Anti-TNF antibodies, compositions, and methods for the treatment of juvenile idiopathic arthritis

Abstract

The present invention relates to compositions and methods using anti-TNF antibodies, such as the anti-TNF antibody golimumab, having a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, for use in the treatment of juvenile idiopathic arthritis (JIA), particularly polyarticular juvenile idiopathic arthritis (pJIA).

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application contains a sequence listing, which has been submitted electronically via EFS-Web as an ASCII sequence listing with the file name "JBI6042USPSP3SeqListing.txt" created on September 10, 2019, and has a size of 25kb. The sequence listing submitted via EFS-Web is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to compositions and methods using anti-TNF antibodies, such as the anti-TNF antibody golimumab having a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:37, for use in the treatment of juvenile idiopathic arthritis (JIA), in particular polyarticular juvenile idiopathic arthritis (pJIA). [Background technology]

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

[0004] Cells other than monocytes or macrophages also produce TNFα, for example, human nonmonocytic tumor cell lines produce TNFα as well as CD4+ and CD8+ peripheral blood T lymphocytes, and several cultured T and B cell lines also produce TNFα.

[0005] TNFα causes proinflammatory effects that lead to tissue damage, such as cartilage and bone degradation, induction of adhesion molecules, induction of procoagulant activity in vascular endothelial cells, increased adhesion of neutrophils and lymphocytes, and stimulating the release of platelet-activating factor from macrophages, neutrophils, and vascular endothelial cells.

[0006] TNFα has been implicated in infections, immune disorders, neoplastic, autoimmune and graft-versus-host pathologies. The association of TNFα with cancer and infectious pathologies is often related to the catabolic state of the host. Cancer patients suffer from weight loss, usually associated with anorexia.

[0007] The profound wasting associated with cancer and other diseases is known as "cachexia". Cachexia involves progressive weight loss, anorexia, and persistent loss of lean body mass in response to malignant tumor growth. The cachectic state is responsible for much of the cancer morbidity and mortality. There is evidence that TNFα is involved in cachexia in cancer, infectious conditions, and other catabolic conditions.

[0008] TNFα is believed to play a central role in gram-negative sepsis and endotoxic shock, which includes fever, fatigue, anorexia, and cachexia. Endotoxin strongly activates monocyte / macrophage production and secretion of TNFα and other cytokines. TNFα and other monocyte-derived cytokines mediate the metabolic and neurohormonal response to endotoxin. Endotoxin administration to human volunteers results in acute illness with flu-like symptoms, including fever, tachycardia, increased metabolic rate, and stress hormone release. Circulating TNFα is increased in patients with gram-negative sepsis.

[0009] Therefore, TNFα is associated with inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections, malignant tumors, and / or neurodegenerative diseases, and is a useful target for certain biological therapy in diseases such as rheumatoid arthritis and Crohn's disease. The beneficial effects of suppressing inflammation and successful retreatment after relapse in rheumatoid arthritis and Crohn's disease have been reported in open-label studies using monoclonal antibodies against TNFα. Beneficial results in rheumatoid arthritis due to suppressing inflammation have also been reported in randomized double-blind placebo-controlled studies.

[0010] Neutralizing antisera or mAbs against TNF have been shown to abrogate deleterious physiological changes and prevent mortality following lethal challenge in experimental endotoxemia and bacteremia in non-human mammals, for example in rodent lethality assays and primate pathology model systems.

[0011] The putative receptor binding locus for hTNF has been disclosed, as has the receptor binding locus for TNFα, which consists of amino acids 11-13, 37-42, 49-57, and 155-157 of TNF.

[0012] Non-human mammalian, chimeric, polyclonal (e.g., antisera), and / or monoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestion or fusion protein products thereof) are potential therapeutics that are being investigated in some cases to attempt to treat certain diseases. However, such antibodies or fragments may induce an immune response when administered to humans. Such immune responses can result in immune complex-mediated clearance of the antibodies or fragments from the blood circulation, making repeated administration unsuitable for therapy, thereby reducing therapeutic benefit to the patient and limiting re-administration of the antibodies or fragments. For example, repeated administration of antibodies or fragments that include non-human portions can result in serum sickness and / or anaphylaxis. To avoid these and other problems, many approaches have been taken to reduce the immunogenicity of such antibodies and portions thereof, including chimerization and humanization, as is well known in the art. However, these and other approaches may still result in antibodies or fragments that have some immunogenicity, low affinity, low binding activity, or are associated with problems in cell culture, scale-up, production and / or low yield. Thus, such antibodies or fragments may not be ideally suited for production or use as therapeutic proteins.

[0013] There has been a need to provide TNF inhibitors that address one or more of these problems, which has led to the development of currently marketed anti-TNF antibodies and other TNF inhibitors, such as REMICADE® (infliximab), HUMIRA® (adalimumab), and SIMPONI® (golimumab). Other TNF inhibitors include, for example, CIMZIA® (certolizumab pegol), a PEGylated antibody fragment, and ENBREL® (etanercept), a soluble TNF receptor fusion protein. For a review of TNF inhibitors, see, for example, Lis et al., Arch Med Sci. 2014 Dec 22;10(6):1175-1185. Summary of the Invention [Means for solving the problem]

[0014] For simplicity, the general and preferred embodiments are defined by the independent and dependent claims appended hereto, which are incorporated herein by reference. Other preferred embodiments, features, and advantages will become apparent from the following detailed description, taken in conjunction with the accompanying drawings.

[0015] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe and clinically proven effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:37.

[0016] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe and clinically effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein the patient is a pediatric patient between 2 and 17 years of age.

[0017] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe and clinically proven effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the patient is a pediatric patient between 2 and 17 years of age, and the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA).

[0018] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven, effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, the patient is a pediatric patient between 2 and 17 years of age, the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA), and the anti-TNFα antibody is administered at 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 In one embodiment, the method comprises administering the compound of formula (I) to the subject in an intravenous (IV) dose of 0.1 mg / kg.

[0019] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe and clinically proven effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and the method further comprises administering methotrexate (MTX) to the patient.

[0020] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven, effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and an anti-TNFα antibody at 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 and the method further comprises administering methotrexate (MTX) to the patient.

[0021] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe and clinically proven effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein the patient meets criteria for inactive disease after 28 weeks of treatment with the anti-TNFα antibody.

[0022] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven, effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and an anti-TNFα antibody at 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 and wherein the patient meets criteria for inactive disease after 28 weeks of treatment with the anti-TNFα antibody.

[0023] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe and clinically proven effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein more than 29% of patients meet criteria for inactive disease after 28 weeks of treatment with the anti-TNFα antibody.

[0024] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven, effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and an anti-TNFα antibody at 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 and the method further comprises administering methotrexate (MTX) to the patient, wherein more than 29% of the patients meet criteria for inactive disease after 28 weeks of treatment with the anti-TNFα antibody.

[0025] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein after 28 weeks of treatment the patient has an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response selected from the group consisting of JIA ACR30, JIA ACR50, JIA ACR70, and JIA ACR90.

[0026] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein after 28 weeks of treatment, more than 83% of patients meet the criteria for JIA ACR30, more than 79% of patients meet the criteria for JIA ACR50, more than 70% of patients meet the criteria for JIA ACR70, and more than 46% of patients meet the criteria for JIA ACR90.

[0027] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven, effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and an anti-TNFα antibody at 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 and wherein the method further comprises administering methotrexate (MTX) to the patient, and wherein after 28 weeks of treatment, more than 83% of the patients meet criteria for JIA ACR30, more than 79% of the patients meet criteria for JIA ACR50, more than 70% of the patients meet criteria for JIA ACR70, and more than 46% of the patients meet criteria for JIA ACR90.

[0028] 6. The method of claim 1, wherein after 28 weeks of treatment with an anti-TNF antibody, the patient has a change from baseline in Juvenile Arthritis Disease Activity Score (JADAS) selected from the group consisting of JADAS10, JADAS27, and JADAS71.

[0029] The method of claim 10, wherein patients with JADAS10 have a median decrease from baseline of greater than 14, patients with JADAS27 have a median decrease from baseline of greater than 16, and patients with JADAS71 have a median decrease from baseline of greater than 20.

[0030] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a patient, comprising administering to the patient an anti-TNF antibody in a clinically safe, clinically proven, effective amount, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and an anti-TNFα antibody at 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 and wherein the method further comprises administering methotrexate (MTX) to the patient, and wherein after 28 weeks of treatment, patients with JADAS10 have a median reduction from baseline of greater than 14, patients with JADAS27 have a median reduction from baseline of greater than 16, and patients with JADAS71 have a median reduction from baseline of greater than 20.

[0031] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein the patient treated with the anti-TNF antibody meets criteria for inactive disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment.

[0032] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein patients treated with the anti-TNF antibody meet criteria for inactive disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment, wherein more than 10% of patients meet criteria for inactive disease after 8 weeks of treatment, more than 20% of patients meet criteria for inactive disease after 16 weeks of treatment, and more than 29% of patients meet criteria for inactive disease after 28 weeks of treatment.

[0033] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the patient treated with the anti-TNF antibody meets criteria for inactive disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment, and wherein the pediatric patient is between 2 and 17 years of age.

[0034] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the patient treated with the anti-TNF antibody meets criteria for inactive disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment, and wherein the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA).

[0035] In a specific embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein the patient treated with the anti-TNF antibody meets criteria for inactive disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment, and wherein the IV dose is 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 The present invention provides a method for

[0036] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the patient treated with the anti-TNF antibody meets criteria for inactive disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment, and the method further comprises administering methotrexate (MTX) to the pediatric patient.

[0037] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein patients treated with the anti-TNF antibody have an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment.

[0038] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein patients treated with the anti-TNF antibody have an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, wherein greater than 50% of patients meet the criteria for JIA ACR30 and JIA ACR50 after 4 weeks of treatment.

[0039] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein patients treated with the anti-TNF antibody have an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, wherein greater than 50% of patients meet criteria for JIA ACR30, JIA ACR50, and JIA ACR70 after 12 weeks of treatment.

[0040] In a particular embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein patients treated with the anti-TNF antibody have an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, wherein greater than 83% of patients meet criteria for JIA ACR30, greater than 79% of patients meet criteria for JIA ACR50, greater than 70% of patients meet criteria for JIA ACR70, and greater than 46% of patients meet criteria for JIA ACR90 after 28 weeks of treatment. A method is provided that meets the ACR90 criteria.

[0041] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the patient treated with the anti-TNF antibody has an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and wherein the pediatric patient is between 2 and 17 years of age.

[0042] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein patients treated with the anti-TNF antibody have an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and wherein the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA).

[0043] In a specific embodiment, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein the patient treated with the anti-TNF antibody has an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, wherein the IV dose is 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 The present invention provides a method for

[0044] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein patients treated with the anti-TNF antibody have an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and the method further comprises administering methotrexate (MTX) to the pediatric patient.

[0045] In certain embodiments, the invention provides a method of treating Juvenile Idiopathic Arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein patients treated with the anti-TNF antibody have a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment.

[0046] In certain embodiments, the invention provides a method of treating Juvenile Idiopathic Arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein patients treated with the anti-TNF antibody have a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and wherein more than 10% of patients have JADAS10, JADAS27, and JADAS71 minimal disease activity disease after 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, and 28 weeks of treatment.

[0047] In certain embodiments, the invention provides a method of treating Juvenile Idiopathic Arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein patients treated with the anti-TNF antibody have a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and wherein after 24 weeks of treatment and 28 weeks of treatment, more than 15% of patients have disease with JADAS10, JADAS27, and JADAS71 minimal disease activity.

[0048] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the patient treated with the anti-TNF antibody has a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and wherein the pediatric patient is 2-17 years of age.

[0049] In certain embodiments, the invention provides a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, wherein the patient treated with the anti-TNF antibody has a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and wherein the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA).

[0050] In a specific embodiment, the invention provides a method of treating Juvenile Idiopathic Arthritis (JIA) in a pediatric patient, comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein the patient treated with the anti-TNF antibody has a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and wherein the IV dose is 80 mg / m at week 0, week 4, and every 8 weeks thereafter. 2 The present invention provides a method for

[0051] In certain embodiments, the invention provides a method of treating Juvenile Idiopathic Arthritis (JIA) in a pediatric patient comprising administering to the patient an intravenous (IV) dose of an anti-TNF antibody, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and wherein patients treated with the anti-TNF antibody have a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 2 weeks of treatment, or 28 weeks of treatment, and the method further comprises administering methotrexate (MTX) to the pediatric patient. [Brief description of the drawings]

