutions are well known in the art. For example, Gennaro's "Remington's Pharmaceutical" "Early Sciences," 18th edition, Mack Publishing Co. (Early Sciences) Examples include, but are not limited to, ton, PA) (1990). As is well known or as described herein, the administration of anti-TNF antibodies, fragments, or mutant compositions A pharmaceutically acceptable carrier suitable for the method of administration, solubility, and / or stability is selected on a daily basis. It is possible.

[0161] Useful pharmaceutical additives and excipients in this composition are not limited to those listed above, but include tan. Proteins, peptides, amino acids, lipids and carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides) and sugars including oligosaccharides, algitol, aldonic acid, esterified sugars and other derivative sugars, This also includes polysaccharides or sugar polymers, which may exist individually or in combination. , present in amounts of 1 to 99.99% by weight or volume, either alone or in combination. Exemplary proteins The additives include serum albumin such as human serum albumin (HSA), recombinant human albumin It contains methyl (rHA), gelatin, casein, etc. It is a representative example that can also function in buffering capacity. The amino acid / antibody components include alanine, glycine, arginine, betaine, and histidine. , glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, barium Examples include methyl phosphate, phenylalanine, and aspartame. Preferred amino acids One of the acids is glycine.

[0162] Suitable carbohydrate additives for use in the present invention include, for example, fructose and maltose. Monosaccharides such as sucrose, galactose, glucose, D-mannose, and sorbose, lactose Disaccharides such as sucrose, trehalose, cellobiose, raffinose, merezito Polysaccharides such as maltodextrin, dextran, starches, mannitol, xylamine Litol, maltitol, lactitol, xylitol, sorbitol (glucitol), Examples include algitols such as myo-inositol. Preferred carbohydrates for use in the present invention The additives are mannitol, trehalose, and raffinose.

[0163] The anti-TNF antibody composition may also contain a buffer or pH adjuster, typically an organic buffer. These are salts prepared from acids or bases. Typical buffering agents include citric acid and ascorbic acid. Organic acid salts such as acids, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, Examples include ris, tromethamine hydrochloride, or phosphate buffer. Preferred for use in this composition. Suitable buffering agents include organic acid salts such as citrate.

[0164] In addition, the anti-TNF antibody composition of the present invention contains polyvinylpyrrolidone, Ficol (polymer). - Sugars), dextroses (for example, 2-hydroxypropyl-β-cyclodextrin) (Cyclodextrin), polyethylene glycol, flavoring agent, antibacterial agent, sweetener, antioxidant Agents, antistatic agents, surfactants (e.g., "TWEEN20" and "TWEEN80") Polysorbates), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol) Includes polymer additives / additives such as terol and chelating agents (e.g., EDTA). obtain.

[0165] These and additionally suitable for use in anti-TNF antibodies, partial or mutant compositions according to the present invention. The known pharmaceutical additives and / or additives are known in the art, for example ``Remington: The Science&Practice of Pha'' rmacy", 19 th ed., Williams & Williams, (1995) , and "Physician's Desk Reference", 52 nd ed, Listed in Medical Economics, Montvale, NJ (1998) These disclosures are incorporated herein by reference in their entirety. Preferred carrier or The additive materials include carbohydrates (e.g., monosaccharides and algitols) and buffering agents (e.g., quae). It is an acid or polymer agent.

[0166] Formulation. As described above, the present invention preferably comprises physiological saline or a selected salt. A stable formulation that is a buffering agent, as well as a preservative solution and formulation containing a preservative, and pharmaceutical Pharmaceutical or veterinary use containing at least one anti-TNF antibody in a suitably acceptable formulation. The present invention provides a versatile preservative formulation suitable for the following purposes: The preservative formulation contains at least one fast-acting compound in an aqueous diluent. Knowledge of, that is, at least one phenol, m-cresol, p-cresol, o-c Resol, chlorocresol, benzyl alcohol, phenylmercury nitrate, phenoxyethanol Nol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate) Alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride , benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof It contains a preservative that is arbitrarily selected from the group consisting of substances known in the art. As such, 0.001 to 5%, or 0.001, 0.003, 0.005, 0.00 9, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0 4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value within those, but not limited to these. Any suitable concentration or mixture of any range or value within that range may be used. A non-limiting example would be a product without preservatives, containing 0.1-2% m-cresol (for example, 0 0.2%, 0.3%, 0.4%, 0.5%, 0.9%, 1.0%, approximately 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.0 0.01-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0 % of phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%) , 0.0005~1.0% alkylparabens (e.g., 0.00075, 0.0009 , 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.0 2, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0 Examples include 0.9%, 1.0%).

[0167] As described above, the present invention relates to a packaging material and a buffering agent optionally formulated in an aqueous diluent. A solution of at least one anti-TNF antibody containing a vi / or preservative We offer products containing Al and, this packaging material contains such solutions in 1, 2, 3, 4, 5, 6, and 9 , 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or more The invention includes a label indicating that it can be retained for a certain period of time. The present invention relates to a packaging material and freeze-dried A first vial containing at least one dried anti-TNF antibody, and a prescribed buffer or The product further includes a second vial containing an aqueous diluent for the preservative, and this packaging material is small At the very least, reconstitute one anti-TNF antibody with an aqueous diluent and maintain it for more than 24 hours. Includes a label instructing the patient to form a solution that can be used.

[0168] At least one anti-TNF antibody used in accordance with the present invention is one of those described herein. Or produced from mammalian cells or transgenic preparations known in the art It may be produced by recombinant means including the following, or it may be purified from other biological sources.

[0169] The range of at least one anti-TNF antibody contained in the product of the present invention is a wet / dry system In this case, the amount obtained during reconstitution will have a concentration in the range of approximately 1.0 μg / ml to approximately 1000 mg / ml. It is included in this, but it is also possible to work with lower and higher concentrations, and these concentrations are intended It depends on the delivery vehicle, for example, in the case of a solution formulation, transdermal patch, lung, transmucosa, This differs from osmotic or micropump methods.

[0170] Preferably, the aqueous diluent further optionally comprises a pharmaceutically acceptable preservative. Common preservatives include phenol, m-cresol, p-cresol, o-cresol, and chloroform. Locresol, benzyl alcohol, alkylparabens (methyl, ethyl, propyl, b) (e.g., tyl), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and This includes thimerosal or a mixture thereof, selected from the group. The concentration of the preservative used is sufficient to produce an antibacterial effect. Such a concentration is selected. This varies depending on the preservative used and can be easily determined by those skilled in the art.

[0171] Other additives, such as isotonic agents, buffers, antioxidants, and preservatives / enhancers, may be added optionally. And preferably it can be added to a diluent. An isotonic agent such as glycerin can be added to a known concentration. It is commonly used. Preferably, a physiologically tolerant buffer is added to improve the pH. It provides control. The formulation has a pH of about 4 to about 10, and preferably about 5 to about 9. The range, and most preferably a wide pH range such as about 6.0 to about 8.0, is targeted. This is possible. Preferably, the formulation of the present invention has a pH of about 6.8 to about 7.8. Suitable buffering agents include phosphate buffering agents, most preferably sodium phosphate, particularly phosphate Contains sodium chloride buffered saline (PBS).

[0172] Other additives, for example, Tween20 (polyoxyethylene (20) sorbitan monolauan). (Rate), Tween40 (Polyoxyethylene (20) Sorbitan Monopalmitate) Tween80 (polyoxyethylene (20) sorbitan monooleate), Plur onic F68 (polyoxyethylene polyoxypropylene block copolymer), and A pharmaceutically acceptable solubilizer such as PEG (polyethylene glycol), or polysorbate Rubate 20 or 80 or Poloxamer 184 or 188, Pluronic (Registered Trademark) Nonionic surfactants such as polyols, other block copolymers, and The addition of chelating agents such as EDTA and EGTA to the formulation or composition is optional. This can reduce aggregation. These additives can be used with pumps or to administer the formulation. This is particularly useful when plastic containers are used. Medicinally acceptable surfactants. The presence of this substance reduces the tendency for proteins to aggregate.

[0173] The formulation of the present invention comprises at least one anti-TNF antibody and phenol, m-cresol, p -Cresol, o-Cresol, Chlorocresol, Benzyl alcohol, Alkyl para Ben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzeth chloride From the group consisting of nium, sodium dehydroacetate, and thimerosal or mixtures thereof To be prepared by a process that includes mixing a selected preservative in an aqueous diluent. This is possible. Mixing at least one anti-TNF antibody with a preservative in an aqueous diluent is possible. This is carried out using dissolution and mixing procedures. To prepare a suitable formulation, for example, a buffer solution is used. A certain amount of at least one anti-TNF antibody in the solution, along with the desired concentration of protein and preservative. Combine with the desired preservative in a sufficient amount of buffer solution. Changes in this process The form will be recognizable to those skilled in the art. For example, the order of adding the components, additional additions. Whether or not a drug is used, the temperature and pH during preparation of the formulation are all related to the dosage concentration and means of administration used. It is a factor that can be optimized.

[0174] The claimed formulation is in the form of a clear solution, or water, a preservative and / or an excipient, Alternatively, a phosphate buffer and / or physiological saline, and the selected salt in an aqueous diluent. A lyophilized at least one anti-TNF compound is reconstituted in a second vial containing It can be provided to the patient as a dual vial containing a body vial. Single-solution vials or dual vials requiring reconstitution can be reused multiple times. It can meet the needs of one or more patient treatment cycles, and therefore, currently We can provide a more convenient treatment regimen than the one currently available.

[0175] The claimed product is useful for administration over a period of time ranging from immediate to 24 hours or more. Therefore, the product claimed by this invention provides significant benefits to patients. The formulation can be optionally safely stored at temperatures of approximately 2 to 40°C, and contains protein. It can retain its biological activity for a long period of time, therefore, on the packaging label, the solution is 6, 1 It may be retained and / or used for 2, 18, 24, 36, 48, 72, or 96 hours or more. It can be indicated. When using stored diluents, such labels should indicate up to 1 This may include use for up to 12 months, 6 months, 1.5 years, and / or 2 years.

[0176] The present invention provides a solution of at least one anti-TNF antibody, wherein at least one antibody is used as an aqueous diluent. It can be prepared by a process that includes mixing in. Mixing is a conventional method of dissolution and This is carried out using a mixing procedure. To prepare a suitable diluent, for example, water or a buffering agent is used. A certain amount of at least one antibody is stored in the desired concentration of protein, and optionally stored. Combine in an amount sufficient to provide an agent or buffer. Variations of this process are used in this industry. This will be recognized by the person(s). For example, the order in which the components are added, and the use of additional additives. None; the temperature and pH during formulation preparation are all optimized in relation to the dosage concentration and administration method used. It is a factor that can be used.

[0177] The claimed product is a clear solution or a second vial containing an aqueous diluent. Dual bags containing vials of at least one lyophilized anti-TNF antibody, which are returned. It can be provided to the patient as an vial. Single solution vial or requiring reconstitution. Both dual vials can be reused multiple times, and are suitable for single or multiple patient treatments. This can satisfy the crust and therefore provide a more convenient treatment regimen than currently available. do.

[0178] The claimed product is returned in a second vial containing a clear solution or an aqueous diluent. A dual vial containing at least one lyophilized anti-TNF antibody. By providing this to pharmacies, clinics, or other such institutions and facilities, It can be provided indirectly. In this case, the clear solution may be up to 1 liter or even less. The volume may be as described above, and a smaller amount of at least one antibody solution may be taken from this larger container. Dispense once or multiple times and transfer to a smaller vial, and then distribute to the customer at the pharmacy or clinic. It can be provided to the patient.

[0179] Approved devices including these single vial systems include BD® (Pen-type injector device), NOVOPEN (registered trademark) (Pen-type injector device Vice), AUTOPEN (registered trademark) (pen-type injector device), OPTIPE N (Registered Trademark) (Pen-type Injector Device), GENOTROPIN PEN (Registered Trademark) (Trademark) (Pen-type injector device), -HUMATROPEN (Registered Trademark) (Pen-type Injector device), BIOJECTOR (registered trademark) (pen-type injector device) ), Reco-Pen, Humaject, J-tip Needle-Free I For delivering solutions such as injectors, Intrajectors, and Medi-Jects. Pen-type injector devices are examples of devices that are manufactured or developed, for example, by: Becton Dickensen (Franklin Lakes, NJ, www (bectondickenson.com) Disetronic (Burgdorf, Switzerland, www.di setronic.com; Bioject,Portland,Oregon(www.bioject.com ), Weston Medical (Peterborough,UK,www.wes ton-medical.com) Medi-Ject Corp (Minneapolis, MN, www.medij Some products are manufactured or developed by ect.com.

[0180] Approved devices, including those using vials, include HUMTROPEN®. Cartridges for delivering reconstituted solutions, such as pen-type injector devices. One example is a pen-type injector system for reconstituting freeze-dried drugs within the body.

[0181] The product claimed herein includes packaging materials. The packaging materials are required by regulatory authorities. In addition to the information provided, the conditions under which the product can be used are also provided. The packaging material of the present invention is less Reconstitute at least one anti-TNF antibody with an aqueous diluent to form a solution, and leave it for 2 to 24 hours or longer. The instructions state that this solution should be used for two vial products, wet and dry, over a specified period. Provide to the patient. For single vial solution products, the label indicates that this solution will last for 2-24 hours. This indicates that it can be used over the above. The claimed product is for use in human pharmaceutical products. It is useful for [something].

[0182] The formulation of the present invention comprises at least one anti-TNF antibody and a selected buffer, preferably a bio-buffer. A process involving mixing a phosphate buffer containing saline solution or a selected salt. It can be prepared by mixing at least one antibody with a buffer in an aqueous diluent. This is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, A certain amount of at least one antibody in water or buffer, and a desired concentration of protein and buffer. Combine with the desired buffer in a sufficient amount of water to provide the desired result. Variations of this process This will be recognized by those skilled in the art. For example, the order of adding the constituent components, the additional additives Whether or not it is used, the temperature and pH during preparation of the formulation, all of these are related to the dosage concentration and means of administration used. These are factors that can be optimized.

[0183] The claimed stable or preservative formulation is a clear solution or an aqueous diluent containing a preservative. Alternatively, a small amount of lyophilized material is reconstituted in a second vial containing buffers and additives. It can be provided to the patient as a dual vial containing at least one vial of anti-TNF antibody. This is possible. Both single-solution vials and dual vials requiring reconstitution can be multiple. It can be reused multiple times and can serve one or more patient treatment cycles. This provides a more convenient treatment regimen than those currently available.