[0052] [Figure 1] 1 is a graphical representation showing an assay of the ability of TNV mAbs in hybridoma cell supernatants to inhibit TNFα binding to recombinant TNF receptors. Various amounts of hybridoma cell supernatants containing known amounts of TNV mAbs were preincubated with a fixed concentration (5 ng / mL) of 125I-labeled TNFα. The mixtures were transferred to 96-well Optiplates precoated with recombinant TNF receptor / IgG fusion protein p55-sf2. The amount of TNFα bound to the p55 receptor in the presence of mAbs was determined after washing away unbound material and counting using a gamma counter. Eight TNV ​​mAb samples were tested in these experiments, but for brevity, three of the mAbs that were shown by DNA sequence analysis to be identical to one of the other TNV mAbs are not shown here. Each sample was tested in duplicate. Results shown are representative of two independent experiments. [Figure 2A]Figure 1 shows the DNA sequence of the TNV mAb heavy chain variable region. The germline gene shown is the DP-46 gene. "TNVs" indicates that the sequence shown is that of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequence define the translation initiation Met codon. The dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides of the TNV sequence (underlined) correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single letter code) starting with the mature mAb is shown only for the germline gene. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. The gap in the germline DNA sequence (CDR3) was due to sequences that were not known at the time or were not present in the germline gene. The TNV mAb heavy chain uses the J6 binding region. [Figure 2B] Figure 1 shows the DNA sequence of the TNV mAb heavy chain variable region. The germline gene shown is the DP-46 gene. "TNVs" indicates that the sequence shown is that of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequence define the translation initiation Met codon. The dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides of the TNV sequence (underlined) correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single letter code) starting with the mature mAb is shown only for the germline gene. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. The gap in the germline DNA sequence (CDR3) was due to sequences that were not known at the time or were not present in the germline gene. The TNV mAb heavy chain uses the J6 binding region. [Diagram 3]Figure 1 shows the DNA sequence of the TNV mAb light chain variable region. The germline gene shown is a representative member of the Vg / 38K family of human kappa germline variable region genes. The dotted lines in the TNV mAb gene sequence indicate that the nucleotide is the same as 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 code) of the mature mAb is shown for the germline gene only. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are for sequences that are unknown or not present in the germline gene. The TNV mAb light chain uses the J3 binding region. [Figure 4] Figure 1 shows the deduced amino acid sequence of the TNV mAb heavy chain variable region. The amino acid sequence shown (single letter code) was deduced from DNA sequence determined from both uncloned and cloned PCR products. The amino acid sequence is shown divided into secretory signal sequence (signal), framework (FW) and complementarity determining region (CDR) domains. The amino acid sequence of the DP-46 germline gene is shown in the line above each domain. Dotted lines indicate that the amino acid in the TNV mAb is identical to the germline gene. TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. "TNV" indicates that the sequence shown is for all TNV mAbs unless a different sequence is shown. The dashed lines in the germline sequence (CDR3) indicate that the sequence is not known or is not present in the germline gene. [Diagram 5]Figure 1 shows the predicted amino acid sequence of the TNV mAb light chain variable region. The amino acid sequence shown (single letter code) was deduced from DNA sequence determined from both uncloned and cloned PCR products. The amino acid sequence is shown divided into secretory signal sequence (signal), framework (FW) and complementarity determining region (CDR) domains. The amino acid sequence of the Vg / 38K type light chain germline gene is shown in the line above each domain. The dotted lines indicate that the amino acid in the TNV mAb is identical to the germline gene. TNV148(B) indicates that the sequence shown relates to both TNV148 and TNV148B. "All" indicates that the sequence shown relates to TNV14, TNV15, TNV148, TNV148B and TNV186. [Figure 6] FIG. 1 is a schematic diagram of the heavy and light chain expression plasmids used to generate rTNV148B expressing C466 cells. p1783 is the heavy chain plasmid and p1776 is the light chain plasmid. The rTNV148B variable and constant region coding domains are indicated in black boxes. The immunoglobulin enhancer in the JC intron is indicated in grey boxes. Relevant restriction sites are indicated. The plasmids are shown oriented such that transcription of the Ab genes proceeds in a clockwise direction. Plasmid p1783 is 19.53 kb in length and plasmid p1776 is 15.06 kb in length. The complete nucleotide sequences of both plasmids are known. The variable region coding sequence of p1783 can be easily 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]Graphical representation of growth curve analysis of five rTNV148B-generated cell lines. Cultures were initiated on day 0 by seeding cells in I5Q+MHX medium in T75 flasks to have a viable cell density of 1.0×105 cells / mL in a volume of 30 mL. The cell cultures used in these studies were continuous cultures as transfections and subcloning were performed. The cells in the T flasks were then thoroughly resuspended and 0.3 mL aliquots of culture were removed. Growth curve studies were terminated when the cell count dropped below 1.5×105 cells / mL. The number of viable cells in the aliquots was determined by trypan blue exclusion and the remaining aliquots were kept for later mAb concentration determination. ELISA for human IgG was performed on all sample aliquots at the same time. [Figure 8] Figure 1 is a graphical representation of a comparison of cell growth rates in the presence of various MHX selection concentrations. Cell subclones C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for an additional 2 days. Both cell cultures were then split into three cultures that contained either no MHX, 0.2xMHX, or 1xMHX. After one day, new T75 flasks were seeded with the cultures at a starting density of 1x105 cells / mL and cells were counted at 24-hour intervals for one week. The doubling times for the first 5 days were calculated using the formula in SOP PD32.025 and are shown above the bars. [Figure 9] Graphical representation of the stability of mAb production over time from two rTNV148B producing cell lines. After transfection and subcloning, subclones of cells that were 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 selection. Cells were serially passaged by splitting cultures every 4-6 days to maintain new viable cultures while simultaneously exhausting previous cultures. Aliquots of exhausted cell supernatants were collected immediately after the cultures were exhausted and stored until mAb concentrations were determined. ELISA for human IgG was performed on all sample aliquots at the same time. [Figure 10]Figure 1 shows the weight change of arthritic mouse model mice Tg197 in response to anti-TNF antibodies of the invention compared to the control of Example 4. Approximately 4-week-old Tg197 study mice were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus of Dulbecco's PBS (D-PBS) or an anti-TNF antibody of the invention (TNV14, TNV148, or TNV196) at either 1 mg / kg or 10 mg / kg. When weight was analyzed as the change from pre-treatment, animals treated with 10 mg / kg cA2 consistently showed higher weight gain than D-PBS-treated animals throughout the study. This weight gain was significant from weeks 3 to 7. Animals treated with 10 mg / kg TNV148 also achieved significant weight gain in week 7 of the study. [Figure 11A] FIG. 1 shows the progression of disease severity based on arthritis index as shown in Example 4. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to show a significant decrease in AI after week 3 when compared to the D-PBS treatment group. There was no significant difference between the 10 mg / kg treatment groups when each was compared to the other at a similar dose (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148 and 196). When comparing 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. TNV148 at 1 mg / kg was also significantly lower than the TNV14 at 1 mg / kg treatment group at weeks 3 and 4. TNV196 showed a significant reduction in AI (when compared to the D-PBS treatment group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment that remained significant at the end of the study. [Figure 11B]FIG. 1 shows the progression of disease severity based on arthritis index as shown in Example 4. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to show a significant decrease in AI after week 3 when compared to the D-PBS treatment group. There was no significant difference between the 10 mg / kg treatment groups when each was compared to the other at a similar dose (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148 and 196). When comparing 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. TNV148 at 1 mg / kg was also significantly lower than the TNV14 at 1 mg / kg treatment group at weeks 3 and 4. TNV196 showed a significant reduction in AI (when compared to the D-PBS treatment group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment that remained significant at the end of the study. [Figure 11C] FIG. 1 shows the progression of disease severity based on arthritis index as shown in Example 4. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to show a significant decrease in AI after week 3 when compared to the D-PBS treatment group. There was no significant difference between the 10 mg / kg treatment groups when each was compared to the other at a similar dose (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148 and 196). When comparing 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. TNV148 at 1 mg / kg was also significantly lower than the TNV14 at 1 mg / kg treatment group at weeks 3 and 4. TNV196 showed a significant reduction in AI (when compared to the D-PBS treatment group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment that remained significant at the end of the study. [Figure 12] Figure 1 shows the weight change of arthritic mouse model mice Tg197 in response to anti-TNF antibodies of the present invention compared to the control of Example 5. Tg197 study mice, approximately 4 weeks of age, were assigned to one of eight treatment groups based on body weight and treated with an intraperitoneal bolus of control article (D-PBS) or 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 efficacy of the test article. Serum samples obtained from animals in groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3 and 4. [Figure 13A] 1 is a graph depicting the progression of disease severity in Example 5 based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS treatment group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and was also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13B]1 is a graph depicting the progression of disease severity in Example 5 based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS treatment group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and was also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13C]1 is a graph depicting the progression of disease severity in Example 5 based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS treatment group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and was also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 14] Figure 1 shows the weight change of arthritic mouse model mice Tg197 in response to anti-TNF antibodies of the present invention compared to the control of Example 6. Tg197 study mice, approximately 4 weeks of age, were assigned to one of six treatment groups based on sex and weight and treated with a single intraperitoneal bolus of either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148). The study utilized D-PBS and 10 mg / kg cA2 control groups. [Figure 15]FIG. 1 depicts the progression of disease severity based on the arthritis index as shown in Example 6. All treatment groups showed some protection at early time points, with 5 mg / kg cA2 and 5 mg / kg TNV148 showing significant reductions in AI at weeks 1-3, and all treatment groups showing significant reductions at week 2. Later in the study, animals treated with 5 mg / kg cA2 showed some protection, with significant reductions at weeks 4, 6, and 7. Both low dose (3 mg / kg) cA2 and TNV148 showed significant reductions at week 6, and all treatment groups showed significant reductions at week 7. None of the treatment groups were able to maintain significant reductions at the end of the study (week 8). There were no significant differences between any of the treatment groups (except the saline control group) at any time point. [Figure 16] Figure 1 shows the weight change of arthritis mouse model mice Tg197 in response to anti-TNF antibodies of the present invention compared to the control of 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 study mice were assigned to one of nine treatment groups based on sex and weight and treated with Dulbecco's PBS (D-PBS) or a single intraperitoneal bolus of 1 mg / kg antibody (TNV148, rTNV148B). [Figure 17]FIG. 1 depicts the progression of disease severity based on arthritis index as shown in Example 7. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 4 and continuing throughout the remainder of the study (week 8). Both the TNV148-treated group and the 1 mg / kg cA2-treated group showed a significant reduction in AI at week 4. A previous study (P-099-017) showed that TNV148 was slightly more effective in reducing the arthritis index after a single 1 mg / kg intraperitoneal bolus, but this study showed slightly higher AI from groups treated with both versions of the TNV antibody. The 1 mg / kg cA2 treated group (except at week 6) did not increase significantly when compared to the 10 mg / kg cA2 group, and the TNV148 treated group was significantly higher at weeks 7 and 8, but there were no significant differences in AI between 1 mg / kg cA2, 1 mg / kg TNV148, and 1 mg / kg TNV148B at any time point in the study. [Figure 18] Diagram of pJIA clinical study design. DBL = database lock, LTE = long-term extension, MSE = major secondary endpoint, PE = primary endpoint. IV infusion of golimumab 80 mg / m2 is marked with an arrow at the indicated times. Patients also received commercially available MTX at the same weekly BSA-based dose as at the time of study enrollment until at least week 28. [Figure 19] Figure 1 shows the percentage of JIA ACR30, 50, 70, and 90 responders by week 28. The symbols for JIA ACR30, 50, 70, and 90 are closed circle, closed square, closed triangle, and closed diamond, respectively. [Figure 20] FIG. 1 shows the proportion of patients with inactive disease by week 28. [Figure 21] Figure 1 shows the proportion of patients with JADAS 10, 27, or 71 minimal disease activity by week 28. *Note: In this analysis, JADAS 10, 27, and 71 endpoint values ​​are the same. [Figure 22]Goodness-of-fit plots of the population PK (PPK) model of individual predictions (μg / mL), population predictions (μg / mL), and days after first dose against observed concentrations (μg / mL) and against conditional weighted residuals (CWRES). [Figure 23] Figure 2 shows the primary endpoint at week 28 in different age categories for observed Ctrof,ss (serum golimumab trough concentration in μg / mL) and post-hocAUC,ss (AUCss of serum golimumab concentration in μg*day / mL) over 8 weeks. The horizontal line within the box represents the median, the lower edge of the box represents the 1st quartile, the upper edge of the box represents the 3rd quartile, and the whiskers are the most extreme observations within a 1.5×IQ range. [Figure 24] Figure 1 shows secondary endpoints at week 52 in different age categories for observed Ctrof,ss (serum golimumab trough concentration in μg / mL) and post-hocAUC,ss (AUCss of serum golimumab concentration in μg*day / mL) over 8 weeks. The horizontal line within the box represents the median, the lower edge of the box represents the 1st quartile, the upper edge of the box represents the 3rd quartile, and the whiskers are the most extreme observations within a 1.5×IQ range. [Diagram 25] FIG. 1 shows PK at week 28 by weight quartile for C trough,ss (serum golimumab trough concentration in μg / mL) and post-hoc AUC,ss (AUCss of serum golimumab concentration in μg*day / mL) over 8 weeks. The horizontal line within the box represents the median, the lower edge of the box represents the 1st quartile, the upper edge of the box represents the 3rd quartile, and the whiskers are the most extreme observations within a 1.5×IQ range. [Figure 26]FIG. 1 shows PK at week 28 by C-reactive protein (CRP) quartiles for Ctrof,ss (serum golimumab trough concentration in μg / mL) and post-hocAUC,ss (AUCss of serum golimumab concentration in μg*day / mL) over 8 weeks. The horizontal line within the box represents the median, the lower edge of the box represents the 1st quartile, the upper edge of the box represents the 3rd quartile, and the whiskers are the most extreme observations within a 1.5×IQ range. [Figure 27] FIG. 1 shows observed Ctrof,ss (serum golimumab trough concentrations in μg / mL) at week 28 in different age categories of pJIA subjects in the GO-VIVA study, and at weeks 20 and 36 in adult RA subjects in the GO-FURTHER study. [Figure 28] FIG. 1 shows post-hoc AUC,ss (AUCss of serum golimumab concentrations in μg*day / mL) over 8 weeks at week 28 in different age categories of pJIA subjects in the GO-VIVA study and in adult RA subjects in the GO-FURTHER study. [Figure 29A] FIG. 1 shows JIA ACR response at week 52 by PK quartiles for serum golimumab concentrations (μg / mL) and depicts JIC ACR30 responders. [Figure 29B] FIG. 1 shows JIA ACR response at week 52 by PK quartiles for serum golimumab concentrations (μg / mL) and depicts JIC ACR50 responders. [Figure 29C] FIG. 1 shows JIA ACR response at week 52 by PK quartiles for serum golimumab concentrations (μg / mL) and depicts JIC ACR70 responders. [Figure 29D] FIG. 1 shows JIA ACR response at week 52 by PK quartiles for serum golimumab concentrations (μg / mL) and depicts JIC ACR90 responders. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention provides a composition comprising an anti-TNF antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37, and a manufacturing process for producing such an anti-TNF antibody.

[0054] As used herein, "anti-tumor necrosis factor alpha antibodies", "anti-TNF antibodies", "anti-TNF antibody portions" or "anti-TNF antibody fragments", and / or "anti-TNF antibody variants", and the like, include any protein- or peptide-containing molecule, including molecules comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) or ligand binding portion thereof of a heavy or light chain, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof, or at least a portion of a TNF receptor or binding protein, that can be incorporated into an antibody of the invention. Such antibodies optionally further affect a particular ligand, including, but not limited to, such antibodies modulate, decrease, increase, antagonize, agonize, reduce, mitigate, block, inhibit, abrogate, and / or interfere with at least one TNF activity or binding, or TNF receptor activity or binding, in vitro, in situ, and / or in vivo. As a non-limiting example, a suitable anti-TNF antibody, specified portion or variant of the present invention can bind to at least one TNF or a specified portion, variant or domain thereof. A suitable anti-TNF antibody, specified portion or variant can also optionally affect at least one of TNF activities or functions, such as, but not limited to, RNA, DNA, or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production, and / or synthesis. The term "antibody" is further intended to encompass antibodies, digested fragments, specified portions and variants thereof, including antibody mimetics, or portions of antibodies or specified fragments or parts thereof that mimic the structure and / or function of an antibody, including single chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to mammalian TNF. For example, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab') 2Antibody fragments capable of binding TNF or portions thereof, including, but not limited to, Fc' (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reassembly), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra).

[0055] Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and / or described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, F(ab') 2 The combination of genes encoding the heavy chain portion is 1 The antibody can be designed to contain DNA sequences encoding the antibody domains and / or hinge region. The various portions of the antibody can be joined chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

[0056] As used herein, the term "human antibody" refers to an antibody that is humanized in a manner that comprises substantially all parts of the protein (e.g., CDRs, frameworks, C L , C H Domain (e.g., C H 1. C H 2, and CH3), hinge (V L , V H)) refers to antibodies that are substantially non-immunogenic in humans with only minor sequence changes or mutations. Similarly, antibodies designated primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), and other mammalian animals refer to antibodies specific for such species, subgenus, genus, subfamily, and family. Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations optionally and preferably retain or reduce immunogenicity in humans or other species compared to the unmodified antibody. Thus, a human antibody is distinct from a chimeric antibody or a humanized antibody. It is noted that a human antibody can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Moreover, when a human antibody is a single chain antibody, it can include a linker peptide not found in naturally occurring human antibodies. For example, an Fv can include a linker peptide, such as 2 to about 8 glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region, such a linker peptide being considered to be of human origin.

[0057] Bispecific, e.g., DuoBody® (bispecific antibodies), heterospecific, heterobinding, or similar antibodies may also be used, which are monoclonal, preferably human or humanized, antibodies with binding specificities for at least two different antigens. In this case, one of the binding specificities is for at least one TNF protein and the other is for any other antigen. Methods for producing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Because purification of the correct molecule, which is usually done by affinity chromatography steps, can be laborious with low product yields, different strategies have been developed to facilitate the production of bispecific antibodies.

[0058] Full-length bispecific antibodies can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, for example, in a cell-free environment in vitro or using co-expression, by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules with different specificities. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an intra-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm may be engineered to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody with two Fab arms or half molecules, each capable of binding a different epitope.

[0059] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0060] As used herein, "heterodimerization" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0061] A "knob-in-hole" strategy (see, for example, WO 2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with the "hole" and the heavy chain with the "knob". Exemplary pairs of CH3 substitutions that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain).

[0062] Other strategies, such as promoting heavy chain heterodimer formation using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, may be used as described in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. Patent Application Publication No. 2011 / 0123532. In another strategy, heterodimerization can be achieved by the following substitutions: L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A ...I_K392M_T394W / F405A_Y407V, T366I_K392M_T394W / F405A_Y407V, T366I_K392M_T394W / F405A_Y407V, T366I_K392M_T394W / F405A_Y407V, T366I_K3 66A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain).