[0184] At least one antibody of any of the stable or preservative formulations or solutions described herein TNF antibodies are administered via SC or IM injection, transdermally, or transpulmonaryly, as is well known in the art. Transmucosal, implantable, osmotic pump, cartridge, micropump, or by those skilled in the art To administer to a subject in accordance with the present invention via various delivery methods, such as other known means. It is possible.

[0185] Therapeutic application. The present invention is known in the art or described herein. Using at least one biintegrin antibody of the present invention, cells, tissues, organs, Methods for modulating or treating at least one TNF-related disorder in animals or patients. provide.

[0186] This invention relates to obesity, immune-related diseases, cardiovascular diseases, infectious diseases, malignant diseases, or neurological diseases. Cells, tissues, organs, animals or patients, including but not limited to one of the following: The present invention also provides methods for modulating or treating at least one TNF-related disorder.

[0187] This invention relates to rheumatoid arthritis, juvenile, systemic juvenile rheumatoid arthritis, ankylosing spondylitis, and severe rheumatoid arthritis. Spondylitis straightens, gastric ulcer, seronegative arthropathy, osteoarthritis, inflammatory bowel disease, ulcerative colitis Systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis / uveitis / optic neuritis, Idiopathic pulmonary fibrosis, systemic vasculitis / Wegner's granulomatosis, sarcoidosis, orchitis / vas deferens Excision and repair surgery, allergic / atopic diseases, asthma, allergic rhinitis, dermatitis, allergies Gheeous contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplantation, organ transplant rejection, transplantation Uni-host disease, systemic inflammatory response syndrome, septic syndrome, Gram-positive bacterial sepsis, Gram-negative bacteria sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, urinary sepsis, meningococcemia, Trauma / bleeding, burns, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, alcoholic hepatitis Chronic inflammatory lesions, sarcoidosis, crohn's disease, sickle cell anemia, diabetes, nephropathy Rose syndrome, atopic diseases, hypersensitivity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis Endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease Thrombocytopenia, rejection of any organ or tissue graft, kidney transplant rejection, heart transplant rejection Rejection reactions, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant (BMT) rejection. Rejections, including skin allograft rejection, cartilage graft rejection, bone graft rejection, and small intestine graft rejection. Fetal thymus transplant rejection, parathyroid transplant rejection, xenograft rejection of any organ or tissue In response to allograft rejection, antireceptor hyperreaction, Graves' disease, Raynaud's disease, and type B insulin. Resistant diabetes, asthma, myasthenia gravis, antibody-mediated cytotoxicity, type III hypersensitivity reaction, systemic Lupus erythematosus, POEMS syndrome (multiple neuropathy, organ enlargement, endocrine disorder, monochlorosis) (Synthetic immunoglobulinemia and cutaneous symptom syndrome), polyneuropathy, organ enlargement, endocrine disorders Disorders, monoclonal immunoglobulinemia, skin symptom syndromes, antiphospholipid syndromes, pemphigus, Scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary biliary tract disease Juicy cirrhosis, vitiligo, vasculitis, post-MI cardiac surgery syndrome, type IV hypersensitivity, contact dermatitis, hyper Sensitivity pneumonitis, allograft rejection, intracellular granuloma, drug hypersensitivity, metabolic / idiopathic virus Luzon disease, hemacromatosis, alpha-1-antitrypsin deficiency, diabetic retinopathy, pons This thyroiditis, osteoporosis, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis Syndrome, neonatal chronic lung disease, chronic obstructive pulmonary disease (COPD), familial hematopoietic lymphohistiocytosis Hyperplasia, dermatological conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerulonephritis, acute Renal failure, hemodialysis, uremia, toxicity, pre-eclampsia, OKT3 therapy, anti-CD3 therapy, cytotoxicity Radiation therapy, chemotherapy, and radiation therapy (for example, asthenia, anemia, cachexia, etc.) (and not limited to these), at least one of the following is a possible cause: chronic salicylate poisoning, etc. Not limited to, at least one immune-related disease in cells, tissues, organs, animals or patients Methods for controlling or treating the disease are also provided. For example, in the "Merck Manual" 12th to 17th editions, Merck & Company, Rahway, NJ (1972, 1 977, 1982, 1987, 1992, 1999), “Pharmacoth "Erapy Handbook," edited by Wells et al., 2nd edition, Appleton and Please refer to Lange, Stamford, Conn. (1998, 2000). Each element is incorporated by referencing the others.

[0188] This invention relates to cardiac stun syndrome, myocardial infarction, and congestive heart failure. Stroke, ischemic attack, bleeding, arteriosclerosis, atherosclerosis, restenosis, diabetic artery Sclerosing diseases, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, cardiovascular syphilis Heart failure, cor pulmonale, primary pulmonary hypertension, arrhythmia, atrial ectopic beat, atrial flutter, atrial fibrillation (sustainable) (Continuing or paroxysmal), post-perfusion syndrome, cardiopulmonary bypass inflammatory response, disorganized or multifocal atrial tachycardia Regular narrow QRS tachycardia, specific arrhythmias Hythmias, ventricular fibrillation, His bundle arrhythmias, atrioventricular block, bundle branch block Locke, myocardial ischemic disease, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy Cardiac disease, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumors, aortic aneurysms and peripheral arteries Aneurysm, aortic incision, inflammation of the aorta, occlusion of the abdominal aorta and its branches, peripheral vascular disease, occlusive Arterial disorders, peripheral atherosclerosis, thromboangiitis obliterans, functional peripheral artery disease, Inno's phenomenon and related diseases, acrocyanosis, erythromelopathy, venous diseases, venous thrombosis, varicose veins, movement and activity Pulsation fistula, lymphedema, fatty edema, unstable angina, reperfusion injury, post-pump syndrome. P syndrome, ischemia-reperfusion injury, etc., including but not limited to at least one of these. i. Regulating or controlling at least one cardiovascular disease in cells, tissues, organs, animals, or patients We also provide methods for treatment. Such methods include at least one anti-TNF antibody. The effective composition or pharmaceutical composition is used to treat cells, tissues, etc. that require such regulation, treatment, or therapy. This may optionally include administering the drug to organs, animals, or patients.

[0189] This invention relates to acute or chronic bacterial infections, including bacterial, viral, and fungal infections. Raw or infectious processes, HIV infection / HIV neurological disorders, meningitis, hepatitis (A, B, or C, etc.) Septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli O157:H7, hemolytic uremic disease Symptoms / embolic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy Mycobacteria, toxic shock syndrome, streptococcal myositis, gas gangrene, tuberculosis bacteria, Mycobacterium Avium intracellulare, Pneumocystis carinii pneumonia, pelvic inflammatory disease, sperm Epididymitis, Legionella, Lyme disease, influenza A, Epstein-Barritis Among the following, fewer than a few are: Viruses, virus-associated hemophagocytic syndrome, viral encephalitis / aseptic meningitis, etc. A small number of cells, tissues, organs, animals or patients, including but not limited to one of these. It also provides methods for controlling or treating at least one infectious disease.

[0190] This invention relates to leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B cells, T cells, or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic leukemia lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndromes Syndromes / hypercalcemia, solid tumors, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic diseases This includes, but is not limited to, at least one of the following: cancer-related bone resorption, cancer-related bone pain, etc. It does not regulate or cure at least one malignant disease in cells, tissues, organs, animals, or patients. We also provide methods for treatment.

[0191] This invention relates to neurodegenerative diseases, multiple sclerosis, migraines, AIDS-related dementia syndrome, demyelinating diseases, for example. For example, multiple sclerosis and acute transverse myelitis, extrapyramidal and cerebellar disorders, such as the corticospinal system Lesions, basal ganglia disorders or cerebellar disorders, hyperactivity disorders, for example, Huntington's dance Diseases and senile chorea, drug-induced movement disorders, for example, by blocking CNS dopamine receptors. Drug-induced and hypokinetic motor disorders, such as Parkinson's disease and progressive supranuclear dysplasia. Paralysis, structural lesions of the cerebellum, spinocerebellar degeneration, for example, spinal ataxia, Friedreich Ataxia, cerebellar degeneration, multiple system degeneration (Mencel, Dejerine-Thomas) s, Shi-Drager and Machado-Joseph), systemic diseases (refsm) Diseases, abetalipoproteinemia, ataxia, telangiectasia, and mitochondrial multiple system disorder (harm), demyelinating core disorder, for example, multiple sclerosis, acute Transverse myelitis and motor unit disorders, such as neurogenic muscular atrophy (anterior horn cell degeneration, for example, Amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy, and juvenile spinal muscular atrophy, Alzheimer's disease Diseases, Down syndrome in middle-aged individuals, diffuse Lewy body disease, Lewy body type senile dementia, Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakob disease, sub Among acute sclerosing panencephalitis, Harerforden-Spats disease, and Boxer's dementia, In cells, tissues, organs, animals, or patients, including but not limited to at least one of the following: The invention also provides methods for modulating or treating at least one neurological disorder. an effective amount of a composition containing at least one TNF antibody or a specified part or variant. This pharmaceutical composition is intended for use in cells, tissues, organs, and animals that require such regulation, treatment, or therapy. The process may include administering a substance or a patient. For example, "Merck Manual 16th Edition, Merck & Company, Rahway, NJ (1992). That.

[0192] Any method of the present invention relates to cells, tissues, etc. that require such regulation, treatment, or therapy. A composition or pharmaceutical combination containing at least one anti-TNF antibody in an effective amount to an organ, animal, or patient. This may include administering the substance. Such methods may optionally be used to treat such immune diseases. This may further include simultaneous administration or combination therapy for at least one anti-TNF antibody, specified Administration of the modified portion or its variant is contraindicated with at least one TNF antagonist (e.g., TNF antibody Alternatively, fragments, soluble TNF receptors or fragments, their fusion proteins, or small molecule TN F antagonists, etc., but not limited to these, antirheumatic drugs (e.g., methotrexate) Auranofin, Aurothioglucose, Azathioprine, Etanercept, Gold Sodium olmalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine) Muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, Local anesthetics, neuromuscular blocking agents, antibacterial agents (e.g., aminoglycosides, antifungal agents, anthelmintic agents, antimicrobial agents) Antiviral drugs, carbapenams, cephalosporins, fluoroquinolones, macrolides, penicillins Sylin, sulfonamides, tetracyclines, and other antibacterial agents), antipsoriatics, corticos Steroids, anabolic steroids, diabetes-related medications, minerals, nutritional supplements, thyroid medications, vitamins Min, calcium-related hormones, antidiarrheals, cough suppressants, antiemetics, anti-ulcer agents, laxatives, anticoagulants , erythropoietin (e.g., epoetin α), filgrastim (e.g., G-CSF) Neupogen, salglamostim (GM-CSF, Leukine), immunosuppressants Immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, dacrizuma) (B), growth hormone, hormone replacement drugs, estrogen receptor modulators, mydriatics, ciliary muscle paralysis Drugs, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanic drugs, anti Psychotic drugs, anti-anxiety drugs, hypnotics, sympathomimetic drugs, stimulants, donepezil, tacrine, asthma drugs β-agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, Epinephrine or its analogues, dorunase α (Pulmozyme), cytokines or Or, before administering at least one selected from cytokine antagonists, and / Or, further, administering it at a later date. Preferred dosages are well known in the art. For example, Wells et al., "Pharmacotherapy Handbook," Vol. 2 Edition, Appleton and Lange, Stamford, CT (2000), “PDR Pharmacopoeia, Tarascon Pocket Pharm acopoeia 2000” special edition, Tarascon Publishing, Lo Please refer to ma Linda, CA (2000), and each of these references is entirely... The body is incorporated into this specification.

[0193] Compositions, combination therapies, simultaneous administrations, devices and / or methods of the present invention (at least one of the present inventions) A suitable TNF antagonist (further comprising one antibody, a specified portion thereof, and its variants) is: Anti-TNF antibody, its antigen-binding fragment, and receptor molecule that specifically binds to TNF, TNF compound Compounds that inhibit and / or block TNF release or its effects on target cells. For example, thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxif) (Ylin and Lolipram), A2b adenosine receptor agonists and A2b adenosine receptor e Compounds that block and / or inhibit TNF receptor signaling, for example, mites Gen-activated protein (MAP) kinase inhibitors block and / or inhibit membrane TNF cleavage. Compounds that do this, for example, metalloproteinase inhibitors, block and / or inhibit TNF activity. Compounds such as angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril) ), as well as compounds that block and / or inhibit TNF production and / or synthesis, for example, MAP This includes, but is not limited to, kinase inhibitors.

[0194] When used herein, the terms "tumor necrosis factor antibody," "TNF antibody," and "TNFα antibody" are used. " or "fragments," etc., are prepared in vitro, in situ, and / or preferably in vivo, TN It reduces, blocks, inhibits, suppresses, or interferes with Fα activity. For example, a suitable human TNF inhibitor of the present invention The body can bind to TNFα, anti-TNF antibodies, their antigen-binding fragments, and TNFα It contains identified variants or their domains that specifically bind to a suitable TNF antibody or decompression agent. One of the components is TNF RNA, DNA, or protein synthesis, TNF release, and TNF receptors. Reduce, block, or suppress Gunal signaling, membrane TNF cleavage, TNF activity, TNF production, and / or synthesis. It can also be stopped, interfered with, blocked, and / or inhibited.

[0195] The chimeric antibody cA2 is a high-affinity neutralizing mouse anti-human TNFαIgG1 antibody called A2. It consists of the antigen-binding variable region and the constant region of human IgG1 κ immunoglobulin. The G1 Fc region improves the effector function of analogous antibodies and increases their circulating serum half-life. It reduces the immunogenicity of the antibody. The binding activity and epitope specificity of the chimeric antibody cA2 are It originates from the variable region of mouse antibody A2. In a particular embodiment, the variable region of mouse antibody A2 A preferred source of nucleic acids encoding the region is the A2 hybridoma cell line.