[0063] In addition to the methods described above, bispecific antibodies can be generated in an in vitro cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions that cause disulfide bonds to isomerize, according to the method described in WO 2011 / 131746. In this method, a first monospecific bivalent antibody and a second monospecific bivalent antibody are engineered to have certain substitutions in the CH3 domain that promote the stability of the heterodimer, and these antibodies are incubated together under reducing conditions sufficient to cause the cysteines in the hinge region to isomerize the disulfide bonds, thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions can be optimally returned to non-reducing conditions. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a temperature of at least 20° C., in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol, at a pH of 5-8, e.g., pH 7.0 or pH 7.4, for at least 90 minutes may be used.

[0064] Anti-TNF antibodies (also referred to as TNF antibodies) useful in the methods and compositions of the invention may optionally be characterized by high affinity binding to TNF, and optionally and preferably low toxicity. In particular, antibodies of the invention, specified fragments or variants thereof, in which the individual components, such as the variable region, constant region and framework, individually and / or collectively, optionally and preferably, have low immunogenicity, are useful in the present invention. Antibodies that can be used in the present invention may optionally be characterized by their ability to treat patients for extended periods with measurable alleviation of symptoms and low and / or acceptable toxicity. Low or acceptable immunogenicity and / or high affinity, as well as other favorable properties, may contribute to the therapeutic results obtained. "Low immunogenicity" is defined herein as a significant increase in HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50%, of treated patients, and / or a low titer increase (less than about 300, preferably less than about 100, as measured by double antigen enzyme immunoassay) in treated patients (Elliott et al., Lancet 344:1125-1127 (1994), incorporated herein by reference in its entirety).

[0065] Utility: The isolated nucleic acids of the present invention may be used to generate at least one anti-TNF antibody or specified variant thereof, which may be used to measure or act in a cell, tissue, organ or animal (including mammals and humans) to diagnose, monitor, regulate, treat, alleviate, help prevent the occurrence of, or reduce the symptoms of at least one TNF condition selected from, but not limited to, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant and / or neurological disorder or disease.

[0066] Such methods may include administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment, mitigation, prevention or reduction of symptoms, effects or mechanisms. An effective amount may include an amount of about 0.001-500 mg / kg per single (e.g., bolus), multiple, or continuous administration, or an amount that achieves a serum concentration of 0.01-5000 μg / mL per single, multiple, or continuous administration, or any effective range or value therein, as determined using known methods described herein or known in the relevant art. References. All publications or patents cited herein are incorporated by reference in their entirety and represent the state of the art at the time of the invention and / or provide a description and enablement of the invention. Publications refer to any scientific publications or patent publications or any other information available in any media format, including all recorded in electronic or printed form. The following publications are incorporated by reference in their entirety: Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

[0067] Antibodies of the invention: At least one anti-TNF antibody of the invention comprising all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3, and / or all of the light chain variable CDR regions of SEQ ID NOs: 4, 5, and 6, may optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, e.g., Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), each of which is incorporated by reference in its entirety.

[0068] Human antibodies specific for human TNF protein or fragments thereof may be raised against suitable immunogenic antigens such as isolated and / or TNF protein, or portions thereof (including synthetic molecules such as synthetic peptides). Other specific or general mammalian antibodies may be raised as well. Preparation of immunogenic antigens and generation of monoclonal antibodies may be carried out using any suitable technique.

[0069] In one approach, hybridomas are generated using a suitable immortalized cell line (e.g., Sp2 / 0, Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or heteromylomas, their fusion products, or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art. See, e.g., www.atcc.org, www.lifetech.com. Antibody-producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptile, fish, mammalian, , rodent, horse, ovine, caprine, sheep, primate, eukaryote, or any other cell that expresses heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, such as genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded or triple-stranded, hybridized, etc., or combinations thereof. See, e.g., Ausubel, supra, and Colligan, supra, Immunology, Chapter 2, both of which are incorporated herein by reference in their entireties.

[0070] Antibody-producing cells can also be obtained from the peripheral blood, or preferably the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. Any other suitable host cells can also be used to express heterologous or endogenous nucleic acid encoding the antibodies of the invention, specified fragments or variants thereof. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods and cloned by limiting dilution or cell sorting or other known methods. Cells producing antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

[0071] Other suitable methods of generating or isolating antibodies of the required specificity can be used, including, but not limited to, recombinant antibody selection from peptide or protein libraries (e.g., but not limited to, display libraries of bacteriophage, ribosomal, oligonucleotide, RNA, cDNA, etc.; see, for example, Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, DE), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden), Dyax Corp., Enzon, Affymax / Biosite, Xoma (Berkeley, CA), Ixsys. See, e.g., European Patent No. 368,684, International Application No. PCT / GB91 / 01134, International Application No. PCT / GB92 / 01755, International Application No. PCT / GB92 / 002240, International Application No. PCT / GB92 / 00883, International Application No. PCT / GB93 / 00605, U.S. Patent Application No. 08 / 350260 (5 / 12 / 94), International Application No. PCT / GB94 / 01422, International Application No. PCT / GB94 / 02662, International Application No. PCT / GB97 / 01835, (CAT / MRC), International Publication No. WO 90 / 14443, International Publication No. WO 90 / 14424, International Publication No. WO 90 / 14430, International Application No. PCT / US94 / 1234, International Publication No. WO 92 / 18619, International Publication No. WO 96 / 07754, (Scripps), European Patent No. 614989 (Morpho Sys), WO 95 / 16027 (BioInvent), WO 88 / 06630, WO 90 / 3809 (Dyax), U.S. Patent No. 4,704,692 (Enzon), International Application PCT / US91 / 02989 (Affymax), WO 89 / 06283, European Patent No. 371998, European Patent No. 550400, (Xoma), European Patent No. 2 29046, International Application PCT / US91 / 07149 (Ixsys), or stochastically generated peptides or proteins - U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86 / 05803, EP 590689 (Ixsys, now Applied Molecular Evolution (AME), each of which is incorporated herein by reference in its entirety), or rely on immunization of transgenic animals capable of generating a repertoire of human antibodies as known in the art and / or described herein (e.g., SCID mice, Nguyen et al., Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998) (each of which is incorporated by reference in its entirety, as are any related patents and applications). Such techniques include ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA, 95:14130-14135 (Nov. 1998)), single cell antibody generation techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Pat. No. 5,627,052; Wen et al., J. Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)), gel microdroplets and flow cytometry (Powell et al., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, MA; Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al., Bio / Technol. 13:787-790 (1995); B cell selectors (Steenbakkers et al., Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers BV, Amsterdam, Netherlands (1988)).

[0072] Methods for engineering or humanizing non-human or human antibodies can be used as well and are well known in the art. Generally, a humanized or engineered antibody has one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable, constant, or other domain of a known human sequence.

[0073] Known human Ig sequences are disclosed in numerous publications and websites, e.g., www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / ; www.abcam.com / ; www.antibodyresource.com / onlinecomp.html; www.public.iastate.edu / ~pedro / research_tools.html; www.mgen.uni-heidelberg.de / SD / IT / IT.html; www.whfreeman.com / immunology / CH05 / kuby05.htm; www.library.thinkquest.org / 12429 / Immune / Antibody.html; www.hhmi.org / grants / lectures / 1996 / vlab / ; www.path.cam.ac.uk / ~mrc7 / mikeimages.html; www.antibodyresource.com / ; www.mcb.harvard.edu / BioLinks / Immunology.html. www.immunologylink.com / ; www.pathbox.wustl.edu / ~hcenter / index.html; 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.html; 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 / AEPStart.html; www.baserv.uci.kun.nl / ~jraats / links1.html; www.recab.uni-hd.de / immuno.bme.nwu.edu / ; www.mrc-cpe.cam.ac.uk / imt-doc / public / INTRO.html; www.ibt.unam.mx / vir / V_mice.html;imgt.cnusc.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 / Slide01.html; www.cryst.bbk.ac.uk / ~ubcg07s / ; www.nimr.mrc.ac.uk / CC / ccaewg / ccaewg.htm; www.path.cam.ac.uk / ~mrc7 / humanisation / TAHHP.html; www.ibt.unam.mx / vir / structure / stat_aim.html; www.biosci.missouri.edu / smithgp / index.html; www.cryst.bioc.cam.ac.uk / ~fmolina / Web-pages / Pept / spottech.html; www.jerini.de / frproducts.html; www.patents.ibm.com / ibm.html.Kabat et al., and Sequences of Proteins of Immunological Interest, USDept. Health (1983), each of which is incorporated herein by reference in its entirety.

[0074] Such imported sequences can be used to reduce immunogenicity or to reduce, enhance or modify binding, affinity, binding rate constants, dissociation rate constants, avidity, specificity, half-life, or any other suitable property, as known in the art. Generally, some or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids. The antibody can also be optionally humanized while retaining high affinity for the antigen and other favorable biological properties. To this end, optionally, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the antigen-binding ability of the candidate immunoglobulin. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues directly and most substantially influence antigen binding.Humanization or engineering of the antibodies of the invention may be carried out by any of the methods described in Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. SCi. USA 89:4285 (1992); Presta et al. al., J.Immunol.151:2623 (1993), U.S. Patent No. 5723323, U.S. Patent No. 5976862, U.S. Patent No. 5824514, U.S. Patent No. 5817483, U.S. Patent No. 5814476, U.S. Patent No. 5763192, U.S. Patent No. 5723323, U.S. Patent No. 5, No. 766886, No. 5714352, No. 6204023, No. 6180370, No. 5693762, No. 5530101, No. 5585089, No. 5225539, No. 4816567, International application PCT / :US98 / 1628 0, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755, WO 90 / 14443, WO 90 / 14424, WO 90 / 14430, EP 229246 (each of which is incorporated by reference in its entirety, including the references cited therein).

[0075] Anti-TNF antibodies can also be produced, optionally, by immunization of transgenic animals (e.g., mice, rats, hamsters, non-human primates, etc.) capable of producing a repertoire of human antibodies as described herein and / or known in the art. Cells that produce human anti-TNF antibodies can be isolated from such animals and immortalized using suitable methods, such as those described herein.

[0076] Transgenic mice capable of producing a repertoire of human antibodies that bind to human antigens can be produced by known methods (see, for example, but not limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016, and 5,789,650 issued to Lonberg et al.; Jakobovits et al., WO 98 / 50433; Jakobovits et al., WO 98 / 24893; Lonberg et al., WO 98 / 24884; Lonberg et al., WO 97 / 13852 ... al., WO 94 / 25585; Kucherlapate et al., WO 96 / 34096; Kucherlapate et al., EP 0463151(B1); Kucherlapate et al., EP 0710719(A1); Surani et al., U.S. Pat. No. 5,545,807; Bruggemann et al., WO 90 / 04036; Bruggemann et al., EP 0438474(B1); Lonberg et al., EP 0814259(A2); Lonberg et al., GB 2272440(A); Lonberg et al. Nature 368:856-859(1994); Taylor et al. al.,Int.Immunol.6(4)579-591(1994), Green et al., Nature Genetics 7:13-21(1994), Mendez et al., Nature Genetics 15:146-156(1997), Taylor et al., Nucleic Acids Research 20(23):6287-6295(1992), Tuaillon et al., Proc Natl Acad Sci USA 90(8)3720-3724(1993), Lonberg et al., Int Rev Immunol 13(1):65-93(1995), and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), each of which is incorporated herein by reference in its entirety. Generally, these mice contain at least one transgene that includes DNA derived from at least one human immunoglobulin locus that is functionally rearranged, or capable of undergoing functional rearrangement. The endogenous immunoglobulin loci of such mice can be disrupted or deleted to eliminate the ability of the mice to produce antibodies encoded by endogenous genes.

[0077] Screening of antibodies for specific binding to similar proteins or fragments can be successfully accomplished using peptide display libraries. This method involves screening a large sampling of peptides for individual members with the desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be 3-5000 or more amino acids in length, frequently 5-100 amino acids long, and often about 8-25 amino acids long. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have also been described. One type involves the display of peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence that encodes a particular displayed peptide sequence. Such methods are described in WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278. Other systems for generating peptide libraries have aspects of both in vitro chemical synthesis and recombinant methods. See WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See also U.S. Patent Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from sources such as Invitrogen (Carlsbad, CA) and Cambridge antibody Technologies (Cambridgeshire, UK).See, e.g., U.S. Patent Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, and 5,856,456, all assigned to Enzon; ​​U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500, all assigned to Dyax; U.S. Patent Nos. 5,427,908 and 5,580,717, all assigned to Affymax; Cambridge antibody See US Patent No. 5,885,793 assigned to Sigma Technologies, US Patent No. 5,750,373 assigned to Genentech, US Patent No. 5,618,920 assigned to Xoma, US Patent No. 5,595,898, US Patent No. 5,576,195, US Patent No. 5,698,435, US Patent No. 5,693,493, US Patent No. 5,698,417 assigned to Xoma, Colligan, supra, Ausubel, supra, or Sambrook, supra, each of which is incorporated herein by reference in its entirety.

[0078] The antibodies of the present invention can also be prepared using at least one anti-TNF antibody encoding nucleic acid to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such antibodies in its milk. Such animals can be prepared using known methods. See, for example, but not limited to, U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, 5,304,489, etc., each of which is incorporated herein by reference in its entirety.

[0079] The antibodies of the present invention can further be prepared using at least one anti-TNF antibody-encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to, tobacco and corn) that produce such antibodies, specified portions or variants in plant parts or cells cultured therefrom. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins using, for example, an inducible promoter have been successfully used to provide large quantities of recombinant proteins. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein. Transgenic corn has also been used to express mammalian proteins at commercial production levels with biological activity equivalent to proteins produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein. Antibodies have also been produced in large quantities from transgenic plant seeds containing antibody fragments, such as single chain antibodies (scFv), including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and references cited therein. Thus, the antibodies of the present invention can also be produced using transgenic plants by known methods. See, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995), Ma et al., Plant Physiol. 109:341-6 (1995), Whitelam et al., Biochem. Soc. Trans. 22:940-944 (1994) and references cited therein. See also, but not limited to, Plant Expression of Antibodies in General, each of the above references is incorporated herein by reference in its entirety.

[0080] The antibodies of the present invention have a wide range of affinities (K D In a preferred embodiment, at least one human mAb of the present invention can optionally bind human TNF with high affinity. For example, a human mAb can bind human TNF with approximately 10 -7 M or less, for example, 0.1 to 9.9 (or any range or value therein) x 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 or any range or value therein, including, but not limited to, K D can be combined with

[0081] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, e.g., Berzofsky, et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992), and methods described therein.) The affinity measured for a particular antibody-antigen interaction may differ when measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other antigen-binding parameters (e.g., K D , K a , K d Measurements of ) are preferably made using standard solutions of antibody and antigen, and standard buffers, such as those described herein.

[0082] Nucleic Acid Molecules. Using the information provided herein, such as a nucleotide sequence encoding at least 70-100% of the contiguous amino acids of at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, an identified fragment, variant, or consensus sequence thereof, or a deposited vector containing at least one of these sequences, a nucleic acid molecule of the invention encoding at least one anti-TNF antibody comprising all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3 and / or all of the light chain variable CDR regions of SEQ ID NOs: 4, 5, and 6 can be obtained using methods described herein or known in the art.

[0083] The nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, called the antisense strand.

[0084] The isolated nucleic acid molecules of the present invention may include an open reading frame (ORF), optionally having one or more introns, for example, but not limited to, a nucleic acid molecule comprising at least one specified portion of at least one CDR, such as CDR1, CDR2, and / or CDR3 of at least one heavy chain (e.g., SEQ ID NOs: 1-3) or light chain (e.g., SEQ ID NOs: 4-6), a nucleic acid molecule comprising a coding sequence of an anti-TNF antibody or variable region (e.g., SEQ ID NOs: 7, 8), as well as a nucleic acid molecule comprising a nucleotide sequence that is substantially different from the above-mentioned nucleic acid molecules, but which still encodes at least one anti-TNF antibody described herein and / or known in the art due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Thus, it would be routine for one of skill in the art to generate such degenerate nucleic acid variants that encode a particular anti-TNF antibody of the present invention. See, e.g., Ausubel et al., supra, and such nucleic acid variants are included in the present invention. Non-limiting examples of isolated nucleic acid molecules of the invention include SEQ ID NOs: 10, 11, 12, 13, 14, and 15, which correspond to non-limiting examples of nucleic acids encoding the HC variable regions and LC variable regions of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, respectively.

[0085] As shown herein, the nucleic acid molecules of the invention, including nucleic acids encoding anti-TNF antibodies, can include those that themselves encode the amino acid sequence of an antibody fragment, sequences encoding full length antibodies or portions of antibodies, coding sequences for antibodies, fragments or portions, and additional sequences, such as at least one intron, with or without the aforementioned additional coding sequences, including, but not limited to, non-coding 5' and 3' sequences, such as transcribed non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and stability of mRNA), as well as coding sequences for at least one signal leader or fusion peptide, additional coding sequences encoding additional amino acids, e.g., amino acids that provide additional functions. Thus, the antibody coding sequence can be fused to a marker sequence, for example, a sequence encoding a peptide that facilitates purification of the antibody containing the antibody fragment or portion to which it is fused.

[0086] A polynucleotide that selectively hybridizes to the polynucleotides described herein. The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids that contain such polynucleotides. For example, the polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotide is a genomic or cDNA sequence that is isolated or otherwise complementary to a cDNA in a human or mammalian nucleic acid library.

[0087] Preferably, the cDNA library contains at least 80% of full-length sequences, preferably at least 85% or 90% of full-length sequences, and more preferably at least 95% of full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are typical, but not limited to, using sequences with low sequence identity to the complementary sequence. Optionally, medium and high stringency conditions can be used for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0088] Optionally, the polynucleotides of the invention will encode at least a portion of the antibodies encoded by the polynucleotides described herein. The polynucleotides of the invention encompass nucleic acid sequences available for selective hybridization to polynucleotides encoding the antibodies of the invention. See, e.g., Ausubel, supra; Colligan, supra. Each is incorporated herein by reference in its entirety.

[0089] Construction of Nucleic Acids The isolated nucleic acids of the invention can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or a combination thereof, as are known in the art.

[0090] The nucleic acid may conveniently contain sequences in addition to the polynucleotide of the invention. For example, a multiple cloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the invention. For example, a hexahistidine marker sequence provides a convenient means for purifying the protein of the invention. The nucleic acid of the invention (excluding the coding sequence) is optionally a vector, adapter or linker for cloning and / or expression of the polynucleotide of the invention.

[0091] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0092] Recombinant methods for constructing nucleic acids. The isolated nucleic acid compositions of the present invention, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning procedures known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to the polynucleotides of the present invention are used to identify desired sequences in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0093] Methods for screening and isolating nucleic acids. Probes based on the sequences of the polynucleotides of the invention as disclosed herein can be used to screen cDNA or genomic libraries. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that various degrees of hybridization stringency can be used in the assay, and either the hybridization or the wash medium can be stringent. The more stringent the hybridization conditions, the greater the degree of complementarity between the probe and the target at which duplex formation occurs. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is successfully altered by changing the polarity of the reaction solution, for example, by manipulating the formamide concentration within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies depending on the stringency of the hybridization medium and / or washing medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therein. However, it should be understood that minor differences in sequences in the probe and primer can be compensated for by reducing the stringency of the hybridization and / or washing medium.

[0094] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present invention without undue experimentation, based on the teachings and guidance provided herein.

[0095] Known methods of DNA or RNA amplification include polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al.; 4,795,699 and 4,921,794 to Tabor, et al.; 5,142,033 to Innis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 to Gyllensten, et al.; 4,889,818 to Gelfand, et al.; Silver et al., No. 4,994,370 to Biswas, No. 4,766,067 to Ringold, No. 4,656,134 to Ringold), and RNA-mediated amplification (U.S. Pat. No. 5,130,238 to Malek et al., having the trade name NASBA), which uses antisense RNA against a target sequence as a template for double-stranded DNA synthesis (the entire contents of which are incorporated herein by reference). (See, e.g., Ausubel, supra, or Sambrook, supra).

[0096] For example, the polymerase chain reaction (PCR) technique can be used to amplify the sequences of the polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, for cloning nucleic acid sequences encoding proteins to be expressed, for generating nucleic acids to be used as probes to detect the presence of desired mRNA in a sample, for sequencing nucleic acids, or for other purposes. Examples of techniques sufficient to guide the skilled artisan through in vitro amplification methods can be found in Berger, supra; Sambrook, supra; and Ausubel, supra; and Mullis, et al., U.S. Patent No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc, San Diego, CA (1990). Commercial kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yields of long PCR products.

[0097] Synthetic methods for constructing nucleic acids. The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide that can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that although chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by ligation of shorter sequences.

[0098] Recombinant Expression Cassette. The present invention further provides a recombinant expression cassette comprising a nucleic acid of the present invention. The nucleic acid sequence of the present invention, e.g., a cDNA or genomic sequence encoding an antibody of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically comprises a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acid of the present invention.

[0099] In some embodiments, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into a suitable location (upstream, downstream, or within an intron) of a non-heterologous form of a polynucleotide of the invention to up- or down-regulate expression of the polynucleotide of the invention. For example, endogenous promoters can be altered by mutation, deletion, and / or substitution in vivo or in vitro.

[0100] Vectors and host cells. The present invention also relates to vectors comprising the isolated nucleic acid molecules of the present invention, host cells engineered with recombinant vectors, and the production of at least one anti-TNF antibody by recombinant techniques well known in the art. See, e.g., Sambrook et al., supra; Ausubel et al., supra, each of which is incorporated herein by reference in its entirety.

[0101] The polynucleotide can be optionally linked to a vector containing a selection marker for propagation in a host.Generally, the plasmid vector is introduced into a precipitate such as a calcium phosphate precipitate, or into a complex with a charged lipid.If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell.

[0102] The DNA insert should be operably linked to a suitable promoter. The expression construct further comprises a transcription initiation site, a transcription termination site, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct will preferably contain a translation initiation site at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the translated mRNA. For expression in mammalian or eukaryotic cells, UAA and UAG are preferred.

[0103] The expression vector preferably includes at least one selectable marker, although this is optional. Such markers include, for example, but are not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos. 4,399,216, 4,634,665, 4,656,134, 4,956,288, 5,149,636, 5,179,017, ampicillin, neomycin (G418), mycophenolic acid or glutamine synthetase (GS) (U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance genes for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria or prokaryotes (see above). (The disclosures herein are incorporated by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to one of skill in the art. Introduction of the vector construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0104] At least one antibody of the invention may be expressed in modified form, such as a fusion protein, and may contain not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the antibody to improve stability and persistence in the host cell during purification or during subsequent processing and storage. Peptide moieties may also be added to the antibody of the invention to facilitate purification. Such regions may be removed prior to final preparation of the antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74, Ausubel, supra, Chapters 16, 17 and 18.

[0105] One of skill in the art is familiar with the numerous expression systems available for expressing nucleic acids encoding the proteins of the invention.

[0106] Alternatively, the nucleic acids of the invention can be expressed in a host cell by switching on (by engineering) in a host cell that contains endogenous DNA encoding an antibody of the invention. Such methods are well known in the art, such as those described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entireties.

[0107] An example of a cell culture useful for producing an antibody, specified portion or variant thereof is a mammalian cell. Mammalian cell lines are often in the form of a monolayer of cells, although suspensions or bioreactors of mammalian cells can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, 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-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection (Manassas, VA). Preferred host cells include CHO cells and cells derived from the lymphatic system, such as myeloma and lymphoma cells. Particularly preferred host cells are CHO cells, P3X63Ag8.653 cells (ATCC Accession No. CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851).

[0108] Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication, a promoter (e.g., the late or early SV40 promoter, a CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1α promoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulin promoter, an enhancer, and / or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., the SV40 large T Ag polyaddition site), and transcription termination sequences. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the invention are known and / or available, for example, from the American Type Culture Collection's catalog of cell lines and hybridomas or other known or commercial sources.

[0109] When eukaryotic host cells are used, typically polyadenylation or transcription termination sequences are incorporated into the vector. An example of a termination sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). In addition, gene sequences for controlling replication in the host cell can be incorporated into the vector, as is known in the art.

[0110] Purification of antibodies. Anti-TNF antibodies can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001), e.g., chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

[0111] The antibodies of the present invention include naturally purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plants, insect and mammalian cells. Depending on the host used in a recombinant production procedure, the antibodies of the present invention may be glycosylated or non-glycosylated, but are preferably glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra; Ausubel, supra, Chapters 10, 12, 13, 16, 18, and 20; Colligan, Protein Science, supra, Chapters 12-14, all of which are incorporated herein by reference in their entireties.

[0112] Exemplary Anti-TNF Antibodies The isolated antibodies of the invention comprising all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3 and / or all of the light chain variable CDR regions of SEQ ID NOs: 4, 5, and 6 include the amino acid sequences of the antibodies disclosed herein encoded by any suitable polynucleotide, or any isolated or prepared antibody. Preferably, the human antibodies or antigen-binding fragments bind to human TNF, thereby partially or substantially neutralizing at least one biological activity of the protein. An antibody or specified portion or variant thereof that partially or preferably substantially neutralizes at least one biological activity of at least one TNF protein or fragment can bind to the protein or fragment, thereby inhibiting an activity mediated through binding of TNF to a TNF receptor or through other TNF-dependent or mediated mechanisms. As used herein, the term "neutralizing antibody" refers to an antibody capable of inhibiting TNF-dependent activity by about 20-120%, preferably at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more, depending on the assay. The ability of an anti-TNF antibody to inhibit TNF-dependent activity is preferably assessed by at least one suitable TNF protein or receptor assay described herein and / or known in the art. The human antibodies of the present invention may be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and may include a κ or λ light chain. In one embodiment, the human antibody includes an IgG heavy chain or a defined fragment, e.g., at least one of the following isotypes: IgG1, IgG2, IgG3, or IgG4. Antibodies of this type can be prepared by using transgenic mice or other transgenic non-human mammals that contain at least one human light chain (e.g., IgG, IgA) and IgM (e.g., γ1, γ2, γ3, γ4) transgene as described herein and / or known in the art. In another embodiment, the anti-human TNF human antibody comprises an IgG1 heavy chain and an IgG1 light chain.

[0113] As used herein, the term "antibody" or "antibodies" includes biosimilar antibody molecules approved under the Biologics Price Competition and Innovation Act of 2009 (BPCI Act) and similar global laws and regulations. Under the BPCI Act, an antibody may be demonstrated to be biosimilar if data show that it is "highly similar" to the reference product, despite minor differences in clinically inactive components, and is "expected to produce equivalent clinical results" as the reference product in terms of safety, purity, and potency (Endocrine Practice: February 2018, Vol. 24, No. 2, pp. 195-204). These biosimilar antibody molecules offer an abbreviated approval pathway, whereby applicants rely on the clinical data of the innovator's reference product to secure regulatory approval. Compared to the original innovator reference antibody approved by the FDA based on successful clinical trials, biosimilar antibody molecules are referred to herein as "follow-on biologics." As presented herein, SIMPONI® (golimumab) is the original innovator reference anti-TNF antibody approved by the FDA based on successful clinical trials. Golimumab has been marketed in the United States since 2009.

[0114] Exemplary Sequences In various embodiments, the TNF inhibitor comprises the anti-TNF antibody SIMPONI® (golimumab), or an antigen-binding fragment thereof comprising the sequence shown below: For more information regarding the anti-TNF antibody SIMPONI® (golimumab) and other anti-TNF antibodies, see, e.g., U.S. Patent Nos. 7,250,165, 7,691,378, 7,521,206, 7,815,909, 7,820,169, 8,241,899, 8,603,778, 9,321,836, and 9,828,424.

[0115] Exemplary Anti-TNF Antibody Sequences Example - SIMPONI® (Golimumab) The heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs) are defined by Kabat.

[0116] Amino acid sequence of Golimumab heavy chain (HC) with CDRs underlined: (SEQ ID NO:36)

[0117] [ka]

[0118] Amino acid sequence of Golimumab light chain (LC) with CDRs underlined: (SEQ ID NO:37)

[0119] [ka]

[0120] Amino acid sequence of Golimumab variable heavy chain (VH) with CDRs underlined: (SEQ ID NO:38)

[0121] [ka]

[0122] Amino acid sequence of the Golimumab variable light chain (VL) with CDRs underlined: (SEQ ID NO:39)

[0123] [ka]

[0124] Amino acid sequence of the complementarity determining region 1 (HCDR1) of the golimumab heavy chain: (SEQ ID NO: 40) SYAMH

[0125] Amino acid sequence of the complementarity determining region 2 (HCDR2) of the golimumab antibody heavy chain: (SEQ ID NO: 41) FMSYDGSNKKYADSVKG

[0126] Amino acid sequence of the complementarity determining region 3 (HCDR3) of the golimumab heavy chain: (SEQ ID NO: 42) DRGIAAGGNYYYYGMDV

[0127] Amino acid sequence of the complementarity determining region 1 (LCDR1) of the golimumab light chain: (SEQ ID NO: 43) RASQSVYSYLA

[0128] Amino acid sequence of the complementarity determining region 2 (LCDR2) of the golimumab light chain: (SEQ ID NO: 44) DASNRAT

[0129] Amino acid sequence of the complementarity determining region 3 (LCDRL) of the golimumab light chain: (SEQ ID NO: 45) QQRSNWPPFT

[0130] At least one antibody of the present invention binds to at least one specific epitope specific to at least one TNF protein, subunit, fragment, portion, or any combination thereof. The at least one epitope may comprise at least one antibody binding region comprising at least one portion of the protein, and the epitope is preferably comprised of at least one extracellular, soluble, hydrophilic, external, or cytoplasmic part of the protein. The at least one specified epitope may comprise any combination of at least one amino acid sequence of at least 1-3 amino acids relative to the entire specified portion of contiguous amino acids of SEQ ID NO:9.

[0131] Generally, a human antibody or antigen-binding fragment of the invention comprises an antigen-binding region comprising at least one human complementarity determining region (CDR1, CDR2, and CDR3) or variant of at least one heavy chain variable region, and at least one human complementarity determining region (CDR1, CDR2, and CDR3) or variant of at least one light chain variable region. As a non-limiting example, an antibody or antigen-binding portion or variant may comprise at least one of a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3 and / or a light chain CDR3 having the amino acid sequence of SEQ ID NO:6. In certain embodiments, an antibody or antigen-binding fragment may have an antigen-binding region comprising at least a portion of at least one heavy chain CDR (i.e., CDR1, CDR2, and / or CDR3) having the corresponding amino acid sequence of CDR1, 2, and / or 3 (e.g., SEQ ID NOs:1, 2, and / or 3). In another specific embodiment, the antibody or antigen-binding portion or variant can have an antigen-binding region that includes at least a portion of at least one light chain CDR (i.e., CDR1, CDR2, and / or CDR3) with the corresponding CDR1, 2, and / or 3 amino acid sequence (e.g., SEQ ID NOs: 4, 5, and / or 6). In a preferred embodiment, the three heavy chain CDRs and the three light chain CDRs of the antibody or antigen-binding fragment have the amino acid sequence of the corresponding CDRs of at least one of mAbs TNV148, TNV14, TNV15, TNV196, TNV118, TNV32, and TNV86, as described herein. Such antibodies can be prepared by preparing and expressing a (i.e., one or more) nucleic acid molecules encoding the antibody using conventional techniques for recombinant DNA technology, or by using any other suitable method, or by chemically linking together the various portions of the antibody (e.g., CDRs, framework) using conventional techniques.