[0196] Chimeric A2 (cA2) utilizes the cytotoxic effects of both natural and recombinant human TNFα. It neutralizes in a dose-dependent manner. From a binding assay between the chimeric antibody cA2 and recombinant human TNFα, The affinity constant of the Mela antibody cA2 is 1.04x10 10 M -1 It was calculated that this is the case. A preferred method for determining the specificity and affinity of monoclonal antibodies due to harm is Ha Rlow et al., "antibodies: A Laboratory Manual", C old Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988), edited by Colligan et al. , “Current Protocols in Immunology”, Green e Publishing Assoc.and Wiley Interscience e, New York (1992-2000), Kozbor et al., "Immunol." Today, Vol. 4, pp. 72-79 (1983), edited by Ausubel et al., "Curre nt Protocols in Molecular Biology” Wiley Interscience, New York (1987–2000), and Mull Seen in "Meth.Enzymol." Vol. 92, pp. 589-601 (1983). These references are incorporated herein in their entirety by reference.

[0197] In certain embodiments, mouse monoclonal antibody A2 is a subset called c134A. It is produced by cell lines. The chimeric antibody cA2 is produced by a cell line called c168A. To be born.

[0198] Further examples of monoclonal anti-TNF antibodies that can be used in the present invention are As described in the technical field (e.g., U.S. Patent No. 5,231,024, Mol er, A. et al., "Cytokine" Vol. 2, No. 3, pp. 162-169 (1990); USA Application No. 07 / 943,852 (filed September 11, 1992), Rathjen et al., International Publication No. 91 / 02078 (published February 21, 1991), Rubin et al., EPO Patent Publication Patent Publication No. 0218868 (Published April 22, 1987), Yone et al., EPO Patent Publication Issue No. 0288088 (October 26, 1988), Liang et al., "Biochem .Biophys.Res.Comm.” Vol. 137, pp. 847-854 (1986) Meager et al., "Hybridoma," Vol. 6, pp. 305-311 (1987). Fendly et al., "Hybridoma," Vol. 6, pp. 359-369 (1987), B Ringman et al., Hybridoma, Vol. 6, pp. 489-507 (1987) and Bi Hirai et al., "J.Immunol.Meth. Vol. 96, pp. 57-62 (1987) See (year). These references are incorporated herein by reference in their entirety.

[0199] TNF receptor molecules. Preferred TNF receptor molecules useful in the present invention have a high affinity for TNFα. They are bound together by sex (for example, Feldmann et al., International Publication No. 92 / 07076 (1992) (Published April 30), Schall et al., "Cell" Vol. 61, pp. 361-370 (199 0 years), and Loetscher et al., "Cell" Vol. 61, pp. 351-359 (199 See (Year 0). These references are incorporated entirely herein by reference. , it has selectively low immunogenicity. In particular, 55kDa(p55 TNF- In the present invention, the TNF cell surface receptors R) and 75kDa (p75 TNF-R) are used. Useful. These receptors include the extracellular domain (ECD) or its functional portion. Body amputation type (for example, Corcoran et al., "Eur.J.Biochem." No. 223) See also pages 831-840 of Volume 1994. EC TNF receptor cleavage forms including D are present in urine and serum, and 30kDa and 40kDa TNF receptors. It has been detected as an α-inhibitory binding protein (Engelmann, H. et al., "J. Bi"). "ol.Chem." Vol. 265, pp. 1531-1536 (1990). TNF receptor Multimeric molecules and TNF immune receptor fusion molecules, as well as their derivatives and fragments or portions, Further examples of TNF receptor molecules useful in the methods and compositions of the present invention. TNF receptor molecules that can be used provide symptom relief ranging from good to excellent and have low toxicity. It is characterized by its ability to treat patients over a long period of time. Low immunogenicity and / or high affinity and other The undefined properties of this may contribute to the resulting treatment outcomes.

[0200] In this invention, the TNF receptor multimer molecule useful is polyethylene glycol (PEG). via one or more polypeptide linkers or other non-peptide linkers, such as The multimeric molecule contains all or the functional portion of the ECD of two or more linked TNF receptors. , further containing signal peptides of secreted proteins to bring about the expression of multimeric molecules This can be done. These polymer molecules and methods for producing them are described in U.S. Patent Application No. 08 / 437,53 It is described in application No. 3 (filed May 9, 1995), and its contents are described in their entirety by reference. It will be included in the detailed specifications.

[0201] The TNF immune receptor fusion molecules useful in the methods and compositions of the present invention are one or two or more. At least one portion of the immunoglobulin molecule and one or more TNF receptors All or functional parts are included. These immunoreceptor fusion molecules are monomers or heteromorphs. They can be assembled as homomultimers. Immune receptor fusion molecules are polyvalent even if they are monovalent. This may also be the case. An example of such a TNF-immune receptor fusion molecule is a TNF receptor / IgG fusion molecule. It is an protein. TNF immune receptor fusion molecules and methods for producing them are in the field of the said technology. (Lesslauer et al., "Eur.J.Immunol." Vol. 21) Volume 2883-2886 (1991), Ashkenazi et al., "Proc. Nat "Acad.Sci.USA," Volume 88, pp. 10535-10539 (1991); Peppel et al., "J.Exp.Med." Vol. 174, pp. 1483-1489 (199 1st year), Kolls et al., "Proc.Natl.Acad.Sci.USA" Vol. 91 Pages 215-219 (1994), Butler et al., "Cytokine" Vol. 6, No. 6. Pages 616-623 (1994), Baker et al., "Eur. J. Immunol." Volume 24, pp. 2040-2048 (1994), Beutler et al., U.S. 5,4 Patent No. 47,851 and U.S. application No. 08 / 442,133 (filed May 16, 1995), Each of these references is incorporated herein in whole by reference. The method for producing the composite molecule is described by Capon et al., U.S. Patent No. 5,116,964, Capon et al. U.S. Patent No. 5,225,538 and Capon et al., Nature, Vol. 337, No. 52 It can also be found on pages 5-531 (1989), and these references are all included by reference. This specification is incorporated herein.

[0202] Functional equivalents, derivatives, fragments, or regions of the TNF receptor molecule are to be used in the present invention. It is of sufficient size and sequence to be functionally similar to the TNF receptor molecule that can perform this function. For example, TNF receptor molecules that bind to TNFα with high affinity and have low immunogenicity. This refers to a portion, or the part of the TNF receptor molecular sequence that encodes the TNF receptor molecule. Functional equivalents of receptor molecules also function as TNF receptor molecules that can be used in the present invention. Modified substances that are similar in nature (for example, those that bind to TNFα with high affinity and have low immunogenicity) It also includes TNF receptor molecules. For example, a functional equivalent of a TNF receptor molecule is "SILEN A "T" codon, or one or more amino acid substitutions, deletions, or additions (e.g., one Use one acidic amino acid in place of another acidic amino acid, or use the same or a different hydrophobic amino acid. One codon that codes for an amino acid is replaced by another codon that codes for a hydrophobic amino acid. It may contain (used in) Ausubel et al., "Current Protocols in Molecular Biology”, Greene Publishing Assoc.and Wiley-Interscience,New York(19 See (1987-2000).

[0203] The term "cytokine" encompasses any known cytokine. For example, Copewith See Cytokines.com. Any antibody can be used as a cytokine antagonist. , fragments or mimetic drugs, any soluble receptor, fragments or mimetic drugs, any small molecule antagonist Examples include, but are not limited to, any combination thereof.

[0204] Treatment by therapy. Any method of the present invention requires such adjustment, treatment or therapy. A composition containing at least one anti-TNF antibody in an effective amount for cells, tissues, organs, animals, or patients. Methods for treating TNF-mediated disorders may include administering a substance or pharmaceutical composition. Such methods may be used as optional concurrent or combination therapies for the treatment of such immune diseases. The present invention may further include at least one anti-TNF antibody, an identified portion or a variant thereof, and the administration of this antibody. The drug contains at least one TNF antagonist (e.g., TNF antibody or fragment, soluble TNF) These include receptors or fragments, their fusion proteins, or small molecule TNF antagonists, etc. (and not limited to), anti-rheumatic drugs (e.g., methotrexate, auranofin, au) Rothioglucose, azathioprine, etanercept, sodium aurthiomalate, sulfuric acid Hydroxychloroquine, leflunomide, sulfasalazine), muscle relaxants, narcotics, non-steroidal drugs Non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers Antibacterial agents (e.g., aminoglycosides, antifungal agents, anthelmintics, antiviral agents, carbapenams) Cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, Tetracyclines (other antibacterial agents), antipsoriasis agents, corticosteroids, anabolic steroids Idoids, diabetes-related medications, minerals, nutritional supplements, thyroid medications, vitamins, calcium-related hormones Antidiarrheal drugs, cough suppressants, antiemetics, anti-ulcer agents, laxatives, anticoagulants, erythropoietin (for example, Epoetin α), filgrastim (e.g., G-CSF, Neupogen), salg Ramotim (GM-CSF, Leukine), vaccination, immunoglobulin, immunosuppressants (For example, basiliximab, cyclosporine, daclizumab), growth hormone, hormone Replacement drugs, estrogen receptor modulators, mydriatics, ciliary muscle paralyzers, alkylating agents, antimetabolites, Mitosis inhibitors, radiopharmaceuticals, antidepressants, antimanic drugs, antipsychotics, anxiolytics, hypnotics, Sympathomimetic drugs, stimulants, donepezil, tacrine, asthma medications, beta-agonists, inhaled corticosteroids, Leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or analogues, Select from dornease α (Pulmozyme), cytokines, or cytokine antagonists. The further includes administering simultaneously, before, and / or after, at least one of the selected treatments.

[0205] As used herein, the term “safe” means that it is the anti-TNF antibody of the present invention (e.g., anti- In the case of compositions, doses, administration regimens, treatments or methods involving the TNF antibody (golimumab). , compared to standard care or other comparator drugs such as other anti-TNF agents for favorable risk. Adverse events (AEs) and serious adverse events (SAs) E) refers to the benefit ratio based on the acceptable frequency and / or acceptable severity. Adverse events are defined as: This refers to an undesirable medical event in a patient who has been administered a drug. Specifically, this Safety of compositions, dosages, administration regimens, treatments or methods using Akira's anti-TNF antibodies These include, for example, infusion reactions, abnormal liver and biliary tract test results, infections including TB, and adverse events including malignancies. This refers to the acceptable frequency and / or acceptable severity of [the condition].

[0206] When used herein in the context of compositions, dosages, administration regimens, treatments or methods The terms "effect" and "effective" refer to the anti-TNF antibody of the present invention (e.g., anti-TNF antibody). This refers to the effectiveness of a specific composition, dose, application, treatment, or method using golimumab. Sex can be measured based on changes during the course of the disease in response to the drug of the present invention. For example, The anti-TNF antibody of the present invention provides at least one indicator that reflects the severity of the disorder being treated. In order to produce improvement, preferably sustained improvement, in the patient, administer a sufficient amount and time to the patient. To determine whether the amount and duration of the treatment are sufficient, the disease in question, Various indicators that reflect the severity of a disease or condition can be evaluated. Such indicators include, for example, disease Clinically recognized information regarding the severity of the condition, symptoms, or manifestation of the disorder in question. Indicators are included. The degree of improvement is generally determined by a physician or other appropriately trained individual. They are determined to have signs, symptoms, biopsy or other test results indicating improvement in clinical symptoms, or disease It can be determined based on any other measure of disease activity. For example, the anti-TNF antibody of the present invention is young Patients with juvenile idiopathic arthritis (JIA), particularly polyarticular juvenile idiopathic arthritis (pJIA) It can be administered to improve the patient's condition. For the treatment of JIA and / or pJIA. The effectiveness in patients meeting the criteria for inactive disease, such as JIA ACR30 and JI A selected from ACR50, JIA ACR70, and / or JIA ACR90 Shows improvement from baseline corresponding to the IA American College of Rheumatology (JIA ACR) response. Patients who are selected from JADAS10, JADAS27, and / or JADAS71 A decrease from baseline in the selected Juvenile Arthritis Disease Activity Score (JADAS) This can be determined by the patient.

[0207] When used herein, unless otherwise specified, the term “clinically proven” (independently) (or used to modify the terms “safety” and / or “effectiveness”) , meeting the approval standards of the U.S. Food and Drug Administration, EMEA, or the corresponding national regulatory agency. This means that it has been proven by a bed test. For example, a clinical trial is a drug test. A randomized, double-blind trial of an appropriate size was used to clinically demonstrate the effect. That's fine.

[0208] Typically, the treatment of the disease is, on average, 1 in total, depending on the specific activity contained in the composition. The dosage per dose ranges from at least approximately 0.01 to 500 milligrams per kg of patient body weight. At least one anti-TNF antibody, preferably 1 / 1 of the patient's body weight per single or multiple doses. At least one antibody in the range of approximately 0.1 to 100 milligrams per kg This is achieved by administering a safe and effective amount or dose of the TNF antibody composition. In other words, the effective serum concentration is 0.1 to 5000 μg / mL of blood per single or multiple doses. It may contain a high concentration. The appropriate dosage is known to medical practitioners, and naturally, It depends on the physical disease state, the specific activity of the composition administered, and the specific patient being treated. In some cases, repeated administration, i.e., specific monitoring, may be required to obtain the desired therapeutic dose. It may be necessary to provide repeated individual doses of the specified amount or quantity, in which case, The separate administration is repeated until the desired daily dose or effect is achieved.

[0209] Preferred doses are, optionally, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9 4, 95, 96, 97, 98, 99 and / or 100-500 mg / kg / administration, It includes any range, value, or fraction, or 0 per single or multiple doses. 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2 0.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6 0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9 0.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5 , 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 15, 15.5, 1 5.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18 0.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 60 0, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3 Serum concentrations of 500, 4000, 4500 and / or 5000 μg / mL, or as appropriate. It may include obtaining a range, value, or fraction of intent.

[0210] Alternatively, the dosage administered depends on the pharmacodynamic characteristics of the specific drug, as well as its method and route of administration. The recipient's age, health status and weight, nature and severity of symptoms, type of concurrent treatment, and treatment The dosage of the active ingredient can vary depending on known factors such as frequency and the desired effect. The dosage can be approximately 0.1 to 100 milligrams per kilogram of body weight. Typically, 1 dose per administration. 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram, or in a sustained-release form. It is effective in obtaining the desired results.

[0211] As a non-limiting example, the treatment of a human or animal may be performed using a single dose, infusion, or repeated dose. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, The least of the 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, or 40th day At least one day, or additionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 5 In at least one week of week 1 or week 52, or additionally, week 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Or, in at least one year during the 20th year, or any combination thereof, per day 0.1 to 100 mg / kg, for example, 0.5, 0.9, 1.0, 1.1, 1.5, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 , 45, 50, 60, 70, 80, 90, or 100 mg / kg, at least the present invention It may be provided as a single or periodic dose of one antibody.

[0212] A dosage form (composition) suitable for internal administration is generally about 0.1 milligrams per unit or container. It contains approximately 500 milligrams of active ingredient. In these pharmaceutical compositions, the active ingredient It is typically present in an amount of approximately 0.5 to 99.999% by weight, based on the total weight of the composition.

[0213] For parenteral administration, the antibody is administered either in combination with a pharmaceutically acceptable parenteral vehicle or separately. It can be formulated as a supplied solution, suspension, emulsion, or lyophilized powder. To do. Examples of such vehicles include water, physiological saline, Ringer's solution, dextrose solution, and It is 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixative oils are used. It can also be used. The vehicle or freeze-dried powder is an additive that maintains isotonicity and chemical stability. Additives (for example, sodium chloride and mannitol for isotonicity, and for chemical stability) It may contain buffering agents and preservatives. The formulation may be dissolved by known or preferred techniques. To be infected with bacteria.

[0214] Suitable pharmaceutical carriers are described in Remington's, a standard reference text in this field. This information is included in the latest edition of Pharmaceutical Sciences, by A. Osol. It is being done.

[0215] Alternative administration. Administer a pharmaceutically effective amount of at least one anti-TNF antibody according to the present invention. To administer, the present invention allows the use of many known and developed administration methods. Although transpulmonary administration is used in the following description, other administration methods can be used in accordance with the present invention. And you may obtain a suitable result.

[0216] The TNF antibody of the present invention can be used in a carrier as a solution, emulsion, colloid, or suspension. or as a dry powder, by inhalation, or by the technical components described herein. Any of the various devices and methods suitable for administration by other known methods in the field It can be used to deliver.

[0217] Parenteral formulations and administration. Parenteral formulations typically contain sterile water or physiological saline as additives. Water, polyalkylene glycols such as polyethylene glycol, plant-derived oils, hydrogen It may contain naphthalene or the like. Aqueous or oily suspensions for injection are prepared according to known methods. It can be prepared by using an appropriate emulsifier or humectant and suspension agent. The injectable preparation is For example, non-toxic, parenterally administered diluents such as aqueous solutions, sterile injection solutions, or suspensions in solvents. It may be so. Suitable vehicles or solvents include water, Ringer's solution, and isotonic saline. These are possible, and as a normal solvent or suspension solvent, a sterile non-volatile oil can be used. This can be done. For these purposes, natural, synthetic, or semi-synthetic fatty oils or fatty acids, natural or containing synthetic or semi-synthetic monoglycerides, diglycerides, or triglycerides. All kinds of non-volatile oils and fatty acids can be used. Parenteral administration is performed using this technology. Known in the field, conventional injection methods, described in U.S. Patent No. 5,851,198 Gas-pressurized needle-free injection devices such as those described in U.S. Patent No. 5,839,446. Examples of laser drilling devices include those described above, but are not limited to these. The entirety is incorporated herein by reference.

[0218] Alternative delivery. The present invention further includes parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, and intra-abdominal delivery. Intracapsule, intracartilage, intrasinus, intracavitary, cerebellum, ventricle, colon, cervical canal, stomach, liver, myocardium, Intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostate, intrapulmonary, intrarectum, intrakidney, intraretina, spinal Intramedullary, synovial sac, thoracic cavity, uterus, bladder, bolus, vagina, rectum, oral cavity, sublingual, nasal cavity The present invention relates to the administration of at least one anti-TNF antibody by internal or transdermal means. The anti-TNF antibody composition is administered parenterally (subcutaneously, intramuscularly, or intravenously) or by any other means. In particular, for use in the form of liquid solutions or suspensions, especially in creams and suppositories. There are, but are not limited to, semi-solid forms for use in vaginal or rectal administration. In the form of tablets or capsules, but not limited to these, administered orally or sublingually. For administration, or in the form of powder, nasal spray or aerosol, or certain drugs, etc. In forms not limited to these, it may be administered within the nasal cavity, or modified in a transdermal patch. Chemical catalysts such as dimethyl sulfoxide are used to either increase the concentration of the drug inside. Using a drug permeation agent (Junginger et al., In "Drug Permeation Enhancement") ancement”;Hsieh,DS,Eds.,pp.59-90(Marce l Dekker, Inc. New York 1994, the entire text is referred to herein by reference. (to be incorporated into) or to enable the application of formulations containing proteins and peptides to the skin. Using an oxidizing agent (International Publication No. 98 / 53847), or electroporation, etc. To create a transient transport pathway, or through the skin such as iontophoresis Application of an electric field to increase the mobility of charged drugs, or application of ultrasound such as ultrasonic introduction ( Using U.S. Patent Nos. 4,309,989 and 4,767,402, gels, ointments This includes, but is not limited to, lotions, suspensions, or patch delivery systems, transdermal delivery. Specifically, it can be prepared (the above publications and patents are incorporated in whole herein by reference). (To be absorbed).

[0219] Intrapulmonary / intranasal administration. For intrapulmonary administration, preferably at least one anti-TNF antibody. The composition is delivered with a particle size effective for reaching the lower airways or sinuses of the lungs. The present invention provides a small amount of At least one anti-TNF antibody is used in the art for administering therapeutic drugs by inhalation. It can be delivered by any of the known inhalation or intranasal devices. These devices allow for the application of aerosolized formulations into the sinus cavity or alveoli. Examples include metered-dose inhalers, nebulizers, dry powder generators, and sprayers. Antibodies are administered transpulmonaryly. Or other devices suitable for intranasal administration are known in the art. These devices all contain formulations suitable for administration to distribute antibodies in aerosols. It can be used. Such aerosols can be used in solutions (both aqueous and non-aqueous) or solids. It can be composed of any of the following body particles, such as VENTOLIN® (a metered-dose inhaler). A typical metered-dose inhaler uses a propellant gas and requires operation during inhalation (for example, in the country See International Publication No. 94 / 16970 and International Publication No. 98 / 35888. Turb Rotahaler (trademark) (Astra), Rotahaler (registered trademark) (Glaxo) DISKUS (registered trademark) (inhaler) (Glaxo), SPIROS (registered trademark) (inhaler) Dura, a device sold by Inhale Therapeutics. Dryness of Vice and Spinhaler® powder inhalers (Fisons), etc. Powder inhalers use exhalation action to dispense mixed powders (U.S. Patent No. 4668218 (Astr a) European Patent No. 237507 (Astra), International Publication No. 97 / 25086 (Gl axo), International Publication No. 94 / 08552 (Dura), U.S. Patent No. 5458135 ( Inhale), International Publication No. 94 / 06498 (Fisons), the entire text is available by reference. (To be incorporated into the specification). AERX® (Nebulizer) (Aradigm), U LTRAVENT® (Nebulizer) (Mallinckrodt), and Ac orn II Nebulizer (Marquest Medical Products) (USA) National Patent No. 5404871 (Aradigm), International Publication No. 97 / 22376) (above) Nebulizers such as (the literature is incorporated entirely herein by reference) use to extract aerosols from solutions. While some devices produce a sol, metered-dose inhalers and dry powder inhalers generate small-particle aerosols. These specific examples of commercially available inhalation devices are specific devices suitable for carrying out the present invention. It is intended to represent and not to limit the scope of the present invention. It is not. Preferably, the composition containing at least one anti-TNF antibody is a dry powder. It is delivered by an inhaler or sprayer. For administering at least one antibody of the present invention Inhalation devices have several desirable characteristics. For example, delivery by an inhalation device is It is advantageously reliable, reproducible, and accurate. The inhalation device is optional. To allow for proper breathing, for example, small particles less than about 10 μm, preferably about 1-5 μm. Dry particles can be delivered.

[0220] Administration of TNF antibody composition by spray. The spray containing the TNF antibody composition protein is ...to pass a nozzle under pressure through a suspension or solution of at least one anti-TNF antibody. This can be achieved by adjusting the nozzle size and configuration, the applied pressure, and the liquid supply. The speed can be selected to achieve the desired output and particle size. For example, capillary An electric spray can be produced by an electric field along with a nozzle feed. Furthermore, particles of at least one anti-TNF antibody composition protein delivered by the sprayer are , less than approximately 10 μm, preferably about 1 μm to about 5 μm, most preferably about 2 μm to about 3 μm It has a particle size within the range of [specify particle size].

[0221] Preparation of at least one anti-TNF antibody composition protein suitable for use with a sprayer. The agent is typically in an aqueous solution, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / mL or 100 mg / gm, but not limited to these, 1 ml or Or, approximately 0.1 mg to approximately 100 mg per 1 mg / gm solution, or any range within that range. Alternatively, the concentration of at least one anti-TNF antibody composition protein of the antibody composition protein This formulation contains additives, buffers, isotonic agents, preservatives, surfactants, and preferably This preparation may contain drugs such as zinc. This preparation may contain additives, reducing agents, bulk proteins or carbohydrates. The antibody composition may also include excipients or drugs to stabilize the antibody protein. Useful bulk proteins in formulating adult proteins include albumin and protamine. Examples include: Typical carbohydrates useful in the formulation of antibody composition proteins include: Examples include sucrose, mannitol, lactose, trehalose, and glucose. Antibody composition Protein preparations are formed by the atomization of the solution during aerosol formation, which causes the antibody composition to... The formulation may also include surfactants that can reduce or prevent surface-induced aggregation of proteins. Polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol Various conventional surfactants such as fatty acid esters can be used. , ranging from 0.001 to 14% by weight of the formulation. A particularly preferred interface for the purposes of the present invention. The activators are polyoxyethylene sorbitan monooleate, polysorbate 80, and polysorbate. Examples include Rubate 20. TNF antibodies or proteins such as identified parts or variants. The formulation may also contain other drugs known in the relevant art.

[0222] Administration of TNF antibody composition via nebulizer. The antibody composition protein is jet nebulized. It can be administered by a nebulizer such as an Iza or ultrasonic nebulizer. Typically, In a jet nebulizer, compressed air is used to create a high-speed jet of air through an orifice. A gas supply source is used. A low-pressure region is created when the gas expands beyond the nozzle, and that The solution of the antibody composition protein is drawn out through a capillary connected to a liquid reservoir. The liquid flow from the tube is sheared into unstable filamentous particles and droplets as it exits the tube, forming aerosols. To create a certain range of configurations, flow rates, and baffle types from a given jet nebulizer. It can be used to achieve the desired performance characteristics. In an ultrasonic nebulizer, Using high-frequency electrical energy, typically using piezoelectric transducers, vibrating mechanical energy It generates energy. This energy is either directly or through the coupling fluid. The antibody composition protein is transferred to the formulation, creating an aerosol containing the antibody composition protein. It is released. Advantageously, the antibody composition protein particles delivered by the nebulizer are approximately Less than 10 μm, preferably about 1 μm to about 5 μm, most preferably in the range of about 2 μm to about 3 μm. It has a particle size of the following size.

[0223] Suitable for use with either a jet or ultrasonic nebulizer, at least one anti-TN F antibody preparations typically contain at least 0.1 mg to 100 mg per 1 ml of solution. It also contains the concentration of another anti-TNF antibody protein. This formulation contains additives, buffers, isotonic agents, It may contain preservatives, surfactants, and preferably drugs such as zinc. This formulation contains buffering agents, A reducing agent, bulk protein, or carbohydrate, or at least one anti-TNF antibody composition It may also include additives or drugs for protein stabilization. At least one anti-TNF antibody bloc. Useful bulk proteins in formulating adult proteins include albumin and protamine. Examples include typical carbohydrates useful in the formulation of at least one anti-TNF antibody. Examples include sucrose, mannitol, lactose, trehalose, and glucose. At least one anti-TNF antibody preparation causes aerosol formation by atomization of the solution. A surfactant capable of reducing or inhibiting surface-induced aggregation of at least one anti-TNF antibody. This may also include: polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene Various conventional surfactants, such as lensorbital fatty acid esters, can be used. The amount is generally in the range of 0.001 to 4% by weight of the formulation. Particularly preferred for the purposes of the present invention The surfactants are polyoxyethylene sorbitan monooleate and polysorbate 80 Examples include polysorbate 20. The same technology is used in preparations of proteins such as antibody proteins. Further drugs known in the field of medicine may also be included in the formulation.

[0224] Administration of TNF antibody composition using a metered-dose inhaler. In a metered-dose inhaler (MDI), the propellant is small. At least one anti-TNF antibody and any additives or other additives containing liquefied compressed gas The mixture is contained within the canister. By operating the throttle valve, it is preferably reduced to about 10 μm. Sizes less than approximately 1 μm to 5 μm, most preferably in the range of approximately 2 μm to 3 μm. A mixture containing particles is released as an aerosol. Jet pulverization, spray drying, clinical Antibody composition proteins produced by various methods known to those skilled in the art, including boundary junction formation. By using this formulation, the desired aerosol particle size can be obtained. Preferred quantitative absorption The input device is manufactured by 3M or Glaxo and contains hydrofluorocarbon propellant. The items to be used are listed below.

[0225] Preparations of at least one anti-TNF antibody for use in metered-dose inhaler devices are generally , suspend at least one anti-TNF antibody in the propellant, for example, with the help of a surfactant. It contains fine powder as a suspension in a non-aqueous solvent. The propellant is trichlorofluoromethyl Tan, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1, 2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), Examples include HFA-227 (hydrofluoroalkane-227), and chlorofluoro Carbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon This can be any conventional substance used for the purpose of this invention. Preferably, the propellant is a It is a difluorocarbon. The surfactant contains at least one anti-TNF antibody in the propellant. Selected for purposes such as stabilizing the suspension and protecting the active agent from chemical degradation. This is possible. Suitable surfactants include sorbitan trioleate and soy lecithin. Examples include oleic acid. In some cases, a solvent such as ethanol is used in the solution. Aerosols are preferred. Protein formulations may be further formulations of drugs known in the art. It can be included inside.

[0226] Those skilled in the art will see that the method of the present invention involves at least one device not described herein. It will be recognized that this can be achieved by transpulmonary administration of an anti-TNF antibody composition.