[0132] The anti-TNF antibody may comprise at least one of a heavy or light chain variable region having a defined amino acid sequence. For example, in a preferred embodiment, the anti-TNF antibody comprises at least one of a heavy chain variable region, optionally having the amino acid sequence of SEQ ID NO: 7, and / or at least one of a light chain variable region, optionally having the amino acid sequence of SEQ ID NO: 8. Antibodies that bind human TNF and comprise defined heavy or light chain variable regions can be prepared using suitable methods, such as phage display (Katsube, Y., et al., Int J Mol. Med, 1(5):863-868 (1998)) or methods employing transgenic animals, as known in the art and / or described herein. For example, a transgenic mouse comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus capable of undergoing functional rearrangement can be immunized with human TNF or a fragment thereof to induce the production of antibodies. If desired, antibody-producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and / or known in the art. Alternatively, antibodies, specified portions or variants can be expressed using the encoding nucleic acid, or a portion thereof, in a suitable host cell.

[0133] The present invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs that comprise amino acids in sequences that are substantially the same as the amino acid sequences described herein. Preferably, such antibodies or antigen-binding fragments and antibodies that comprise such chains or CDRs have high affinity (e.g., K D About 10 -9M or less) to human TNF. Amino acid sequences that are substantially the same as those described herein include sequences containing conservative amino acid substitutions as well as amino acid deletions and / or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid with a second amino acid that has chemical and / or physical properties (e.g., charge, structure, polarity, hydrophobicity / hydrophilicity) similar to those of the first amino acid. Conservative substitutions include replacing one amino acid with another within the following groups: lysine (K), arginine (R), and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D, and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine ​​(C), and glycine (G); F, W, and Y; C, S, and T.

[0134] Amino acid codes. The amino acids constituting the anti-TNF antibodies of the present invention are often abbreviated. Amino acid designations can be provided by designating the amino acid by its one-letter code, its three-letter code, name, or three-nucleotide codon, and are well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994).

[0135] [Table 1]

[0136] The anti-TNF antibodies of the invention may contain one or more amino acid substitutions, deletions, or additions, either by natural mutation or by human manipulation, as specified herein.

[0137] Of course, the number of amino acid substitutions that one of skill in the art may make will depend on a number of factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions, or deletions for any given anti-TNF antibody, fragment, or variant will not exceed 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, e.g., 1-30, or any range or value therein, as specified herein.

[0138] Amino acids within the anti-TNF antibodies of the invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, supra, Chapter 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one TNF-neutralizing activity. Sites critical for antibody binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[0139] The anti-TNF antibodies of the present invention can include, but are not limited to, at least one portion, sequence or combination selected from one to all of the contiguous amino acids of at least one of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

[0140] The anti-TNF antibody may further optionally comprise at least one polypeptide of 70-100% of the contiguous amino acids of at least one of SEQ ID NOs: 7 and 8.

[0141] In one embodiment, the amino acid sequence of an immunoglobulin chain or a portion thereof (e.g., variable region, CDR) has about 70-100% identity (e.g., 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 range or value therein) to the amino acid sequence of at least one corresponding chain of SEQ ID NO: 7, 8. For example, the amino acid sequence of the light chain variable region can be compared to the sequence of SEQ ID NO: 8, or the amino acid sequence of the heavy chain CDR3 can be compared to SEQ ID NO: 7. Preferably, 70-100% amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein) is determined using a suitable computer algorithm, as known in the art.

[0142] Exemplary heavy and light chain variable region sequences are shown in SEQ ID NOs: 7 and 8. An antibody of the invention, or a specified variant thereof, can include any number of contiguous amino acid residues from an antibody of the invention, the number being selected from the group of integers consisting of 10-100% of the number of contiguous residues in an anti-TNF antibody. Optionally, this subsequence of contiguous amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or more amino acids in length, or any range or value therein. Additionally, the number of such subsequences can be any integer selected from the group consisting of 1-20, such as at least 2, 3, 4, or 5.

[0143] As will be apparent to one of skill in the art, the present invention includes at least one biologically active antibody of the present invention. A biologically active antibody has a specific activity that is at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95% to 1000% of that of a natural (non-synthetic), endogenous or related and known antibody. Methods for assaying and quantifying enzyme activity and substrate specificity are well known to those of skill in the art.

[0144] In another aspect, the invention relates to human antibodies and antigen-binding fragments described herein that are modified by the covalent attachment of an organic moiety. Such modifications can generate antibodies or antigen-binding fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In certain embodiments, the hydrophilic polymer group can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, with a molecular weight of about 800 to about 120,000 daltons, and the fatty acid group or fatty acid ester group can contain about 8 to about 40 carbon atoms.

[0145] The modified antibodies and antigen-binding fragments of the present invention may include one or more organic moieties that are directly or indirectly covalently attached to the antibody. Each organic moiety attached to the antibody or antigen-binding fragment of the present invention may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes monocarboxylic and dicarboxylic acids. As used herein, the term "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than octane. For example, polylysine is more soluble in water than octane. Thus, antibodies modified by the covalent attachment of polylysine are encompassed by the present invention. Suitable hydrophilic polymers for modifying the antibodies of the present invention may be linear or branched, and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers for modifying the antibodies of the present invention have a molecular weight of about 800 to about 150,000 daltons as individual molecular entities. For example, PEG 5000 and PEG 20,000 can be used, where the subscript is the average molecular weight of the polymer in Daltons. The hydrophilic polymer group can be substituted with 1 to about 6 alkyl groups, fatty acid groups, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by utilizing a suitable method. For example, a polymer containing an amine group can be linked to a carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be linked to a hydroxyl group on the polymer.

[0146] Fatty acids and fatty acid esters suitable for modifying antibodies of the invention may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying antibodies of the invention include, for example, n-dodecanoate (C 12 , laurate), n-tetradecanoate (C 14 , myristate), n-octadecanoate (C 18 , stearate), n-eicosanoate (C 20 , arachidate), n-docosanoate (C 22 , behenic acid), n-triacontanoate (C 30 ), n-tetracontanoate (C 40 ), cis-Δ9-octadecanoate (C 18 oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C 20 , arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight or branched chain lower alkyl group. The lower alkyl group can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0147] Modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. As used herein, the term "modifying agent" refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) that contains an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be linked to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). Activating groups can be attached directly to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or to linker moieties, such as divalent C 1 ~C 12 The linkage can be via a group in which one or more carbon atoms can be replaced with a heteroatom such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH 2 ) 3 -, -NH-(CH 2 ) 6 -NH-, -(CH 2 ) 2 -NH- and -CH 2 -O-CH 2 -CH 2 -O-CH 2 -CH 2-O-CH-NH-. Modifiers containing linker moieties can be generated by forming an amide bond between the free amine and the fatty acid carboxylate, for example by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or it can be reacted with maleic anhydride and the resulting product cyclized to generate an activated maleimide derivative of the fatty acid. (See, for example, Thompson et al., WO 92 / 16221, the teachings of which are incorporated herein by reference in their entirety.)

[0148] The modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, an organic moiety can be attached to the antibody in a non-site-specific manner using an amine-reactive modifying agent, such as an NHS ester of PEG. Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of an antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibody of the invention. Modified human antibodies and antigen-binding fragments containing organic moieties attached to specific sites of the antibodies of the invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)) and those described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996).

[0149] Anti-idiotypic antibodies to anti-Tnf antibody compositions. In addition to monoclonal or chimeric anti-TNF antibodies, the present invention also relates to anti-idiotypic (anti-Id) antibodies specific for such antibodies of the present invention. Anti-Id antibodies are antibodies that recognize unique determinants generally associated with the antigen-binding region of another antibody. Anti-Ids can be prepared by immunizing an animal (e.g., a mouse strain) of the same species and genotype as the Id antibody source with the antibody or its CDR-containing region. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody, producing anti-Id antibodies. Anti-Id antibodies can also be used as "immunogens" to induce an immune response in yet another animal, producing so-called anti-anti-Id antibodies.

[0150] Anti-TNF antibody compositions. The present invention also provides at least one anti-TNF antibody composition comprising at least one, at least two, at least three, at least four, at least five, at least six, or more of said anti-TNF antibodies provided in a non-naturally occurring composition, mixture, or form as described herein and / or known in the art. Such compositions include non-naturally occurring compositions comprising at least one or two full-length, C- and / or N-terminal deletion variants, domains, fragments, or specified variants of an amino acid sequence of an anti-TNF antibody selected from the group consisting of 70-100% of the contiguous amino acids of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or a specified fragment, domain, or variant thereof. A preferred anti-TNF antibody composition comprises at least one or two full-length, fragment, domain, or variant as at least one CDR- or LBR-containing portion of 70-100% of an anti-TNF antibody of SEQ ID NO: 1, 2, 3, 4, 5, 6, or a specified fragment, domain, or variant thereof. More preferred compositions comprise 70-100% of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 40-99% of at least one of the specified fragments, domains, or variants thereof. Such composition percentages are by weight, volume, concentration, molality, or molality as a liquid or dry solution, mixture, suspension, emulsion, or colloid, as known in the art or as described herein.

[0151] The anti-TNF antibody compositions of the present invention further comprise at least one anti-TNF antibody directed to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, and optionally at least one TNF antagonist (e.g., but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, a fusion protein thereof, or a small molecule TNF antagonist), an anti-rheumatic drug (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, aurantiome, bronchodilator, rifampicin, ribavir ... sodium benzoate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, other antibacterials), antipsoriatics, corticosteroids, anabolic steroids, diabetes-related medications , minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropieitin (e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunizing agents, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormone, hormone replacement drugs, estrogen receptor modulators, mydriatics, ciliary muscle The composition may include at least one of any suitable and effective amount of compositions or pharmaceutical compositions further comprising at least one selected from paralytics, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanic agents, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or analogs, dornase alpha (Pulmozyme), cytokines or cytokine antagonists. Non-limiting examples of such cytokines include, but are not limited to, any of IL-1 to IL-23.Suitable dosages are well known in the art, see, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2. nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein by reference in its entirety.

[0152] Such anti-cancer or anti-infective agents may also include toxin molecules associated, conjugated, co-formulated, or combined with at least one antibody of the invention. The toxin may optionally act to selectively kill diseased cells or tissues. The diseased cells may be cancer cells or other cells. Such toxins may be purified or recombinant toxins or toxin fragments that contain at least one functional cytotoxic domain of a toxin, for example, but are not limited to, selected from at least one of ricin, diphtheria toxin, snake toxin, or bacterial toxin. The term toxin also includes both endotoxins and exotoxins produced by any naturally occurring or mutated or recombinant bacteria or viruses that may cause any pathology, including toxic shock, which may lead to death, in humans and other mammals. Such toxins may include, but are not limited to, enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxin, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxin, and the like. Such bacteria include enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (e.g., strains of serotype 0157:H7), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus pyogenes), Shigella spp. (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Salmonella spp. (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella enteritidis), Clostridium spp. (e.g., Clostridium perfringens, Clostridium difficile, Clostridium typhi, and Clostridium pyogenes), and Examples of suitable strains of Streptococcus botulinum, Campylobacter spp. (e.g., Campylobacter jejuni, Campylobacter fetus), Helicobacter spp. (e.g., Helicobacter pylori), Aeromonas spp. (e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Plesiomonas shigelloides, Yersinia enterocolitica, Vibrio spp. (e.g., Vibrio cholerae, Vibrio parahaemolyticus), Klebsiella spp., Pseudomonas aeruginosa, and Streptococcus spp.For example, Stein, ed., INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990), Evans et al., eds., Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., New York (1991), Mandell et al. al, Principles and Practice of Infectious Diseases, 3d.Ed., Churchill Livingstone, New York (1990), Berkow et al, eds., The Merck Manual, 16th edition, Merck and Co., Rahway, NJ, 1992, Wood et al, FEMS Microbiology Immunology, 76:121-134 (1991), Marrack et al. al, Science, 248:705-711 (1990), the contents of which are incorporated herein by reference in their entireties.

[0153] The anti-TNF antibody compounds, compositions or mixtures of the present invention may further comprise at least one of any suitable auxiliary agent, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of and methods for preparing such sterile solutions are well known in the art and are described, for example, in Gennaro, Ed., Remington's Pharmaceutical Sciences, 1896, ed., "Preparation of sterilized solutions of TNF antibodies," ... th Edition, Mack Publishing Co. (Easton, PA), 1990. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility, and / or stability of the anti-TNF antibody, fragment, or variant composition can be routinely selected as known in the art or described herein.

[0154] Pharmaceutical excipients and additives useful in the present compositions include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., saccharides including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / antibody components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0155] Carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc., disaccharides such as lactose, sucrose, trehalose, cellobiose, etc., polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starches, etc., alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.

[0156] The anti-TNF antibody composition may also include a buffer or pH adjuster, typically a salt prepared from an organic acid or base.Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, Tris, tromethamine hydrochloride, or phosphate buffers.Preferred buffers for use in the composition are organic acid salts such as citrate.

[0157] In addition, the anti-TNF antibody compositions of the invention may include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavorings, antimicrobials, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0158] These and additional known pharmaceutical excipients and / or additives suitable for use in the anti-TNF antibody, portion or variant compositions according to the invention are known in the art and are described, for example, in "Remington: The Science & Practice of Pharmacy," 1999. th ed., Williams&Williams, (1995) and “Physician's Desk Reference”, 52 nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are incorporated herein by reference in their entireties. Preferred carrier or excipient materials are carbohydrates (e.g., monosaccharides and alditols) and buffers (e.g., citric acid) or polymeric agents.

[0159] Formulations. As mentioned above, the present invention provides stable formulations, preferably saline or phosphate buffer with selected salts, as well as preservative-containing preservative solutions and formulations, and versatile preservative formulations suitable for pharmaceutical or veterinary use, comprising at least one anti-TNF antibody in a pharma- ceutically acceptable formulation. The preservative formulations contain at least one known preservative, optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. As known in the art, the range of concentrations may be from 0.001 to 5%, or 0.001, 0.003, 0.005, 0.009, 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 therein may be used.Non-limiting examples include no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., Examples of suitable alkylparabens include 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0% alkylparaben (for example, 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0160] As discussed above, the present invention provides an article of manufacture comprising packaging and at least one vial containing a solution of at least one anti-TNF antibody, optionally with a buffer and / or preservative formulated in an aqueous diluent, the packaging comprising a label indicating that such solution can be preserved for 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or more. The present invention further comprises an article of manufacture comprising packaging and a first vial containing at least one lyophilized anti-TNF antibody and a second vial containing an aqueous diluent of the formulated buffer or preservative, the packaging comprising a label instructing a patient to reconstitute the at least one anti-TNF antibody in the aqueous diluent to form a solution that can be preserved for 24 hours or more.

[0161] At least one anti-TNF antibody used in accordance with the present invention may be produced by recombinant means, including from mammalian cells or transgenic preparations, or may be purified from other biological sources, as described herein or known in the art.

[0162] The range of at least one anti-TNF antibody included in the products of the invention is in an amount that, upon reconstitution, results in a concentration ranging from about 1.0 μg / mL to about 1000 mg / mL for wet / dry systems, although lower and higher concentrations are workable, depending on the intended delivery vehicle, e.g., for solution formulations, as opposed to transdermal patch, pulmonary, transmucosal, or osmotic or micropump methods.

[0163] Preferably, the aqueous diluent optionally further comprises a pharma- ceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of the preservative used in the formulation is sufficient to produce an antimicrobial effect. Such concentration will vary depending on the preservative selected and can be easily determined by one skilled in the art.

[0164] Other excipients, such as isotonicity agents, buffers, antioxidants, preservative enhancers, can be optionally and preferably added to the diluent. An isotonicity agent, such as glycerin, is commonly used at a known concentration. Preferably, a physiologically tolerable buffer is added to provide improved pH control. The formulation can cover a wide range of pH, such as from about pH 4 to about pH 10, and preferably from about pH 5 to about pH 9, and most preferably from about 6.0 to about 8.0. Preferably, the formulation of the present invention has a pH of about 6.8 to about 7.8. Suitable buffers include phosphate buffers, most preferably sodium phosphate, especially phosphate buffered saline (PBS).