[0227] Oral formulations and administration. Oral formulations are designed to artificially increase the permeability of the intestinal wall. Juvant (for example, resorcinol, and polyoxyethylene oleyl ether and Simultaneous administration of nonionic surfactants such as n-hexadecyl polyethylene ether, and Enzyme inhibitors to inhibit enzymatic degradation (e.g., pancreatic trypsin inhibitors, diisopropyl alcohol) By simultaneous administration of fluorophosphate (DFF) and transilol. For oral administration. The active ingredient compounds in the standard dosage form are sucrose, lactose, cellulose, mannitol, and trehalose. Raffinose, maltitol, dextran, starch, agar, alginate, chitin Chitosan, pectin, tragacanth gum, acacia gum, gelatin, collagen, cold at least one such as in, albumin, synthetic or semi-synthetic polymer, and glyceride These dosage forms can also be mixed with other types of additives, such as inactivated ones. Diluent, lubricant (magnesium stearate), parabens, preservative (sorbic acid, asphalt) Corbic acid, α-tocopherol, etc.), antioxidants (cysteine, etc.), disintegrants, binders It may contain thickeners, buffers, sweeteners, flavorings, fragrances, etc.

[0228] Tablets and pills can be further processed into enteric-coated preparations for oral administration. The liquid preparations include emulsions, syrups, elixirs, and suspensions that are acceptable for medical use. Examples include turbidants and solution preparations. These preparations are inactivations commonly used in the art. It may contain a diluent, such as water. Liposomes are used for insulin and heparin drugs. It is also described as a delivery system (U.S. Patent No. 4,239,754). More recently, Microspheres (proteinoids), artificial polymers of mixed amino acids, deliver pharmaceuticals. It is used for (U.S. Patent No. 4,925,673). Furthermore, U.S. Patent No. 5,8 The carrier compounds described in U.S. Patent No. 79,681 and U.S. Patent No. 5,5871,753 are produced It is known in the art that it is used to deliver physically active drugs orally. ru.

[0229] Mucosal preparation and administration. At least one anti-TNF antibody is administered for absorption through the mucosal surface. The composition and method for this purpose comprises a plurality of submicron particles, a macromolecule that adheres to mucous membranes, and a biological Absorption through the mucosal surface is achieved by ensuring that the active peptide and emulsion particles adhere to the mucous membrane. An emulsion comprising an aqueous continuous phase that promotes the action (U.S. No. 5,514,670 (No.) Mucosal surfaces suitable for application of the emulsion of the present invention include the cornea, conjunctiva, oral cavity, sublingual area, Routes of administration include nasal, vaginal, lung, gastric, intestinal, and rectal administration. For vaginal or rectal administration The preparation, for example, suppositories, may contain, for example, polyalkylene glycol, petrolatum, and other additives. It may contain cocoa butter, etc. The formulation for intranasal administration may be solid, and may contain additives. For example, it may contain lactose, or it may be an aqueous or oily nasal spray. It can be administered orally. For oral administration, the additives include sugar, calcium stearate, and stearic acid. Examples include magnesium and alpha-starch (pregelinatined starch) (U.S. Patent No. 5). , No. 849, 695).

[0230] Transdermal preparation and administration. For transdermal administration, at least one anti-TNF antibody is administered via liposomes. or polymer nanoparticles, fine particles, microcapsules or microspheres (especially as specified) Unless otherwise specified, they are encapsulated within delivery devices such as poly (collectively referred to as microparticles). Polyhydroxy acids such as lactic acid, polyglycolic acid and their copolymers, polyorthoethyl Synthetic polymers such as sterols, polyanhydrides and polyphosphazenes, as well as collagen and poly Natural polymers such as amino acids, albumin and other proteins, alginates and so Many suitable devastations include fine particles made from other polysaccharides and combinations thereof. The chair is known (U.S. Patent No. 5,814,599).

[0231] Long-term administration and formulation. The compound of the present invention can be administered as a single dose over a long period of time, for example, In some cases, it may be desirable to deliver the drug to the subject for 1 week to 1 year. Various sustained-release, depot, or Embedding formulations can be used. For example, formulations with low solubility in body fluids can be used. pharmaceutically acceptable non-toxic salts of the compound, e.g., (a) phosphoric acid, sulfuric acid, citric acid, tartaric acid, Tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfone Acids, acid addition salts with polybasic acids such as polygalacturonic acid, (b) zinc, calcium, bismuth Sodium, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc. Polyvalent metal cations, or for example, N,N'-dibenzyl ethylenediamine or A salt having an organic cation formed from ethylenediamine, or (c)(a) and (b ) may contain a combination of, for example, zinc tannate. In addition, the compound of the present invention, Preferably, a relatively insoluble salt, such as those mentioned above, is used in combination with sesame oil, which is suitable for injection. For example, it can be formulated in a gel such as aluminum monostearate gel. Particularly preferred salts are zinc salts, zinc tannates, and pamoates. Injectable sustained-release depot formulations include, for example, polylactic acid / described in U.S. Patent No. 3,773,919. Capsules are found in slowly degrading, non-toxic, non-antigenic polymers such as polyglycolic acid polymers. It contains a compound or salt dispersed for cellularization. The compound, or preferably the above Relatively insoluble salts, such as those found in cholesterol matrix, are particularly suitable for use in animals. It can also be formulated in custard pellets. Further sustained-release depot or infusion treatments are available. For example, gaseous or liquid liposomes are described in the literature (U.S. Patent No. 5,770,222, and “Sustained and Controlled Release Drug D "Elivery Systems," edited by JR Robinson, Marcel De This is known from kker, Inc., NY, 1978.

[0232] Since the present invention has been described in general terms, similar examples are provided for specific details, but are not limited to them. This will be more easily understood by referring to the following examples, which are not intended to do so. Ro.

[0233] Example 1: Cloning and expression of TNF antibodies in mammalian cells. A typical mammalian expression vector mediates the initiation of mRNA transcription by at least one p Lomota elements, antibody coding sequences, and the necessary components for transcription termination and polyadenylation of the transcript. Includes signal. Additional elements include enhancers, Kozak sequences, and RNA splices. It includes intervening sequences adjacent to the donor and acceptor sites for transplantation. The images show early and late promoters from SV40, retroviruses, such as RSV, HT. LVI, long-terminal repeats (LTRS) from HIVI, and cytomegalovirus (CMV) This can be achieved with the initial promoter. However, it is also possible to use cellular elements. (e.g., human actin promoter). Suitable for use in the implementation of the present invention. Current Vector products include, for example, pIRES1neo, pRetro-Off, and pRetro -On, PLXSN or pLNCX (Clonetech Labs, Palo A lto,CA), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -) or pc DNA3.1 / Hygro(+ / -)(Invitrogen), PSVL, and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (A Examples of vectors include TCC 67109). Mammalian hosts that can be used: The cells include human Hela293, H9, and Jurkat cells, and mouse NIH3T3 and C cells. 127 cells, Cos1, Cos7 and CV1, quail QC1-3 cells, mouse L cells and Contains Chinese hamster ovary (CHO) cells.

[0234] Alternatively, in a stable cell line containing genes integrated into chromosomes, the gene is introduced. These can be expressed by DHFR, GPT, neomycin, or hygromycin, etc. Co-transfection with a selection marker allows for the identification of transfected cells and single Making separation possible.

[0235] The transfected gene also increases in expression of large amounts of encoding antibodies. It can be widened. The DHFR (dihydrofolate reductase) marker is hundreds or Furthermore, it is useful for developing cell lines with thousands of copies. Another useful selection marker is The enzyme glutamine synthase (GS) (Murphy et al., "Biochem. J."). "Vol. 227, pp. 277-279 (1991), Bebbington et al., "Bio / "Technology," Vol. 10, pp. 169-175 (1992). These markers Using this method, mammalian cells are grown in a selective medium, and the cells with the highest resistance are selected. These cell lines contain amplified genes integrated into the chromosomes. Hamster ovary (CHO) and NSO cells are often used for antibody production.

[0236] Expression vectors pC1 and pC4 are strong promoters (LTRs) of Roussarcoma virus (C) Ullen et al., "Molec. Cell. Biol." Vol. 5, pp. 438-447 (19 In addition to 1985, CMV enhancer (Boshart et al., "Cell" Vol. 41, p. 52) It contains fragments from pages 1-530 (1985). For example, restriction enzyme cleavage site BamHI Multiple cloning sites, including XbaI and Asp7l8, are cloning sites for the target gene. To facilitate the process. The vector further contains the 3' intron of the rat preproinsulin gene. It contains polyadenylation and termination signals.

[0237] Cloning and expression in CHO cells. Vector pC4 was used for the expression of TNF antibodies. Plasmid pC4 is plasmid pSV2-dhfr (ATCC accession number 371). It is a derivative of 46). This plasmid was introduced into mouse D under the control of the SV40 initial promoter. Contains HFR genes. Dihydrofolate is transfected with these plasmids. Chinese hamster ovary cells or other cells lacking activity are treated with the chemotherapeutic agent methotrexate. Selective media supplemented with alpha-MEM (e.g., Life Technologies) Selection can be achieved by growing cells in (Gaithersburg, MD). Yes, it is possible. Amplification of the DHFR gene in methotrexate (MTX)-resistant cells is sufficient. This is documented in minutes (for example, FWAlt et al., "J. Biol. Chem." 25 3:1357-1370 (1978); JL Hamlin and C. Ma, "Bio chem.et Biophys.Acta” Vol. 1097, pp. 107-143 (199 0 years), and MJPage and MASydenham, "Biotechnol See "ogy" Vol. 9, pp. 64-68 (1991). As a result of the amplification of the DHFR gene, the cells that have grown have an excess of the target enzyme DHFR. Overproduction leads to the development of drug resistance. The second gene is linked to the DHFR gene. If present, it is usually co-amplified and overexpressed. Using this approach, the amplified gene The ability to develop cell lines with more than 1,000 copies of the gene is a key aspect of this technology. It is known in the field of technology. Next, when recovering methotrexate, one of the host cells Alternatively, a cell line containing amplified genes integrated into two or more chromosomes can be obtained.

[0238] Plasmid pC4 is the long-terminal receptor of Roussarcoma virus, which is used to express the target gene. Strong promoter of recovery (LTR) (Cullen et al., "Molec.Cell.Biol.") In addition to "Volume 5, pages 438-447 (1985)", human cytomegalovirus (CM) V) Enhancers of the immediate early genes (Boshart et al., "Cell" Vol. 41, p. 521~ It contains a fragment isolated from page 530 (1985). Downstream of the promoter is the gene These are the BamHI, XbaI, and Asp718 restriction enzyme cleavage sites that enable the incorporation of the enzymes. Following these cloning sites, the plasmid contains the rat preproinsulin gene. It contains 3'-introns and polyadenylated moieties. Other highly efficient promoters, for example, Human β-actin promoter, SV40 early or late promoter, or other retroactive Long-term repeats from HIV and HTLVI, for example, can also be used for expression. Clontech's Tet-Off and Tet-On gene expression systems, and similar systems Using this method, TNF can be expressed in mammalian cells in a controlled manner. M. Gossen and H. Bujard, “Proc. Natl. Acad. Sci. U. SA, Vol. 89, pp. 5547-5551 (1992). Polyadenylation of mRNA For this purpose, other signals from, for example, human growth hormone or globin genes may also be used. This is possible. A stable cell line containing the target gene integrated into the chromosome is called GPT, G Select when co-transfecting with a select marker such as 418 or hygromycin. It is also possible to select more than one marker first, for example G418, in addition to methotrex. Using Cert is advantageous.

[0239] After digesting this plasmid pC4 with restriction enzymes, follow a procedure known in the art Next, dephosphorylation is performed using calf intestinal phosphatase. Then, the vector is treated with 1% agarose. It is isolated from sgel.

[0240] Next, the isolated variable and constant region coding DNA and the dephosphorylated vector are transferred to T4 DNA They are then ligated together. Next, E. coli HB101 or XL-1 Blue cells are transformed. For example, restriction enzyme analysis can be used to identify bacteria containing a fragment inserted into plasmid pC4. Identify.

[0241] The transfection involves Chinese hamsters lacking the active DHFR gene. Ovarian (CHO) cells are used. 5 μg of the expression plasmid pC4 is used to express lipofectin. It is used and co-transfected with 0.5 μg of plasmid pSV2-neo. Sumid pSV2-neo is a group of antibiotics that include G418, a dominant selection marker. In contrast, it contains neogenes from Tn5 that encode an enzyme that confers resistance. 1 μg / Cells are seeded in α-negative MEM supplemented with mL of G418. After 2 days, the cells are triple-saturated. Processed and placed in a hybridoma cloning plate (Greiner, Germany) Supplementation with 10, 25, or 50 ng / mL methotrexate + 1 μg / mL G418. Seeds are sown in α-negative MEM. After approximately 10-14 days, a single clone is treated with trypsin. After that, different concentrations of methotrexate (50nM, 100nM, 200nM, 400n Sow seeds in a 6-well petri dish or 10 mL flask using M (800 nM). Clones grown with the highest concentration of methotrexate are then subjected to an even higher concentration of methotrexate. New 6-well plates including Cert (1mM, 2mM, 5mM, 10mM, 20mM) Transfer to [location]. Repeat the same procedure until you obtain clones that grow at a concentration of 100-200 mM. The expression of the desired gene product can be determined, for example, by SDS-PAGE and Western blotting. Therefore, it is analyzed by reverse-phase HPLC analysis.

[0242] Example 2: High affinity human TNF-responsive transgenic mice Production of IgG monoclonal antibodies. Overview. Transgenic mice containing human heavy chain and light chain immunoglobulin genes. To use for the treatment of one or more TNF-mediated diseases, inhibit TNF activity. To produce high-affinity, fully human monoclonal antibodies that can be used therapeutically. Contains human variable and constant region antibody transgenes for both heavy and light chains (CBA / JxC 57 / BL6 / J) Immunizing F2 hybrid mice with human recombinant TNF (Taylo r et al., "Intl. Immunol." Vol. 6, pp. 579-591 (1993); Lo nberg et al., "Nature" Vol. 368, pp. 856-859 (1994), Neu Berger, M., "Nature Biotech," Vol. 14, p. 826 (199 6th year), Fishwild et al., "Nature Biotechnology" Vol. 14 Pages 845-851 (1996). Several fusions are fully human TNF-reactive IgG. One or more panels of monoclonal antibodies were generated. Fully human anti-TNF antibodies were produced. Further characterization: They are all IgG1κ. Such antibodies are approximately 1 x 10⁶ 9 ~9x1 0 12 These fully human monoclonal antibodies are known to have an affinity constant. Due to their unexpectedly high affinity, they have therapeutic applications in TNF-related diseases, conditions, or disorders. This would be a suitable candidate for the purpose.

[0243] Abbreviations: BSA - Bovine serum albumin, CO2 - Carbon dioxide, DMSO - Dimethyl sulfonate. Hydrogen peroxide, EIA-enzyme immunoassay, FBS-fetal bovine serum, H2O2-hydrogen peroxide, HRP - Horseradish peroxidase, ID - Intradermal, Ig - Immunoglobulin, TNF - Compound Tissue necrosis factor α, IP - intraperitoneal, IV - intravenous, Mab or mAb - monoclonal antibody, OD-Optical density, OPD-o-phenylenediamine dihydrochloride, PEG-Polyethylene glycoside PSA-Penicillin, Streptomycin, Amphotericin, RT-Room Temperature, SQ- Subcutaneous, v / v - volume per unit volume, w / v - weight per unit volume.

[0244] Materials and methods Animals. Transgenic mice capable of expressing human antibodies are known in the art. (For example, GenPharm International, San Jose (Commercially available from CA, Abgenix, Freemont, CA, etc.) This is It expresses immunoglobulins but does not express mouse IgM or Igκ. For example, such Transgenic mice undergo V(D)J binding, heavy chain class switching, and somatic mutations. It contains a human sequence transgene that generates a repertoire of human sequence immunoglobulins. Lonberg et al., *Nature*, Vol. 368, pp. 856-859 (1994). Light chain transgenes, for example, partially contain nearly half of the germline human Vκ region in yeast artificially. It can originate from chromosomal clones. In addition, heavy chain transgenes can be derived from both human μ and human γ1. Fishwild et al., Nature Biotechnology, Vol. 14, p. 845 (Pages ~851 (1996)) and / or the γ3 constant region can be coded. Appropriate Mice derived from specific genotype strains are used in the immunization and fusion processes to target TNF. It is possible to produce fully human monoclonal antibodies.

[0245] Immunization. One or more immunization schedules are used to treat anti-TNF human hybridomas. It can be used to generate [something]. According to the following exemplary immunization protocol, While it is possible to perform the first few fusions, it is not possible to use other similar known protocols. It is also possible to use several transgenic females and / or surgically castrated males aged 14-20 weeks. For each mouth, add an equal volume of TITE to a final volume of 100-400 μL (e.g., 200 μL). 1-1000 μg of recombinant human TN emulsified with RMAX or complete Freund's adjuvant Inoculate F into the IP or ID. Each mouse is then randomly inoculated with 100 μg each at the 2SQ site. They can also receive 1-10 μg of physiological saline in L. After that, the mice 1- IP (1-400μ) after 7, 5-12, 10-18, 17-25 and / or 21-34 days. g) and SQ (1-400 μg x 2) equal amounts of TITERMAX or complete Freund's TNF emulsified with an adjuvant can be immunized. 12-25 and 25 without anticoagulant. After approximately 40 days, bleeding can be induced in the mouse by posterior orbital puncture. Next, the blood can be collected at room temperature. After allowing to coagulate for 1 hour, the serum was collected and the droplets were used in a TNF EIA assay by a known method. Determine. If repeated injections do not result in an increase in potency, perform fusion. At that time, 100 in the mouse A final IV booster injection of 1-400 μg of TNF diluted in μL of physiological saline. It can be administered. Three days later, the mice are euthanized by cervical dislocation and their spleens are aseptically removed. Furthermore, 100 U / mL of penicillin, 100 μg / mL of streptomycin and 0.25 10 mL of cold phosphate-buffered diet containing μg / mL amphotericin B (PSA) It can be immersed in saline solution (PBS). The spleen is to be sterile perfused with PSA-PBS. Spleen cells are then collected. The cells are washed once in cold PSA-PBS and stained with trypan blue dye. Count using exclusion and resuspend in RPMI 1640 medium containing 25 mM HEPES. .

[0246] Cell fusion. According to known, for example, conventional methods in the art, a 1:1 to 1:10 ratio Fusion can be performed in a ratio of myeloma cells to living spleen cells. As a non-limiting example, the spleen Cells and myeloma cells can be pelleted together. Then, pellet over 30 seconds. Slowly infuse 1 mL of 50% (w / v) PEG / PBS solution (PEG molecular weight 1,4) at 37°C. It can be resuspended in 50 (Sigma). Then, add 25 mM Hepez over 1 minute. By slowly adding 10.5 mL of RPMI 1640 medium (37°C) that is present... This allows you to stop the fusion. Centrifuge the fused cells at 500-1500 rpm for 5 minutes. Separate the cells. Then, place them in HAT medium (25 mM HEPES, 10% fetal clone I serum (Hy (clone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 μg / mL Gentamicin, 2.5% Origen culture supplement (Fisher), 10% 653 Prepared RPMI1640 / Hepes medium, 50 μM 2-mercaptoethanol, 10 RP containing 0 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine After resuspending in MI 1640 medium, transfer 200 to 15 96-well flat-bottom tissue culture plates. Incubate in a plate at μL / well. Then, incubate in a mixture containing 5% CO2 and 95% air for 7-10 days. Place the plate in a humidified incubator at 37°C.

[0247] Detection of human IgG anti-TNF antibodies in mouse serum. Using solid-phase EIA, human TN Mouse serum can be screened for human IgG antibodies specific to F. The plate can be cleanly coated overnight with 2 μg / mL TNF in PBS. After washing with 0.15M physiological saline containing 0.02% (v / v) Tween20, The wells are blocked in PBS with 1% (w / v) BSA, 200 μL / well, at room temperature for 1 hour. This can be done. The plate can be used immediately or frozen at -20°C for later use. The mouse serum dilution is placed in a TNF-coated plate at a rate of 50 μL / well. Incubate at warm temperature for 1 hour. After washing the plate, add to 1% BSA-PBS in a 1:3 ratio. By using 50 μL / well of Fc-specific HRP-labeled goat anti-human IgG diluted to 0,000 Probe at room temperature for 1 hour. The plate can be washed again with 100 μL / well. citrate-phosphate basic solution (0.1M citric acid and 0.2M sodium phosphate, 0 Add 0.01% H2O2 and 1 mg / mL OPD) over 15 minutes at room temperature. Next, Add 25 μL / well of stop solution (4N sulfuric acid) and measure with an automated plate spectrophotometer. Read the OD at 490nm.

[0248] Detection of complete human immunoglobulins in hybridoma supernatant using a suitable EIA. This allows for the detection of growth-positive hybridomas that secrete fully human immunoglobulin. In short, a 96-well pop-out plate (VWR, 610744) is left overnight at 4°C. Coated with 10 μg / mL of goat anti-human IgG Fc in a sodium carbonate buffer. This can be done. Wash the plate, block it with 1% BSA-PBS at 37°C for 1 hour, and immediately... Use or freeze at -20°C. Place the undiluted hybridoma supernatant on a plate. Incubate at 7°C for 1 hour. Wash the plate and immerse in 1:10 of 1% BSA-PBS. Probe with HRP-labeled goat anti-human κ diluted at 000,000 for 1 hour at 37°C. Next, As described above, the plate is incubated with the substrate solution.

[0249] Determination of complete human anti-TNF responsiveness. The above hybridomas are suitable for RIA or other applications. Using this method, it is possible to simultaneously assay the responsiveness to TNF. For example, As described above, the supernatant was incubated on a goat anti-human IgG Fc plate and washed. Afterwards, probe with radiolabeled TNF at room temperature for 1 hour, using an appropriate count per well. The wells are washed twice with PBS, and the bound radiolabeled cells are measured using a suitable counter. Quantify TNF.

[0250] Human IgG1κ anti-TNF secretory hybridomas were expanded in cell culture and, at limited dilution, Subcloning can be performed sequentially. The resulting clone population can then be expanded. The samples are then frozen and stored in a freezing medium (95% FBS, 5% DMSO) and kept in liquid nitrogen.

[0251] Isotype. The determination of antibody isotype involves testing mouse immunoserum against a specific titration. This can be achieved using an EIA of a similar format to the one used for cleaning. Yes, it is possible. As mentioned above, TNF can be coated onto a 96-well plate, and 2 The purified antibody at μg / mL was incubated on a plate at room temperature for 1 hour. Yes, it's possible. Wash the plate and label it with HRP diluted 1:4000 in 1% BSA-PBS. Probe with goat anti-human IgG1 or HRP-labeled goat anti-human IgG3 for 1 hour at room temperature. Wash the plate again and incubate it with the substrate solution as described above.

[0252] Binding dynamics of human anti-human TNF antibodies to human TNF. Antibody binding characteristics include, for example, T It can be suitably evaluated using NF capture EIA and BIAcore technology. As mentioned above, the stepwise concentrations of the human TNF antibody used in the assay were 2 μg / mL. This allows us to evaluate the binding to TNF-coated EIA plates. Subsequently, the OD can be represented as a semi-logarithmic plot showing the relative coupling efficiency.

[0253] The quantitative coupling constant can be determined, for example, as follows, or by any other known preferred method. This can be obtained. BIAcore CM-5 (carboxymethyl) chip BIA Place in core 2000 unit. HBS buffer (0.01M HEPES, 0. 15M NaCl, 3mM EDTA, 0.005%v / v P20 surfactant, pH7 4) Add to the flow cell of the tip at 5 μl / min until a stable baseline is obtained. Dissolve in water. Dissolve 15 mg of EDC(N-ethyl-N'-(3-dimethyl-amino) in 200 μL of water. Dissolve 100 μL of propyl carbodiimide hydrochloride in 200 μL of water. Add mg to 100 μL of NHS (N-hydroxysuccinimide) solution. Results and Inject 40 μL of the resulting solution onto the tip. Add 6 μL of human TNF solution (10 ml). Inject 15 μg / mL of sodium acetate (pH 4.8) onto the tip, and then apply approximately 500 RU. This will increase the buffer. Use TBS / Ca / Mg / BSA electrophoresis buffer (20mM Tris, 0 0.15M sodium chloride, 2mM calcium chloride, 2mM magnesium acetate, 0.5% Change to Riton X-100 (25 μg / mL BSA, pH 7.4) and run it on the tip overnight. Then equilibrate the mixture and hydrolyze or cap all unreacted succinate esters.

[0254] Dissolve the antibodies in electrophoresis buffers at concentrations of 33.33, 16.67, 8.33, and 4.17 nM. Adjust the flow rate to 30 μL / min and the temperature of the instrument to 25°C. One of them has fixed TNF. The first is a derivatized flow cell (sample), and the second is a non-derivatized flow cell (blank). Used for dynamic testing. Inject 120 μL of each antibody concentration onto the flow cell at a rate of 30 μL / min ( (Association phase), followed by continuous flow of buffer for 360 seconds (dissociation phase). 30 μL each of 2M thiocyanate. By injecting guanidine anate twice in sequence, the surface of the chip is regenerated (cause of tissue necrosis). Dissociation of the α-antibody complex).

[0255] The data analysis is performed using BIA 3.0 or CLAMP2, which are known in the relevant technical field. This is done using 0.0. For each antibody concentration, a blank sensorogram is used for the sample sensorogram. It is reduced from Mu. Dissociation (k d, sec -1 ) and meetings (k a mol -1 sec -1 )of Perform a global fit on both and dissociate constant (K D Calculate (k) mol d / k a ). Because the antibody affinity is sufficiently high, if the RU of the captured antibody is greater than 100, Further dilution of the body is performed.

[0256] Results and Discussion Generation of anti-human TNF monoclonal antibodies. Several fusions were performed to create antibodies specific to human TNF. Each fusion that produces dozens of antibodies is seeded into 15 plates (1440 wells / fusion). It has been found that some of these consist of combinations of human and mouse Ig chains. The remaining hybridomas secrete anti-TNF antibodies consisting only of human heavy and light chains (se (cret) It is expected that all human hybridomas will be IgG1κ.

[0257] Binding kinetics of human anti-human TNF antibodies. ELISA analysis is used to analyze these hybridomas as well. Confirm that purified antibodies from TNF bind to TNF in a concentration-dependent manner. Figures 1 and 2A-2B show the results of the relative binding efficiencies of these antibodies. In this case, The binding activity of an antibody to its congener antigen (epitope) is measured. TNF is directly subjected to EIA. When it binds to a plate, it can cause protein denaturation, and the apparent binding affinity is unknown. It should be noted that this cannot reflect binding to denatured proteins. 50 percent Scent binding is observed across a wide range of concentrations.

[0258] Quantitative binding constants were obtained using BIAcore analysis of human antibodies, and human monoclonal Some of the antibodies are 1x10 -9 ~7x10 -12 K in the range D Possessing a very high affinity To reveal one's gender.

[0259] Conclusion. Some fusions involve human variable and constant region antibody transgenes that are immunized with human TNF. This is performed using splenocytes from hybrid mice containing the IgG1κ isotype. We generated a set of several fully human TNF-reactive IgG monoclonal antibodies. Further characterization of human anti-TNF antibodies. Some of the generated antibodies were 1 x 10⁻¹⁶. 9 ~9x10 12 It has an affinity constant. Unexpectedly, these fully human monoclonal antibodies Due to their high affinity, they are suitable for therapeutic use in TNF-dependent diseases, conditions, or related states. To become something.

[0260] Example 3: Generation of human IgG monoclonal antibodies reactive to human TNFα. Summary. Contains human variable and constant region antibody transgenes for both heavy and light chains (CBA / JxC57BL / 6J)F2 hybrid mice (1-4) were immunized with recombinant human TNFα. It transformed. One fusion, named GenTNV, was linked to immobilized recombinant human TNFα. Eight fully human IgG1κ monoclonal antibodies were produced that matched. Immediately after identification, eight cells... The strain was transferred to Molecular Biology for further characterization. Since the sequence of Mab is completely human, they are the same as cA2(Remicad) in humans. It is expected to be less immunogenic than e).