[0165] Other additives, such as pharma- ceutically acceptable solubilizers, such as Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol), or non-ionic surfactants, such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyols, other block copolymers, and chelating agents, such as EDTA and EGTA, can be optionally added to the formulation or composition to reduce aggregation. These additives are particularly useful when pumps or plastic containers are used to administer the formulation. The presence of pharma-ceutically acceptable surfactants reduces the tendency of proteins to aggregate.

[0166] The formulations of the present invention can be prepared by a process that includes mixing at least one anti-TNF antibody with a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal or mixtures thereof in an aqueous diluent. Mixing at least one anti-TNF antibody with a preservative in an aqueous diluent is performed using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a quantity of at least one anti-TNF antibody in a buffer solution is combined with a desired preservative in a sufficient amount of buffer solution to provide the desired concentration of protein and preservative. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of the components, the use or non-use of additional additives, the temperature and pH during preparation of the formulation are all factors that can be optimized for the administration concentration and administration means used.

[0167] The claimed formulations can be provided to patients as clear solutions or as dual vials containing a vial of at least one lyophilized anti-TNF antibody that is reconstituted with a second vial containing water, preservatives and / or excipients, preferably phosphate buffer and / or saline, and the selected salt in an aqueous diluent. Either the single solution vial or the combination vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0168] The claimed products are useful for administration from immediate to over a period of 24 hours or more. Thus, the products claimed by the present invention provide significant benefits to patients. The formulations of the present invention can optionally be safely stored at temperatures of about 2 to about 40° C. and retain the biological activity of the protein for extended periods of time, and thus the packaging label can indicate that the solution can be retained and / or used for periods of 6, 12, 18, 24, 36, 48, 72, or 96 hours or more. In cases where a preserved diluent is used, such label can include use up to 1-12 months, half a year, one and a half years, and / or up to two years.

[0169] A solution of at least one anti-TNF antibody of the present invention can be prepared by a process that includes mixing at least one antibody in an aqueous diluent. The mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a certain amount of at least one antibody in water or a buffer is combined in an amount sufficient to provide the desired concentration of protein, and optionally a preservative or buffer. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of the components, whether or not additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the administration concentration and administration means used.

[0170] The claimed products can be provided to patients as clear solutions or as combination vials containing a vial of at least one lyophilized anti-TNF antibody that is reconstituted with a second vial containing an aqueous diluent. Either the single solution vial or the combination vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0171] The claimed products can be provided indirectly to patients by providing a pharmacy, clinic, or other such institution or facility with a combination vial containing a clear solution or a vial of at least one lyophilized anti-TNF antibody reconstituted with a second vial containing an aqueous diluent, where the clear solution can be up to a liter or even more in volume, from which smaller amounts of the at least one antibody solution can be removed one or more times from the larger container and transferred to smaller vials and provided to customers and / or patients by the pharmacy or clinic.

[0172] Recognized devices that include these single vial systems include pen injector devices for delivering solutions such as BD® (pen injector device), NOVOPEN® (pen injector device), AUTOPEN® (pen injector device), OPTIPEN® (pen injector device), GENOTROPIN PEN® (pen injector device), -HUMATROPEN® (pen injector device), BIOJECTOR® (pen injector device), Reco-Pen, Humaject, J tip Needle-Free Injector, Intraject, Medi-Ject, and the like, manufactured or developed by, for example, Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject,Portland,Oregon(www.bioject.com); Weston Medical (Peterborough,UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, MN, www.mediject.com).

[0173] Approved devices that contain combination vial systems include pen injector systems for reconstituting lyophilized medications in cartridges for delivery of the reconstituted solution, such as the HUMATROPEN® (pen injector device).

[0174] The products claimed herein include packaging. The packaging provides the conditions under which the product may be used, in addition to any information required by regulatory agencies. The packaging of the present invention provides instructions to the patient to reconstitute at least one anti-TNF antibody in an aqueous diluent to form a solution and use the solution for a period of 2-24 hours or more in a wet / dry two vial product. In the case of a single vial solution product, the label indicates that the solution may be used for a period of 2-24 hours or more. The products claimed herein are useful for human pharmaceutical product applications.

[0175] The formulations of the present invention can be prepared by a process that includes mixing at least one anti-TNF antibody and a selected buffer, preferably saline or a phosphate buffer containing a selected salt. Mixing at least one antibody and a buffer in an aqueous diluent is performed 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 is combined with a desired buffer in a sufficient amount of water to provide the desired concentration of protein and buffer. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of the components, whether or not additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the administration concentration and administration means used.

[0176] The claimed stable or preserved formulations can be provided to patients as clear solutions or as combination vials containing a vial of at least one lyophilized anti-TNF antibody that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent. Either the single solution vial or the combination vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0177] At least one anti-TNF antibody in any of the stable or preserved formulations or solutions described herein can be administered to a patient according to the present invention via a variety of delivery methods, as known in the art, such as SC or IM injection, transdermal, transpulmonary, transmucosal, implant, osmotic pump, cartridge, micropump, or other means well known in the art and understood by the skilled artisan.

[0178] Therapeutic Applications The present invention also provides methods for modulating or treating at least one TNF-related disease in a cell, tissue, organ, animal or patient using at least one dual integrin antibody of the present invention, as known in the art or described herein.

[0179] The present invention also provides methods for modulating or treating at least one TNF-related disorder in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of obesity, an immune-related disorder, a cardiovascular disorder, an infectious disease, a malignant disease or a neurological disorder.

[0180] The present invention also relates to the treatment of rheumatoid arthritis, juvenile, systemic onset juvenile rheumatoid arthritis, ankylosing spondylitis, ankylosing spondylitis, gastric ulcer, seronegative arthropathy, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis / uveitis / optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis / Wegener's granulomatosis, sarcoidosis, orchitis / vasectomy repair, allergic / atopic diseases, asthma, allergic rhinitis, dermatitis, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplantation, organ transplant rejection, graft versus host disease, systemic Inflammatory response syndrome, sepsis syndrome, gram-positive sepsis, gram-negative sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia, trauma / hemorrhage, burns, exposure to ionizing radiation, acute pancreatitis, adult respiratory distress syndrome, alcoholic hepatitis, chronic inflammatory disease, sarcoidosis, crohn's disease, sickle cell anemia, diabetes, nephrosis, atopic disease, hypersensitivity reaction, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease, thrombocytopenia, of any organ or tissue Allograft rejection, kidney transplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant (BMT) rejection, skin allograft rejection, cartilage graft rejection, bone graft rejection, small intestine transplant rejection, fetal thymus graft rejection, parathyroid graft rejection, xenograft rejection of any organ or tissue, allograft rejection, antireceptor hyperreaction, Graves' disease, Raynaud's disease, type B insulin-resistant diabetes mellitus, asthma, myasthenia gravis, antibody-mediated cytotoxicity, type III hypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organ Hypertrophy, endocrinopathy, monoclonal gammopathy, and skin syndromes), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin syndromes, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary biliary cirrhosis, vitiligo, vasculitis, post-MI open-heart syndrome, type IV hypersensitivity, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, granulomas due to intracellular organisms, drug hypersensitivity, metabolic / idiopathic Wilson's disease, hemacromatosis, alpha-1-antitrypsin deficiency, diabetic retinopathy,Also provided are methods for modulating or treating at least one immune related disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of Hashimoto's thyroiditis, osteoporosis, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, chronic obstructive pulmonary disease (COPD), familial hemophagocytosis, dermatological conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerulonephritis, acute renal failure, hemodialysis, uremia, toxicity, pre-eclampsia, OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy (including, but not limited to, asthenia, anemia, cachexia, etc.), chronic salicylate intoxication, and the like. See, e.g., Merck Manual, 12th-17th Editions, Merck & Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999); Pharmacotherapy Handbook, Wells et al., eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each of which is incorporated by reference in its entirety.

[0181] The present invention relates to a method for treating cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic arteriosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, cardiovascular syphilis, heart failure, cor pulmonale, primary pulmonary hypertension, arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal), post-perfusion syndrome, cardiopulmonary bypass inflammatory response, chaotic or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrhythmias, ventricular fibrillation, His bundle arrhythmias, Also provided are methods for regulating or treating cardiovascular disease in a cell, tissue, organ, animal or patient, including at least one of the following: arrhythmias, atrioventricular block, bundle branch block, myocardial ischemic disease, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumors, aortic and peripheral aneurysms, aortic dissection, inflammation of the aorta, occlusion of the abdominal aorta and its branches, peripheral vascular disorders, obliterative arterial disorders, peripheral atherosclerosis, thromboangiitis obliterans, functional peripheral arterial disorders, Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venous disease, venous thrombosis, varicose veins, arteriovenous fistula, lymphedema, lipedema, unstable angina, reperfusion injury, post pump syndrome, ischemia-reperfusion injury, and the like. Such methods may optionally include administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.

[0182] The present invention also relates to acute and chronic parasitic or infectious processes including acute or chronic bacterial infections, bacterial, viral and fungal infections, HIV infection / HIV neuropathy, meningitis, hepatitis (such as A, B or C), septic arthritis, peritonitis, pneumonia, epiglottitis, Escherichia coli 0157:h7, hemolytic uremic syndrome / embolic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, tuberculosis, mycobacterium ... Also provided are methods for modulating or treating at least one infectious disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: Mycobacterium avium intracellulare, Pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis / epididymitis, Legionella, Lyme disease, influenza type a, Epstein-Barr virus, viral associated hemophagocytic syndrome, viral encephalitis / aseptic meningitis, and the like.

[0183] The present invention also relates to leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B cell, T cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CML), chronic lymphocytic leukemia (CML), leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (myelodysplastic syndrome) Also provided are methods for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of the following: myeloma, lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal carcinoma, malignant histiocytosis, malignant paraneoplastic syndrome / hypercalcemia, solid tumors, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, and the like.

[0184] The present invention also relates to the treatment of neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia syndrome, demyelinating diseases such as multiple sclerosis and acute transverse myelitis, extrapyramidal and cerebellar disorders such as lesions of the corticospinal system, lesions of the basal ganglia or cerebellar disorders, hyperkinetic movement disorders such as Huntington's chorea and senile chorea, drug-induced movement disorders such as those induced by drugs that block CNS dopamine receptors, hypokinetic movement disorders such as Parkinson's disease, progressive supranuclear palsy, structural lesions of the cerebellum, spinocerebellar degenerations such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degeneration, multisystem degeneration (Mencel, Dejerine-Thomas, Shi-Drager and Machado-Joseph), systemic diseases (Refsum's disease, abetalipoproteinemia, ataxia, telangiectasia and mitochondrial multisystem disorders), demyelinating core disorders. Also provided are methods for modulating or treating at least one neurological disorder in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of the following: multiple sclerosis, acute transverse myelitis and motor unit' disorders, such as neuromuscular atrophy (anterior horn cell degeneration, e.g., amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy), Alzheimer's disease, Down's syndrome in middle age, diffuse Lewy body disease, senile dementia with Lewy bodies, Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakob disease, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and dementia pugilistica. Such methods may optionally comprise administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or identified portion or variant. For example, see Merck Manual, 16 th Edition, Merck & Company, Rahway, NJ (1992).

[0185] Any of the methods of the invention may comprise administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody. Such methods may optionally further comprise the co-administration or combination therapy for the treatment of such immune disorders, where the administration of at least one anti-TNF antibody, specified portion or variant thereof may be combined with at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, a fusion protein thereof, or a small molecule TNF antagonist), an anti-rheumatic drug (such as, but not limited to, methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, aurothioprine ... sodium malate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, other antibacterials), antipsoriatics, corticosteroids, anabolic steroids drugs, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietin (e.g., epoetin alpha), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunizing drugs, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptors The method further comprises administering before, simultaneously with, and / or after at least one selected from a volume regulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, an antimanic, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or an analogue, dornase alfa (Pulmozyme), a cytokine, or a cytokine antagonist.Suitable dosages are well known in the art, see, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2. nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein by reference in its entirety.

[0186] TNF antagonists suitable for the compositions, combination therapies, co-administration devices and / or methods of the invention (which further include at least one antibody, specified portion and variants thereof of the invention) include anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules that specifically bind to TNF, compounds that block and / or inhibit TNF synthesis, TNF release, or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b agonists. These include, but are not limited to, adenosine receptor enhancers, compounds that block and / or inhibit TNF receptor signaling, such as mitogen-activated protein (MAP) kinase inhibitors, compounds that block and / or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors, compounds that block and / or inhibit TNF activity, such as angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril), and compounds that block and / or inhibit TNF production and / or synthesis, such as MAP kinase inhibitors.

[0187] As used herein, a "tumor necrosis factor antibody", "TNF antibody", "TNFα antibody" or fragment or the like reduces, blocks, inhibits, abrogates or interferes with TNFα activity in vitro, in situ, and / or preferably in vivo. For example, suitable TNF human antibodies of the present invention are capable of binding to TNFα and include anti-TNF antibodies, antigen-binding fragments thereof, and specified variants or domains thereof that specifically bind to TNFα. Suitable TNF antibodies or fragments may also reduce, block, abrogate, interfere with, prevent and / or inhibit TNF RNA, DNA, or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and / or synthesis.

[0188] Chimeric antibody cA2 consists of the antigen-binding variable region of a high-affinity neutralizing murine anti-human TNFα IgG1 antibody designated A2 and the constant region of a human IgG1 kappa immunoglobulin. The human IgG1 Fc region improves the effector function of the cognate antibody, increases the circulating serum half-life, and reduces the immunogenicity of the antibody. The avidity and epitope specificity of chimeric antibody cA2 are derived from the variable region of murine antibody A2. In certain embodiments, the preferred source of nucleic acid encoding the variable region of murine antibody A2 is the A2 hybridoma cell line.

[0189] Chimeric A2 (cA2) neutralizes the cytotoxic effects of both natural and recombinant human TNFα in a dose-dependent manner. Binding assays of chimeric antibody cA2 with recombinant human TNFα revealed that the affinity constant of chimeric antibody cA2 was 1.04x10 10 M -1Preferred methods for determining the specificity and affinity of monoclonal antibodies by competitive inhibition can be found in Harlow, et al., antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992-2000); Kozbor et al., Immunol. Today, 4:72-79 (1983); Ausubel et al., eds. Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-2000); and Muller, Meth. Enzymol., 92:589-601 (1983), which are incorporated herein by reference in their entireties.

[0190] In a specific embodiment, the murine monoclonal antibody A2 is produced by a cell line designated c134A. The chimeric antibody cA2 is produced by a cell line designated c168A.

[0191] Further examples of monoclonal anti-TNF antibodies that can be used in the present invention are described in the art (e.g., U.S. Pat. No. 5,231,024; Moller, A. et al., Cytokine 2(3):162-169 (1990); U.S. Application Serial No. 07 / 943,852, filed September 11, 1992; Rathjen et al., WO 91 / 02078, published February 21, 1991; Rubin et al., EPO Patent Publication No. 0 218 868, published April 22, 1987; Yone et al., EPO Patent Publication No. 0 288 088, published October 26, 1988; Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, et al. (see, e.g., Fendly et al., Hybridoma 6:305-311 (1987), Fendly et al., Hybridoma 6:359-369 (1987), Bringman, et al., Hybridoma 6:489-507 (1987), and Hirai, et al., J. Immunol. Meth. 96:57-62 (1987), each of which is incorporated herein by reference in its entirety.