[0261] Abbreviations: BSA - Bovine serum albumin, CO2 - Carbon dioxide, DMSO - Dimethyl sulfonate. Hydrogen peroxide, EIA-enzyme immunoassay, FBS-fetal bovine serum, H2O2-hydrogen peroxide, HC-heavy chain, HRP-horseradish peroxidase, ID-intradermal, Ig-immunoglobulin TNF (Tissue Necrosis Factor α), IP (Intraperitoneal Immunodeficiency Intravenous OD-optical density, OPD-o-phenylenediamine dihydrochloride, PEG-polyethylene glycol Call, PSA-penicillin, streptomycin, amphotericin, RT-room temperature, SQ -Subcutaneous, TNFα-tumor necrosis factor α, v / v-volume per unit volume, w / v-volume per unit volume Weight per unit.

[0262] Introduction. Transgenic mice containing human heavy chain and light chain immunoglobulin genes Using this method, a fully human monoclonal antibody specific to recombinant human TNFα was generated. 2 (Remicade) has the advantage of increasing the serum half-life and reducing immunogenic side effects. It has beneficial properties and is used to therapeutically inhibit inflammatory processes involved in TNFα-mediated diseases. Since these specific antibodies are used, it is expected that they can be utilized.

[0263] When defined herein, the term “half-life” means the plasma concentration of a drug (e.g., the half-life of a therapeutic antimicrobial agent). This shows that the TNFα antibody is halved after one elimination half-life. Therefore, each subsequent With a half-life of 5, less of the drug is eliminated. The amount of drug remaining in the body after one half-life is 5 It is 0%, and after two half-lives it is 25%, and so on. The half-life of a drug is its clear It depends on the lance and volume of distribution. The elimination half-life is considered independent of the amount of drug in the body. It is possible.

[0264] Materials and methods. Animals. Transylamines that express human immunoglobulins but do not express mouse IgM or Igκ. Sgenic mice were developed by GenPharm International. These mice have undergone V(D)J binding, heavy chain class switching, and somatic mutations. This introduces functional human antibodies that generate a repertoire of antigen-specific human immunoglobulins (1). It contains genes. The light chain transgene partially occupies nearly half of the germline human Vκ locus. It is derived from a yeast artificial chromosome clone. In addition to several VH genes, heavy chain (HC) The transgene encodes both the human μ and human γ1(2), and / or the γ3 constant region. The immunoconjugation and fusion processes for generating monoclonal antibodies described herein In this study, mice derived from the HCo12 / KCo5 genotype strain were used.

[0265] Purification of human TNFα. TNFα linked to Sepharose 4B (Pharmacia). Using a column packed with receptor-Fc fusion protein (p55-sf2)(5), Infinity chromatography was used to determine human TNFα in tissue cultures from C237A cells. Purified from the supernatant. The cell supernatant was mixed with 1 / 9 of its volume in 10x Dulbecco PBS. Mix with D-PBS and pass through the column at 4 mL / min at 4°C. Next, color with PBS. Wash the tubing, elute TNFα with 0.1M sodium citrate, pH 3.5, and then use 2M Tris The solution was neutralized with HCl, pH 8.5. The purified TNFα was treated with 10 mM Tris, 0.12 M salt. The buffer was replaced with sodium hydroxide and pH 7.5, and filtered through a 0.2 μm syringe filter. It was over.

[0266] Immunization. Female GenPharm mice, approximately 16 weeks old, were immunized on days 0, 12, and 28. A total of 100 μg of TNFα (Lot JG) emulsified with an equal volume of Titermax adjuvant. 102298 or JG102098) IP (200 μL) and ID (at the base of the tail) Immunization was performed with 100 μL. Without anticoagulants, posterior orbital puncture was performed on days 21 and 35. Mice were induced to bleed. The blood was allowed to coagulate at room temperature for 1 hour, and serum was collected. The assay was performed using titration. After injection on day 28, the mice were allowed to rest for 7 weeks before being given Gen A fusion called TNV was performed. Subsequently, a specific human I was subjected to TNFα at a ratio of 1:160. Mice with a γ titer were given 50 μg of TN diluted in 100 μL of physiological saline. The final IV booster injection of Fα was administered. Three days later, the mice were euthanized by cervical vertebral dislocation and spleen was removed. The organs were aseptically removed, and 100 U / mL of penicillin and 100 μg / mL of streptomycin were administered. A 10 mL chilled solution containing cin and 0.25 μg / mL amphotericin B (PSA) The spleen was immersed in phosphate-buffered saline (PBS). The spleen was then sterile-perfused with PSA-PBS. Splenocytes were collected by this method. The cells were washed once in cold PSA-PBS and then Coutt The counting was performed using an r counter, and the solution was resuspended in RPMI 1640 medium containing 25 mM hepes. did.

[0267] Cell line. The Cell Biology Services (CBS) group was established in 1997. On May 14th, Centocor's Product Development Group We received non-secreting mouse myeloma fusion cell line 653 from P. )FBS (Cell Culture Labs), 1 mM sodium pyruvate, 0. 1 mM NEAA, 2 mM L-Glutamine (all from JRH Biosciences) Expanded in RPMI medium (JRH Biosciences) supplemented with 95% FB After freezing and storing in S and 5% DMSO (Sigma), store in a CBS vapor phase liquid nitrogen freezer. The cells were stored in a sterile cell bank (Quality Control Center). or, Malvern), Mycoplasma (Bionique Laboratorie) s) was absent. The cells were maintained in the logarithmic growth phase culture until fusion. Before fusion, they The specimens were washed in PBS, counted, and survival rates were determined by removing trypan blue dye (over 95%). ).

[0268] Human TNFα is produced by recombinant cell lines and named C237A, and is used in Centoc The cell line was produced in Molecular Biology. BS (Cell Culture Labs), 2mM L-Glutamine (all JRH) (from Biosciences) and I supplemented with 0.5 g / mL mycophenolic acid Expanded in MDM medium (JRH Biosciences), with 95% FBS and 5% DM After being frozen in SO(Sigma), it is stored in a CBS(13) vapor phase liquid nitrogen freezer. The cell bank is sterile (Quality Control Center, Malvern, Mycoplasma (Bionique Laboratories) It wasn't there.

[0269] Cell fusion. Cell fusion involves 653 mouse myeloma cells and 1:1 mouse spleen cells. The procedure was performed using [tool name]. In short, spleen cells and myeloma cells were pelleted together. 30 seconds Pour the mixture over the pellet and slowly infuse it at 37°C with 1 mL of 50% (w / v) PEG / PBS solution (PE The granules were resuspended in 1,450 g / mol G (Sigma) solution. 10.5 mL of R solution was added over 1 minute. By slowly adding PMI medium (without additives) (JRH) (37°C), fusion occurs. The process was stopped. The fused cells were centrifuged at 750 rpm for 5 minutes. After that, the cells were placed in HAT medium. (10% fetal bovine serum (JRH), 1 mM sodium pyruvate, 2 mM L-glutamic acid N, 10 μg / mL gentamicin, 2.5% Origen culture supplement (Fis her), 50 μM 2-mercaptoethanol, 1% 653 modified RPMI medium, 10 RP containing 0 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine After resuspending in MI / HEPES medium, transfer to 5 96-well flat-bottom tissue culture plates. The cultures were then incubated in plates with 0 μL / well. Next, they were incubated for 7-10 days in a mixture containing 5% CO2 and 95% air. The plates were placed in a humidified incubator at 37°C.

[0270] Detection of human IgG anti-TNFα antibodies in mouse serum. Using solid-phase EIA, human T Mouse serum was screened for human IgG antibodies specific to NFα. In short, P The plate was coated overnight with 1 μg / mL TNFα in BS. 0.02% (v / v After washing with 0.15M saline containing Tween20, the wells were placed in 1% PBS. (w / v) BSA, 200 μL / well, was blocked at room temperature for 1 hour. The plates were immediately... The mice were either used immediately or frozen at -20°C for later use. Serum was divided into 50 μL / wells and 2-fold serial dilutions at room temperature for 1 hour to test for human TNFα coating. Incubated on a plate. After washing the plate, 1% BSA-PB 50 μL / well of Fc-specific (Accurate) solution diluted 1:30,000 in S Probe with HRP-labeled goat anti-human IgG at room temperature for 1 hour. Wash the plate again. And, 100 μL / well of citrate-phosphate basic solution (0.1 M citrate and 0.2 Sodium M phosphate, 0.01% H2O2, and 1 mg / mL OPD) are administered over 15 minutes. It was added at room temperature. Then, 25 μL / well of stop solution (4N sulfuric acid) was added, and the reaction was automated. The OD was read at 490 nm using a plate spectrophotometer.

[0271] Detection of fully human immunoglobulins in hybridoma supernatant. GenPharm mouse. It can produce both mouse and human immunoglobulin chains, thus two distinct Using the EIA assay, growth-positive hives were detected for the presence of both human light chains and human heavy chains. Ridomas clones were tested. Plates were coated as described above, and undiluted hives were used. The lidoma supernatant was incubated on a plate at 37°C for 1 hour. The plate was washed, HRP conjugated yams diluted 1:10,000 in 1% BSA-HBSS at 37°C for 1 hour. In 1% BSA-HBSS, the anti-human κ (Southern Biotech) antibody is present. Either of the HRP-conjugated goat anti-human IgG Fc-specific antibodies diluted 1:30,000. The probe was then performed. Next, the plate was incubated with the substrate solution as described above. Neither anti-human κ nor anti-human IgG Fc EIA format produced a positive signal. The hybridoma clone was discarded.

[0272] Isotype. Determining the isotype of an antibody involves testing mouse immunoserum against a specific titer. This was achieved using an EIA of a similar format to the one used for cleaning. 4E E100% of goat anti-human IgG(H+L) was tested overnight at °C in a sodium carbonate buffer. Plate A was coated and blocked as described above. No dilution was used from the 24-well culture. The supernatant was incubated on a plate at room temperature for 1 hour. The plate was washed and 1% B HRP-labeled goat anti-human IgG1, IgG2, and I diluted 1:4000 in SA-PBS The probe was performed at room temperature for 1 hour using either IgG3 or IgG4 (Binding Site). The sheets were washed again and incubated with the substrate solution as described above.

[0273] Results and Discussion. Generation of fully human anti-human TNFα monoclonal antibody. Recombinant human TNF GenPharm mice immunized with α-protein were named GenTNV. One fusion was performed. From this fusion, 196 growth-positive hybrids were screened. Eight hybridoma cell lines secreting fully human IgG antibodies reactive with human TNFα were used. Identified. Each of these eight cell lines is a human IgG1κ isotype immunoglobulin. By secreting phosphorus and subcloning all cells twice using limiting dilution, a stable cell line was obtained. (Over 90% homogeneous). The cell line names and their respective C code notations are listed in Table 1. These cells were frozen in a 12-vial research cell bank stored in liquid nitrogen.

[0274] Parental cells collected from the wells of each of the eight cell lines' 24-well culture dishes were transferred. For analysis and further characterization, Molecular B It was handed over to the iology group.

[0275] [Table 2]

[0276] Conclusion. GenTNV fusion was immunized with recombinant human TNFα prepared in Centocor. Splenocytes from hybrid mice containing human variable and constant region antibody transgenes were utilized. The study was conducted using eight fully human TNFα-reactive IgG monoclonal IgG1κ isotypes. Ronal antibodies were generated. For further characterization and development, the parent cell line was modified for Molecul It was transferred to the ar Biology group. One of these new human antibodies is Re Compared to micades, it has the potential benefit of reducing immunogenicity and allergic complications. Therefore, it may be useful for anti-inflammatory purposes.

[0277] References: Taylor et al. International Immunology 6:579- 591 (1993). Lonberg et al., *Nature*, Vol. 368, pp. 856-859 (1994). Neuberger, M., "Nature Biotechnology," Vol. 14 No. 826 (1996). Fishwild et al., Nature Biotechnology, Vol. 14, p. 84 pp. 5-851 (1996). Scallon et al., "Cytokine," Vol. 7, pp. 759-770 (1995).

[0278] Example 4: Cloning and preparation of a cell line expressing human anti-TNFα antibody. Summary. A panel of eight human monoclonal antibodies (mAbs) with TNV notation clearly shows high Of the eight mAbs, it was found to bind to immobilized human TNFα due to its binding activity. These seven studies demonstrated efficient blocking of human TNFα binding to recombinant TNF receptors. Sequence analysis of the DNA encoding the seven mAbs revealed that all mAbs possess the human V region. We confirmed that the DNA sequence shows that the three pairs of mAbs are identical to each other, and therefore there are eight m The original panels of Ab are represented by TNV14, TNV15, TNV148, and TNV196. It was also revealed that it contains only four distinct mAbs. The estimated amino acid composition of the mAbs Based on the analysis of the columns and the results of the in vitro TNFα neutralization data, mAb TNV148 and We selected TNV14 for further testing.

[0279] Database search for the proline residue at position 75 (framework 3) of the TNV148 heavy chain. Because it was not found in that position in other human antibodies of the same subgroup, it was identified as a known reproductive antibody. To match the series framework e sequence, site-directed DNA mutagenesis is performed, The serine residue was encoded at this position. The serine-modified mAb was denoted as TNV148B. PCR-amplified DNA encoding the heavy and light chain variable regions of TNV148B and TNV14. This is based on the recently cloned heavy and light chain genes of another human mAb (12B75). It was published as International Publication No. 02 / 12500, "IL-12 Antibod Titled "ies, Compositions, Methods and Uses", U.S. Patent Application No. 60 / 236,827, filed October 7, 2000, is referenced in its entirety. It was cloned into a newly prepared expression vector (as incorporated herein).

[0280] P3X63Ag8.653(653) cells or Sp2 / 0-Ag14(Sp2 / 0) Myeloma cells were transfected with heavy chain and light chain expression plasmids, and high-level Bell's recombinant TNV148B and TNV14 (rTNV148B and rTNV14) mA Cell lines that produce b were screened using two subcloning techniques. (Time course) The growth curve and stability of mAb production were evaluated using 653 transfectant clone C4. 66D and C466C are stable in used cultures at approximately 125 g / mL of rTN. It produces V148B mAb, while the Sp2 / 0 transfectant 1.73-12-1 22(C467A) is stable in used cultures at approximately 25 g / mL of rTNV1 It was shown to produce 48B mAb. Similar analysis showed that Sp2 / 0 transfectan Toclone C476A produced rTNV14 at a concentration of 18 g / mL in used cultures. This demonstrated that.

[0281] Introduction. Human TNFα immunized GenPharm / Medarex mouse (HCo12 / K A panel of eight mAbs derived from the CO5 genotype binds to human TNFα and is fully human. It was previously shown to have the gG1κ isotype. Using a simple binding assay, TN By evaluating the ability to block Fα from binding to recombinant TNF receptors, the present invention We determined whether exemplary mAbs may possess TNFα neutralizing activity. Based on the results, DNA sequencing findings, and several in vitro characterizations of the mAb, further specialization was performed. TNV148 was selected as the mAb to be labeled.