[0192] TNF receptor molecules. Preferred TNF receptor molecules useful in the present invention are those that bind TNFα with high affinity (see, e.g., Feldmann et al., WO 92 / 07076, published April 30, 1992; Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell 61:351-359 (1990), which are incorporated herein by reference in their entireties), and optionally have low immunogenicity. In particular, the 55 kDa (p55 TNF-R) and 75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the present invention. Truncated forms of these receptors, including the extracellular domain (ECD) of the receptor or a functional portion thereof (see, e.g., Corcoran et al., Eur. J. Biochem. 223:831-840 (1994)), are also useful in the present invention. Truncated forms of TNF receptors including the ECD have been detected in urine and serum as 30 kDa and 40 kDa TNFα inhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)). TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules, as well as derivatives and fragments or portions thereof, are further examples of TNF receptor molecules that are useful in the methods and compositions of the present invention. TNF receptor molecules that can be used in the present invention are characterized by good to excellent relief of symptoms and the ability to treat patients for long periods with low toxicity. Low immunogenicity and / or high affinity as well as other yet undefined properties may contribute to the therapeutic results obtained.

[0193] TNF receptor multimeric molecules useful in the present invention comprise all or functional portions of the ECDs of two or more TNF receptors linked via one or more polypeptide linkers or other non-peptide linkers, such as polyethylene glycol (PEG). The multimeric molecules may further comprise a signal peptide of a secretory protein to effect expression of the multimeric molecule. These multimeric molecules and methods for their production are described in U.S. Application Serial No. 08 / 437,533, filed May 9, 1995, the contents of which are incorporated herein by reference in their entirety.

[0194] TNF immunoreceptor fusion molecules useful in the methods and compositions of the invention comprise at least a portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. These immunoreceptor fusion molecules can be assembled as monomers or hetero- or homo-multimers. The immunoreceptor fusion molecules can also be monovalent or multivalent. An example of such a TNF immunoreceptor fusion molecule is a TNF receptor / IgG fusion protein. TNF immunoreceptor fusion molecules and methods for their production have been described in the art (Lesslauer et al., Eur. J. Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994); Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al., J. Immunol. 24:2040-2048 (1994 ... (e.g., Capon et al., U.S. Patent No. 5,447,851 and U.S. Application Serial No. 08 / 442,133, filed May 16, 1995, each of which references is incorporated herein by reference in its entirety.) Methods for producing immune receptor fusion molecules can also be found in 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 337:525-531 (1989), each of which references is incorporated herein by reference in its entirety.

[0195] A functional equivalent, derivative, fragment, or region of a TNF receptor molecule refers to a portion of a TNF receptor molecule, or a portion of a TNF receptor molecule sequence that encodes a TNF receptor molecule, that is of sufficient size and sequence to be functionally similar to the TNF receptor molecule that can be used in the present invention (e.g., binds TNFα with high affinity and has low immunogenicity). A functional equivalent of a TNF receptor molecule also includes modified TNF receptor molecules that are functionally similar to the TNF receptor molecule that can be used in the present invention (e.g., binds TNFα with high affinity and has low immunogenicity). For example, a functional equivalent of a TNF receptor molecule may contain a "SILENT" codon, or one or more amino acid substitutions, deletions, or additions (e.g., one acidic amino acid is substituted for another acidic amino acid, or one codon encoding the same or different hydrophobic amino acid is substituted for another codon encoding a hydrophobic amino acid). See Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2000).

[0196] Cytokines include any known cytokine. See, for example, CopewithCytokines.com. Cytokine antagonists include, but are not limited to, any antibody, fragment or mimetic, any soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination thereof.

[0197] Therapeutic Treatment Any of the methods of the invention may include a method for treating a TNF-mediated disorder comprising administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody.Such methods may optionally further comprise co-administration or combination therapy for the treatment of such immune disorders, where the administration of at least one anti-TNF antibody, specified portion or variant thereof is in combination with at least one TNF antagonist (e.g., but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, a fusion protein thereof, or a small molecule TNF antagonist), an anti-rheumatic drug (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, aurothioprine, bromoglucose ... sodium malate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, other antibacterials), antipsoriatics, corticosteroids, anabolic steroids drugs, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietin (e.g., epoetin alpha), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunizing drugs, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptors The method further comprises administering before, simultaneously with, and / or after at least one selected from a volume regulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, an antimanic, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or an analogue, dornase alfa (Pulmozyme), a cytokine, or a cytokine antagonist.

[0198] The term "safety" as used herein with respect to a composition, dose, dosing regimen, treatment or method with an anti-TNF antibody of the invention (e.g., the anti-TNF antibody golimumab) refers to a favorable risk to benefit ratio with an acceptable frequency and / or severity of adverse events (AEs) and serious adverse events (SAEs) compared to standard of care or another comparator drug, such as another anti-TNF agent. An adverse event is an untoward medical occurrence in a patient administered a pharmaceutical product. Specifically, safety with respect to a composition, dose, dosing regimen, treatment or method with an anti-TNF antibody of the invention refers to an acceptable frequency and / or severity of adverse events including, for example, infusion reactions, abnormal hepatic and biliary tests, infections including TB, and malignancies.

[0199] The terms "efficacy" and "effective" as used herein in the context of a composition, dose, dosing regimen, treatment or method refer to the efficacy of a particular composition, dose, administration, treatment or method with an anti-TNF antibody of the invention (e.g., the anti-TNF antibody golimumab). Efficacy can be measured based on changes in the course of a disease in response to an agent of the invention. For example, an anti-TNF antibody of the invention is administered to a patient in an amount and for a time sufficient to cause an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the disorder being treated. To determine whether the amount and time of treatment is sufficient, various indicators reflecting the extent of the subject's illness, disease or condition can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestations of the disorder of interest. The degree of improvement is generally determined by a physician or other appropriately trained individual, who may determine based on signs, symptoms, biopsy, or other test results indicating improvement in clinical symptoms, or any other measure of disease activity. For example, anti-TNF antibodies of the invention can be administered to obtain improvement in a patient's condition with respect to juvenile idiopathic arthritis (JIA), particularly polyarticular juvenile idiopathic arthritis (pJIA). Efficacy in treating JIA and / or pJIA can be determined, for example, by patients meeting criteria for inactive disease, having an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response selected from JIA ACR30, JIA ACR50, JIA ACR70, and / or JIA ACR90, and / or having a reduction from baseline in a Juvenile Arthritis Disease Activity Score (JADAS) selected from JADAS10, JADAS27, and / or JADAS71.

[0200] As used herein, unless otherwise indicated, the term "clinically proven" (used independently or to modify the terms "safety" and / or "efficacy") shall mean proven by clinical trials that meet the approval standards of the U.S. Food and Drug Administration, EMEA, or a corresponding national regulatory agency. For example, a clinical study may be a randomized, double-blind trial of appropriate size used to clinically prove the efficacy of a drug.

[0201] Typically, treatment of a condition is achieved by administering a safe and effective amount or dose of at least one anti-TNF antibody composition, on average, in the range of at least about 0.01-500 milligrams of at least one anti-TNF antibody per kg of patient body weight per dose, preferably at least about 0.1-100 milligrams of antibody per kg of patient body weight per single or multiple doses, depending on the specific activity contained in the composition. Alternatively, an effective serum concentration may include a serum concentration of 0.1-5000 μg / mL per single or multiple doses. Suitable dosages are known to medical practitioners and will, of course, depend on the specific disease state, the specific activity of the composition being administered, and the specific patient undergoing treatment. In some cases, it may be necessary to provide repeated administrations, i.e., repeated individual administrations of a specific monitored amount or quantity, to obtain the desired therapeutic dose, where the individual administrations are repeated until the desired daily dose or effect is obtained.

[0202] 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, 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, 51, 52, 53, 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, 94, 95, 96, 97, 98, 99 and / or 100-500 mg / kg / dose, or any range, value, or fraction thereof, or a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1. 9, 2.0, 2.5, 2.9, 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, 9.5, 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, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, The concentration may include to obtain a serum concentration of 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, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and / or 5000 μg / mL, or any range, value or fraction thereof.

[0203] Alternatively, the dose administered may vary depending on known factors such as the pharmacodynamic characteristics of the particular agent and its method and route of administration, the age, health and weight of the recipient, the nature and extent of the condition, type of concurrent treatment, frequency of treatment, and the desired effect. The dosage of active ingredient may typically be about 0.1 to 100 milligrams per kilogram of body weight. Typically, 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain the desired results.

[0204] As a non-limiting example, treatment of humans or animals may be performed using a single, infusion or multiple doses on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, or 40th day of administration. or additionally on at least one of the following days: 1, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 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, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 101, 104, 105, 106, 107, 108, 109, 109, 110, 1 0.1 to 100 mg / kg per day, for example 0.5, 0.9, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, or 20.0 years, or any combination thereof. , 1.1, 1.5, 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, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg of at least one antibody of the invention, either once or periodically.

[0205] Dosage forms (compositions) suitable for internal administration generally contain about 0.1 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.5 to 99.999% by weight based on the total weight of the composition.

[0206] For parenteral administration, the antibody can be formulated as a solution, suspension, emulsion, or lyophilized powder, either in combination with a pharma- ceutically acceptable parenteral vehicle or provided separately. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity and chemical stability (e.g., sodium chloride, mannitol for isotonicity; buffers and preservatives for chemical stability). The formulation is sterilized by known or suitable techniques.

[0207] Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

[0208] Alternative Administration: Many known and developed administration methods can be used according to the invention to administer a pharma- ceutically effective amount of at least one anti-TNF antibody according to the invention. Although pulmonary administration is used in the following description, other modes of administration may be used according to the invention with suitable results.

[0209] The TNF antibodies of the invention can be delivered in a carrier, as a solution, emulsion, colloid or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation, or by other methods described herein or known in the art.

[0210] Parenteral formulations and administration. Preparations for parenteral administration may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils derived from plants, hydrogenated naphthalenes, etc. as common excipients. Aqueous or oily suspensions for injection can be prepared by using appropriate emulsifiers or wetting agents and suspending agents according to known methods. Injections may be non-toxic parenterally administrable diluents such as aqueous solutions, sterile injections, or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, etc., and sterile fixed oils can be used as normal solvents or suspension solvents. For these purposes, any kind of fixed oils and fatty acids, including natural, synthetic, or semi-synthetic fatty oils or fatty acids, natural, synthetic, or semi-synthetic mono- or di- or triglycerides, can be used. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforator devices such as those described in U.S. Pat. No. 5,839,446, which are incorporated herein by reference in their entireties.

[0211] Alternative Delivery: The present invention further relates to administration of at least one anti-TNF antibody by parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, intravaginal, rectal, buccal, sublingual, intranasal or transdermal means. At least one anti-TNF antibody composition may be formulated for parenteral (subcutaneous, intramuscular or intravenous) or any other administration, particularly for use in the form of a liquid solution or suspension, particularly in semi-solid forms such as, but not limited to, creams and suppositories, for use in vaginal or rectal administration, in forms such as, but not limited to, tablets or capsules, for buccal or sublingual administration, or intranasally, in forms such as, but not limited to, powders, nasal drops or aerosols, or certain drugs, or with the use of chemical enhancers such as dimethylsulfoxide to either modify the skin structure or increase the drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement"; Hsieh, DS, Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, incorporated herein by reference in its entirety), or using oxidizing agents to allow application of protein and peptide containing formulations to the skin (WO 98 / 53847), or using the application of an electric field to create a transient transport pathway, such as electroporation, or to increase the mobility of a charged drug through the skin, such as iontophoresis, or the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) transdermally, such as, but not limited to, gels, ointments, lotions, suspensions, or patch delivery systems (the above publications and patents are incorporated herein by reference in their entireties).

[0212] Pulmonary / Intranasal Administration. For pulmonary administration, preferably, at least one anti-TNF antibody composition is delivered in a particle size effective to reach the lower airways or sinuses of the lungs. In accordance with the present invention, at least one anti-TNF antibody can be delivered by any of a variety of inhalation or intranasal devices known in the art for administering therapeutics by inhalation. These devices that can deposit an aerosolized formulation in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for pulmonary or intranasal administration of antibodies are also known in the art. All such devices can use formulations suitable for administration to dispense the antibody in an aerosol. Such aerosols can be composed of either solutions (both aqueous and non-aqueous) or solid particles. Metered dose inhalers, such as VENTOLIN® (metered dose inhaler), typically use a propellant gas and require actuation upon inspiration (see, e.g., WO 94 / 16970, WO 98 / 35888). Dry powder inhalers such as Turbuhaler™ (Astra), Rotahaler® (Glaxo), DISKUS® (inhaler) (Glaxo), SPIROS® (inhaler) (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler® powder inhaler (Fisons) use breath actuation of a mixed powder (U.S. Pat. No. 4,668,218 (Astra), EP 237507 (Astra), WO 97 / 25086 (Glaxo), WO 94 / 08552 (Dura), U.S. Pat. No. 5,458,135 (Inhale), WO 94 / 06498 (Fisons), all of which are incorporated herein by reference in their entireties).Nebulizers such as AERX® (nebulizer) (Aradigm), ULTRAVENT® (nebulizer) (Mallinckrodt), and Acorn II nebulizer (Marquest Medical Products) (U.S. Pat. No. 5,404,871 (Aradigm), WO 97 / 22376), which are incorporated herein by reference in their entirety, generate aerosols from solutions, whereas metered dose inhalers, dry powder inhalers, and the like generate small particle aerosols. These specific examples of commercially available inhalation devices are intended as representative of particular devices suitable for practicing the invention, and are not intended as limiting the scope of the invention. Preferably, compositions comprising at least one anti-TNF antibody are delivered by a dry powder inhaler or nebulizer. There are several desirable features of an inhalation device for administering at least one antibody of the invention. For example, delivery by an inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device can optionally deliver small dry particles, for example less than about 10 μm, preferably about 1-5 μm, for good respirability.

[0213] Administration of TNF antibody composition in a spray. A spray containing TNF antibody composition proteins can be generated by passing a suspension or solution of at least one anti-TNF antibody through a nozzle under pressure. The size and configuration of the nozzle, the applied pressure and the liquid feed rate can be selected to achieve the desired output and particle size. For example, an electrostatic spray can be generated by an electric field in conjunction with a capillary or nozzle feed. Advantageously, the particles of at least one anti-TNF antibody composition protein delivered by the sprayer have a particle size of less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

[0214] Formulations of at least one anti-TNF antibody composition protein suitable for use with a nebulizer typically include antibody composition protein in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one anti-TNF antibody composition protein per mL or mg / gm of solution, or any range or value therein, for example, but not limited to, 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, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / mL or mg / gm. The formulation may include agents such as excipients, buffers, isotonicity agents, preservatives, surfactants, and preferably zinc. The formulation may also include excipients or agents for stabilizing the antibody composition protein, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful in formulating antibody composition proteins include albumin, protamine, and the like. Exemplary carbohydrates useful in formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose, glucose, and the like. The antibody composition protein formulation may also include a surfactant that can reduce or prevent surface-induced aggregation of the antibody composition protein caused by atomization of the solution during aerosol formation. A variety of conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts generally range from 0.001 to 14% by weight of the formulation. Particularly preferred surfactants for purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art for formulation of a protein, such as a TNF antibody, or specified portion or variant, can also be included in the formulation.