[0282] The DNA sequence encoding TNV148 mAb was cloned, and a suitable constant region was selected. Modified and well-characterized 653 and Sp to fit within the gene expression vector. Transfected cell lines were introduced into 2 / 0 mouse myeloma cells, resulting in the obtained transfected cell lines. Until a subclone producing 40 times more mAbs than the original hybridoma cell line was identified... Screening was performed using this method.

[0283] Materials and methods. Reagents and cells. TRIZOL reagents were purchased from Gibco BRL. Proteinase K was obtained from Sigma Chemical Company. Reverse transcriptase is Life Obtained from Sciences, Inc. Taq DNA polymerase is Perkin E The restriction enzyme was obtained from either LMER Cetus or Gibco BRL. Purchased from England Biolabs. QIAquick PCR Purif The ication kit was obtained from Qiagen. QuikChange Site-D I purchased the corrected Mutagenesis Kit from Stratagene. Wizard Plasmid Miniprep Kit and RNasin are from Promega. Yes, I found it. I got Optiplates from Packard. 125 Iodine is Ame Purchased from rsham. Custom oligonucleotides are Keystone / Bioso Purchased from Urce International. The Oligonucle used in this work. Table 2 shows the name, identification number, and sequence of Otido.

[0284] Table 2. Uses for cloning, engineering, or sequencing TNV mAb genes A modified oligonucleotide. The sequence of amino acids encoded by oligonucleotides 5'14s and HuH-J6 As shown above, the "M" amino acid residue represents the translation start codon. Oligonucleotide 5'14 The underlined sequences s and HuH-J6 represent the BsiWI and BstBI restriction sites, respectively. As shown, the diagonal lines in HuH-J6 correspond to the exon / intron boundary. The sequence is on the negative strand. Note that the corresponding oligonucleotides are written in 3'-5' orientation.

[0285] [Table 3]

[0286] One frozen vial of 653 mouse myeloma cells was obtained. The vial was thawed on the same day, and Tf The cells were expanded in IMDM, 5% FBS, and 2 mM glutamine (culture medium) in Lasco. The cells were transfected with anti-TNF DNA as described herein after 2-3 weeks. It was maintained in continuous culture until [the end of the process]. Five days after thawing, some of the cultures were taken. The material is pelletized by centrifugation, resuspended in 95% FBS and 5% DMSO, and then processed into 30 vials. The cells were divided into equal portions, frozen, and stored for later use. Similarly, Sp2 / 0 mouse myeloma cells were used. One frozen vial of cells was obtained. The vial was thawed, and the new frozen product (freeze-d) was prepared as described above. (own) was prepared and the frozen vials were stored in CBC's freezer boxes AA and AB. These cells are thawed and used for all Sp2 / 0 transfections described herein. I used it.

[0287] Assay for inhibiting the binding of TNF to receptors. Hybrid containing TNV mAb Using doma cell supernatant, the mAb was recombinant TNF receptor fusion protein p55-sf2 to 125 The ability to block the binding of I-labeled TNFα was assayed (Scallon (1995) "Cytokine" Vol. 7, pp. 759-770). 37°C for 1 hour. During incubation, Opt 50 L of p55-sf2 at 0.5 g / mL in PBS. The solution was added to iplates and used to coat the wells. PBS / 0.1% BSA was used as the diluent. As a result, a series of dilutions of eight TNV ​​cell supernatants were used in a 96-well round-bottom plate. Prepared. Cell supernatant containing anti-IL-18 mAb was included as a negative control, and cA2( Anti-TNF chimeric antibody, Remicade, U.S. Patent No. 5,770,198, by reference. The same anti-IL-18 supernatant spiked with (the entire sample is incorporated herein) was used as a positive control. It was included so that the final TNFα concentration would be 5 ng / mL. 125 I-labeled TNFα(5 8 (Ci / g, D.Shealy) was added to 100 L of cell supernatant. The mixture was left at room temperature. They were pre-incubated for 1 hour. The coated Optiplates were washed. Remove unbound p55-sf2 and 50:L 125 O I-TNFα / cell supernatant mixture Transferred to ptiplates. After 2 hours at room temperature, Optip was used three times with PBS-Tween. The latex was washed. 100:L of Microscint-20 was added, and TopCo The coupled cpm was determined using an unt-γ counter.

[0288] Amplification and DNA sequence analysis of the V gene. Hybridoma cells were prepared for RNA preparation. After washing once with BS, TRIZOL reagent was added. 7×10 6 ~1.7×10 7 The fine The cells were resuspended in 1 mL of TRIZOL. After adding 200 μL of chloroform, the tube was vigorously shaken. Shake well. Centrifuge the sample at 4°C for 10 minutes. Transfer the aqueous phase to a new microfuge tube. Transferred to another container and an equal amount of isopropanol was added. The tube was shaken vigorously and incubated at room temperature for 10 minutes. Next, the sample was centrifuged at 4°C for 10 minutes. The pellet was divided into 1 ml of 70% esters. The RNA pellet was washed once with tanol and dried briefly in a vacuum dryer. 40 μL of D The RNA preparation was resuspended in EPC-treated water. The quality of the RNA preparation was assessed in 0.5 μL of 1% agarose gel. This was determined by fractionation. The RNA was stored in a -80°C freezer until use. Ta.

[0289] To prepare the heavy and light chain cDNA, 3 μL of RNA and 3 μL of light chain cDNA are added to an 11.5 μL volume. 1 μg of oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light chain) A mixture containing one of the following (see Table 1) was prepared. The mixture was heated in a water bath at 70°C for 10 minutes. Incubate for 1 minute, then cool on ice for 10 minutes. 2.5 μL of 10× reverse transcriptase solution. Acupuncture agent, 10 μL of 2.5 mM dNTPs, 1 μL of reverse transcriptase (20 units), and 0.4 Prepare a separate mixture consisting of μL of the ribonuclease inhibitor RNasin (1 unit). Prepared. Add 13.5 μL of this mixture to 11.5 μL of cold RNA / oligonucleotide mixture. The mixture was added, and the reaction was incubated at 42°C for 40 minutes. Next, the cDNA synthesis reaction was carried out. The solution was stored in a freezer at -20°C until use.

[0290] Unpurified heavy and light chain cDNA are used as templates to code for the variable region. The following was PCR amplified. Five oligonucleotides were used to prime the amplification of heavy-chain DNA. Ochido vs (366 / 354, 367 / 354, 368 / 354, 369 / 354, and 3 70 / 354 (Table 1) were tested simultaneously. Regarding the ability to prime light chain DNA amplification: Two oligonucleotide pairs (362 / 208 and 363 / 208) were tested simultaneously. . 2 units of PLATINUM® High Fidelity (HIFI) Ta in a total volume of 50 μL PCR was performed using q DNA polymerase. Each reactant was mixed with 2 μL of cDN. Reactant A: 10 picomoles of each oligonucleotide, 0.2 mM dNTP, 5 μL of 10 ×Contained HIFI buffer and 2 mM magnesium sulfate. Thermal CyclaPro Gram was heated at 95°C for 5 minutes, followed by 30 cycles (30 seconds at 94°C, 3 seconds at 62°C). The incubation period was 0 seconds, followed by 1.5 minutes at 68°C. Next, the final incubation period was 10 minutes at 68°C. We performed the procedure.

[0291] To prepare PCR products for direct DNA sequencing, follow the manufacturer's protocol. Therefore, using the QIAquick(trademark) PCR Purification Kit They were purified. After eluting the DNA from the spin column using 50 μL of sterile water, Next, it was dried to a volume of 10 μL using a vacuum dryer. Then, 1 μL was added to the total volume of 20 μL. Purified PCR product of L, 10 μM oligonucleotide primer, 4 μL BigD ye Terminator (trademark) ready reaction mix, and 14 The DNA sequencing reaction mixture was prepared in μL of sterile water. Oligonucleotide vs. 367 / 354 The heavy chain PCR products prepared were obtained using oligonucleotide primers 159 and 360. The sequence was determined. Light chain PCR product prepared with oligonucleotide pair 363 / 208. The sequences were determined using oligonucleotides 34 and 163. Thermal sequencing was performed. The Cycla program consists of 25 cycles (30 seconds at 96°C, 15 seconds at 50°C, 60°C). The reaction was carried out at 4°C for 4 minutes, followed by overnight at 4°C. The reaction product was polyacrylamide gel. The DNA was fractionated via [method / process] and detected using an ABI 377 DNA sequencer.

[0292] Site-directed mutagenesis to alter amino acids. Pro 75 To replace the serine residue, a single nucleus of the TNV148 heavy chain variable region DNA sequence The rheotide was changed. Complementary oligonucleotides 399 and 400 (Table 1) were designed and manufactured. As explained by the manufacturer, QuikChange™ site-directed mutagenesis is used This change was caused by using a 15% polyacrylamide gel. The creotide was first fractionated, and the main band was purified. Either 10 ng or 50 ng was used. TNV148 heavy chain plasmid template (p1753), 5 μL of 10× reaction buffer, 1 μL of dNTP mix, 125 ng of primer 399, 125 ng of primer 400 Mutagenic reaction products were prepared using 1 μL of Pfu DNA polymerase. Bacterial water was added to bring the total volume to 50 μL. Next, the reaction mixture was incubated at 95°C for 30 seconds. Incubate in a thermal cycler programmed to incubate, then 9 5°C for 30 seconds, 55°C for 1 minute, 64°C for 1 minute, 68°C for 7 minutes, followed by 3 The incubation process consisted of 14 cycles, each lasting 2 minutes at 0°C. The reactants produced a mutagenic oligonucleotide, which was otherwise identical to a newly synthesized one. Designed to be incorporated into a plasmid. To remove the original TNV148 plasmid. Then, add 1 μL of DpnI endonuclease to cleave only the original methylated plasmid. Afterward, the sample was incubated at 37°C for 1 hour. Next, using 1 μL of the reactant, ...

Claims

1. Juvenile idiopathic arthritis (JIA) in pediatric patients A method of treatment, wherein the method involves administering an intravenous (IV) dose of anti-TNF antibody to the pediatric patient. The administration includes administering the anti-TNF antibody to a heavy chain containing the amino acid sequence of SEQ ID NO:

36. The HC) and light chain (LC) containing the amino acid sequence of SEQ ID NO: 37, for 50 of the pediatric patients Over % of patients had a positive test result 52 weeks after treatment at the JIA (Japanese Association of Rheumatology). Matology: JIA ACR) 30, JIA ACR 50, and JIA ACR 70 A method to meet the criteria.

2. The method according to claim 1, wherein the pediatric patient is between 2 and under 18 years of age.

3. The aforementioned juvenile idiopathic arthritis (JIA) is also known as polyarticular juvenile idiopathic arthritis (polyarticular). The method according to claim 1, wherein the condition is juvenile idiopathic arthritis (pJIA).

4. The aforementioned IV dose is administered in week 0, week 4, and thereafter every 8 weeks, using the anti-TNF antibody. 80 mg / m² ２ The method according to claim 1.

5. The method further comprises administering methotrexate (MTX) to the pediatric patient. The method according to claim 1.

6. A method for treating juvenile idiopathic arthritis (JIA) in pediatric patients, wherein the method is , comprising administering an intravenous (IV) dose of anti-TNF antibody to the pediatric patient, the anti-TN The F antibody contains a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 36 and the amino acids of SEQ ID NO:

37. The light chain (LC) containing the sequence was found to have a low incidence of disease in more than 20% of the pediatric patients 52 weeks after treatment. Regarding disease activity, the Juvenile Arthritis Disease Activity Score (JADAS 71) was used for 71 joints. How to do it.

7. The method according to claim 6, wherein the pediatric patient is between 2 and 18 years of age.

8. The aforementioned juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA). The method according to claim 6.

9. The aforementioned IV dose is administered in week 0, week 4, and thereafter every 8 weeks, using the anti-TNF antibody. 80 mg / m² ２ The method according to claim 6.

10. The method further comprises administering methotrexate (MTX) to the pediatric patient. The method according to claim 6.

11. A method for treating juvenile idiopathic arthritis (JIA) in pediatric patients, wherein the method is , comprising administering an intravenous (IV) dose of anti-TNF antibody to the pediatric patient, the anti-TN The F antibody contains a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 36 and the amino acids of SEQ ID NO:

37. The light chain (LC) containing the sequence was used, and the pediatric patient, 52 weeks after treatment, was treated by JIA USA Liu. A method that meets the criteria for inactive disease as defined by the Japanese Association of Machia (JIA ACR).

12. More than 30% of the aforementioned pediatric patients met the criteria for JIA ACR inactive disease 52 weeks after treatment. The method according to claim 11, which satisfies the requirements.

13. The method according to claim 11, wherein the pediatric patient is between 2 and 18 years of age.

14. The aforementioned juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA). The method according to claim 11.

15. The aforementioned IV dose is administered in week 0, week 4, and thereafter every 8 weeks, using the anti-TNF antibody. 80 mg / m² ２ The method according to claim 11.

16. The method further comprises administering methotrexate (MTX) to the pediatric patient. The method according to claim 11.

17. A method for treating juvenile idiopathic arthritis (JIA) in pediatric patients, wherein the method is , comprising administering an intravenous (IV) dose of anti-TNF antibody to the pediatric patient, the anti-TN The F antibody contains a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 36 and the amino acids of SEQ ID NO:

37. The light chain (LC) containing the sequence was used, and the pediatric patient, 52 weeks after treatment, was treated by JIA USA Liu. A method that meets the clinical remission criteria of the Japanese Association of Mathematics Research (JIA ACR).

18. More than 10% of the aforementioned pediatric patients met the criteria for JIA ACR clinical remission after 52 weeks of treatment. In addition, the method according to claim 17.

19. The method according to claim 17, wherein the pediatric patient is between 2 and 18 years of age.

20. The aforementioned juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA). The method according to claim 17.

21. The aforementioned IV dose is administered in week 0, week 4, and thereafter every 8 weeks, using the anti-TNF antibody. 80 mg / m² ２ The method according to claim 17.

22. The method further comprises administering methotrexate (MTX) to the pediatric patient. The method according to claim 17.

Images