[0215] Administration of TNF antibody compositions by nebulizers. Antibody composition proteins can be administered by nebulizers, such as jet nebulizers or ultrasonic nebulizers. Typically, jet nebulizers use a compressed air source to create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created, which draws the antibody composition protein solution through a capillary tube connected to a liquid reservoir. The liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube to create an aerosol. A range of configurations, flow rates, and baffle types can be used to achieve the desired performance characteristics from a given jet nebulizer. In ultrasonic nebulizers, high-frequency electrical energy is used to create vibrational mechanical energy, typically using a piezoelectric transducer. This energy is transmitted to the antibody composition protein formulation, either directly or through a coupling liquid, to create an aerosol containing the antibody composition protein. Advantageously, particles of antibody composition protein delivered by a nebulizer have a particle size of less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

[0216] A formulation of at least one anti-TNF antibody suitable for use in either a jet or ultrasonic nebulizer typically contains a concentration of at least one anti-TNF antibody protein of about 0.1 mg to about 100 mg per mL of solution. The formulation may contain agents such as excipients, buffers, isotonicity agents, preservatives, surfactants, and preferably zinc. The formulation may also contain an excipient or agent for stabilization of the at least one anti-TNF antibody composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating at least one anti-TNF antibody composition protein include albumin, protamine, and the like. Exemplary carbohydrates useful in formulating at least one anti-TNF antibody include sucrose, mannitol, lactose, trehalose, glucose, and the like. The at least one anti-TNF antibody formulation may also contain a surfactant that may reduce or prevent surface-induced aggregation of the at least one anti-TNF antibody caused by atomization of the solution upon aerosol formation. A variety of conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. The amount generally ranges from 0.001 to 4% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. For formulations of proteins, such as antibody proteins, additional agents known in the art can also be included in the formulation.

[0217] Administration of TNF antibody compositions by metered dose inhalers. In metered dose inhalers (MDIs), a propellant, at least one anti-TNF antibody, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol containing particles with sizes less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. The desired aerosol particle size can be obtained by using formulations of the antibody composition proteins produced by a variety of methods known to those skilled in the art, including jet milling, spray drying, critical point condensation, and the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and use hydrofluorocarbon propellants.

[0218] Formulations of at least one anti-TNF antibody for use in a metered dose inhaler device generally comprise a fine powder containing at least one anti-TNF antibody as a suspension in a non-aqueous solvent, for example suspended in a propellant with the aid of a surfactant. The propellant can be any conventional substance used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), and the like. Preferably, the propellant is a hydrofluorocarbon. The surfactant can be selected to stabilize the at least one anti-TNF antibody as a suspension in the propellant, to protect the active agent against chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soy lecithin, oleic acid, and the like. In some cases, a solution aerosol using a solvent such as ethanol is preferred. Additional agents known in the art for formulation of proteins may be included in the formulation.

[0219] Those skilled in the art will recognize that the methods of the present invention can be accomplished by pulmonary administration of at least one anti-TNF antibody composition via devices not described herein.

[0220] Oral formulation and administration. Formulation for oral administration is by simultaneous administration of adjuvants (e.g., resorcinol, and non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and simultaneous administration of enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. The active ingredient compound in the solid dosage form for oral administration can be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer, and glyceride. These dosage forms may also contain other types of additives, such as inert diluents, lubricants (magnesium stearate), paraben preservatives (sorbic acid), ascorbic acid, α-tocopherol, antioxidants (cysteine), disintegrants, binders, thickeners, buffers, sweeteners, flavorings, perfumes, and the like.

[0221] Tablets and pills can be further processed into enteric coated preparations. Liquid preparations for oral administration include emulsions, syrups, elixirs, suspensions and solution preparations that are acceptable for medical use. These preparations can contain inert diluents commonly used in the field, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, it is known in the art that carrier compounds described in U.S. Pat. Nos. 5,879,681 and 5,5,871,753 are used to deliver biologically active agents orally.

[0222] Mucosal Formulations and Administration. Compositions and methods for administering at least one anti-TNF antibody for absorption through mucosal surfaces include emulsions comprising a plurality of submicron particles, mucoadhesive macromolecules, bioactive peptides, and an aqueous continuous phase that facilitates absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present invention include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for intranasal administration may be solid and may contain excipients such as lactose, or may be aqueous or oily solution nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (US Pat. No. 5,849,695).

[0223] Transdermal formulations and administration. For transdermal administration, at least one anti-TNF antibody is encapsulated in a delivery device such as a liposome or polymeric nanoparticle, microparticle, microcapsule, or microsphere (collectively referred to as microparticle unless otherwise specified). Many suitable devices are known, including microparticles made from polyhydroxy acids such as polylactic acid, polyglycolic acid and their copolymers, synthetic polymers such as polyorthoesters, polyanhydrides and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599).

[0224] Extended Administration and Formulations. It may sometimes be desirable to deliver the compounds of the invention to a subject over extended periods of time, e.g., from one week to one year, with a single dose. A variety of sustained release, depot, or implantable dosage forms can be utilized. For example, the dosage form may contain a pharma- ceutically acceptable non-toxic salt of a compound that is poorly soluble in body fluids, such as (a) an acid addition salt with a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, polygalacturonic acid, (b) a salt with a polyvalent metal cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or an organic cation formed, e.g., from N,N'-dibenzyl-ethylenediamine or ethylenediamine, or (c) a combination of (a) and (b), e.g., zinc tannate salt. In addition, the compounds of the present invention, or preferably relatively insoluble salts such as those mentioned above, can be formulated in gels, such as aluminum monostearate gels, with, for example, sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate, pamoate, and the like. Another type of injectable sustained release depot formulation contains the compound or salt dispersed for encapsulation in a slowly degrading non-toxic non-antigenic polymer, such as polylactic acid / polyglycolic acid polymers, as described in U.S. Pat. No. 3,773,919. The compounds, or preferably relatively insoluble salts such as those mentioned above, can also be formulated in silastic pellets of a cholesterol matrix, particularly for use in animals. Additional sustained release, depot or implant formulations, such as gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222, and "Sustained and Controlled Release Drug Delivery Systems", edited by JR Robinson, Marcel Dekker, Inc., NY, 1978).

[0225] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

[0226] Example 1: Cloning and expression of TNF antibodies in mammalian cells.

[0227] A typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the antibody coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanking donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with early and late promoters from SV40, long terminal repeats (LTRS) from retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Suitable expression vectors for use in the practice of the present invention include, for example, vectors such as pIRES1neo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -) or pcDNA3.1 / Hygro(+ / -) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that can be used include human Hela293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[0228] Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. Co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows for the identification and isolation of transfected cells.

[0229] The transfected gene can also be amplified to express large amounts of the encoded antibody. The DHFR (dihydrofolate reductase) marker is useful for developing cell lines that have hundreds or even thousands of copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al., Bio / Technology 10:169-175 (1992)). Using these markers, mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for antibody production.

[0230] The expression vectors pC1 and pC4 contain a fragment of the CMV enhancer (Boshart, et al., Cell 41:521-530 (1985)) in addition to the Rous sarcoma virus strong promoter (LTR) (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)). Multiple cloning sites with, for example, restriction enzyme cleavage sites BamHI, XbaI, and Asp718 facilitate cloning of genes of interest. The vectors further contain the 3' intron, polyadenylation, and termination signals of the rat preproinsulin gene.

[0231] Cloning and expression in CHO cells. Vector pC4 is used for expression of TNF antibodies. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). This plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells transfected with these plasmids that lack dihydrofolate activity can be selected by growing the cells in selective medium (e.g., α-MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic drug methotrexate. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) has been well documented (see, e.g., FW Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); JL Hamlin and C Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and MJ Page and MASydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasing MTX concentrations develop resistance to the drug due to overproduction of the target enzyme, DHFR, as a result of amplification of the DHFR gene. When a second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this approach can be used to develop cell lines with more than 1,000 copies of the amplified gene. Subsequently, upon recovery of the methotrexate, cell lines are obtained which contain the amplified gene integrated into one or more chromosomes of the host cell.

[0232] Plasmid pC4 contains the strong promoter of the long terminal repeat (LTR) of Rous sarcoma virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) as well as the fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)) to express the gene of interest. Downstream of the promoter are BamHI, XbaI and Asp718 restriction enzyme cleavage sites that allow integration of the gene. After these cloning sites, the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene. Other highly efficient promoters, such as the human β-actin promoter, the SV40 early or late promoters, or the long terminal repeats from other retroviruses, such as HIV and HTLVI, can also be used for expression. TNF can be expressed in a regulated manner in mammalian cells using Clontech's Tet-Off and Tet-On gene expression systems and similar systems (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)). For polyadenylation of mRNA, other signals, for example from human growth hormone or globin genes, can also be used. Stable cell lines with the gene of interest integrated into the chromosome can also be selected upon cotransfection with a selection marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selection marker, for example G418, plus methotrexate at the outset.

[0233] The plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art, and the vector is then isolated from a 1% agarose gel.

[0234] The isolated variable and constant region encoding DNA and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria that contain the fragment inserted into plasmid pC4 are identified, for example, using restriction enzyme analysis.

[0235] For transfection, Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used. 5 μg of the expression plasmid pC4 are cotransfected with 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmid pSV2neo contains the dominant selection marker, the neo gene from Tn5, which encodes an enzyme that confers resistance to a group of antibiotics, including G418. The cells are seeded in alpha minus MEM supplemented with 1 μg / mL G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25 or 50 ng / mL methotrexate + 1 μg / mL G418. After about 10-14 days, single clones are trypsinized and then seeded in 6-well Petri dishes or 10 mL flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentration of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones growing at a concentration of 100-200 mM are obtained. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot or by reverse-phase HPLC analysis.

[0236] Example 2: Generation of high affinity human IgG monoclonal antibodies reactive with human TNF using transgenic mice.

[0237] Summary. Transgenic mice containing human heavy and light chain immunoglobulin genes are used to generate high affinity, fully human monoclonal antibodies that can be used therapeutically to inhibit the action of TNF for the treatment of one or more TNF-mediated diseases. (CBA / JxC57 / BL6 / J)F contains both heavy and light chain human variable and constant region antibody transgenes. 2 Hybrid mice are immunized with human recombinant TNF (Taylor et al., Intl. Immunol. 6:579-591 (1993); Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M., Nature Biotech. 14:826 (1996); Fishwild, et al., Nature Biotechnology 14:845-851 (1996)). Several fusions have yielded one or more panels of fully human TNF-reactive IgG monoclonal antibodies. The fully human anti-TNF antibodies are further characterized. All are IgG1κ. Such antibodies are approximately 1x10 9 ~9x10 12 The unexpected high affinity of these fully human monoclonal antibodies makes them excellent candidates for therapeutic use in TNF-related diseases, conditions, or disorders.

[0238] Abbreviations: BSA-bovine serum albumin, Co 2 - Carbon dioxide, DMSO - Dimethyl sulfoxide, EIA - Enzyme immunoassay, FBS - Fetal bovine serum, H 2 O 2 - hydrogen peroxide, HRP - horseradish peroxidase, ID - interadermal, Ig - immunoglobulin, TNF - tissue necrosis factor alpha, IP - intraperitoneal, IV - intravenous, Mab or mAb - monoclonal antibody, OD - optical density, OPD - o-phenylenediamine dihydrochloride, PEG - polyethylene glycol, PSA - penicillin, streptomycin, amphotericin, RT - room temperature, SQ - subcutaneous, v / v - volume per unit volume, w / v - weight per unit volume.

[0239] Materials and Methods Animals. Transgenic mice capable of expressing human antibodies are known in the art and are commercially available (e.g., from GenPharm International, San Jose, CA, Abgenix, Freemont, CA, etc.) that express human immunoglobulins but do not express mouse IgM or Igκ. For example, such transgenic mice contain human sequence transgenes that undergo V(D)J joining, heavy chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins (Lonberg, et al., Nature 368:856-859 (1994)). The light chain transgene can be derived, for example, in part, from a yeast artificial chromosome clone that contains approximately half of the germline human Vκ region. In addition, the heavy chain transgene can encode both human μ and human γ1 (Fishwild, et al., Nature Biotechnology 14:845-851 (1996)) and / or γ3 constant regions. Mice from the appropriate genotype strain can be used in the immunization and fusion process to generate fully human monoclonal antibodies against TNF.

[0240] Immunization. One or more immunization schedules can be used to generate anti-TNF human hybridomas. The following exemplary immunization protocol can be followed for the first few fusions, although other similar known protocols can also be used. Several 14-20 week old female and / or surgically castrated male transgenic mice are inoculated IP or ID with 1-1000 μg of recombinant human TNF emulsified with an equal volume of TITERMAX or complete Freund's adjuvant in a final volume of 100-400 μL (e.g., 200). Each mouse can also optionally receive 1-10 μg in 100 μL of physiological saline in each of 2 SQ sites. Mice may then be immunized 1-7, 5-12, 10-18, 17-25, and / or 21-34 days later IP (1-400 μg) and SQ (1-400 μg x 2) with equal amounts of TNF emulsified in TITERMAX or complete Freund's adjuvant. Mice may be bled by retro-orbital puncture 12-25 and 25-40 days later without anticoagulant. Blood is then allowed to clot for 1 hour at room temperature and serum is collected and titrated using a TNF EIA assay by known methods. If repeated injections do not result in increased titers, fusions are performed. Mice may then be given a final IV booster injection of 1-400 μg of TNF diluted in 100 μL of physiological saline. After 3 days, mice can be euthanized by cervical dislocation and spleens can be aseptically removed and immersed in 10 mL of cold phosphate buffered saline (PBS) containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B (PSA). Splenocytes are harvested by aseptically perfusing the spleen with PSA-PBS. Cells are washed once in cold PSA-PBS, counted using trypan blue dye exclusion, and resuspended in RPMI 1640 medium containing 25 mM Hepes.

[0241] Cell fusion. Fusion can be performed according to known, e.g., methods known in the art, at a mouse myeloma cell to live spleen cell ratio of 1:1 to 1:10. As a non-limiting example, spleen cells and myeloma cells can be pelleted together. The pellet can then be slowly resuspended in 1 mL of 50% (w / v) PEG / PBS solution (PEG molecular weight 1,450, Sigma) at 37°C for 30 seconds. Fusion can then be stopped by slowly adding 10.5 mL of RPMI 1640 medium (37°C) containing 25 mM Hepes for 1 minute. The fused cells are centrifuged at 500 to 1500 rpm for 5 minutes. The cells are then resuspended in HAT medium (RPMI 1640 medium containing 25 mM Hepes, 10% fetal clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 μg / mL gentamicin, 2.5% Origen culture supplement (Fisher), 10% 653-adjusted RPMI 1640 / Hepes medium, 50 μM 2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine) and then plated at 200 μL / well into fifteen 96-well flat-bottom tissue culture plates. Then, incubate for 7-10 days in 5% CO. 2 Place the plate in a humidified 37° C. incubator containing 5% CO and 95% air.

[0242] Detection of human IgG anti-TNF antibodies in mouse serum. A solid-phase EIA can be used to screen mouse serum for human IgG antibodies specific for human TNF. Briefly, plates can be coated overnight with 2 μg / mL TNF in PBS. After washing with 0.15 M saline containing 0.02% (v / v) Tween 20, wells can be blocked with 1% (w / v) BSA in PBS, 200 μL / well for 1 hour at room temperature. Plates are used immediately or frozen at -20°C for later use. Mouse serum dilutions are incubated on the TNF-coated plates at 50 μL / well for 1 hour at room temperature. Plates are washed and then probed with 50 μL / well Fc-specific HRP-labeled goat anti-human IgG diluted 1:30,000 in 1% BSA-PBS for 1 hour at room temperature. The plate can be washed again and 100 μL / well of citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0.01% H 2 O 2 and 1 mg / mL OPD) is added over 15 minutes at room temperature. Then, 25 μL / well of stop solution (4N sulfuric acid) is added and the OD is read at 490 nm on an automated plate spectrophotometer.

[0243] Detection of fully human immunoglobulins in hybridoma supernatants. A suitable EIA can be used to detect growth positive hybridomas secreting fully human immunoglobulins. Briefly, 96-well pop-out plates (VWR, 610744) can be coated with 10 μg / mL goat anti-human IgG Fc in sodium carbonate buffer overnight at 4° C. The plates are washed and blocked with 1% BSA-PBS for 1 hour at 37° C. and used immediately or frozen at −20° C. Undiluted hybridoma supernatant is incubated on the plates for 1 hour at 37° C. The plates are washed and probed with HRP-labeled goat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for 1 hour at 37° C. The plates are then incubated with substrate solution as described above.

[0244] Determination of fully human anti-TNF reactivity. The above hybridomas can be simultaneously assayed for reactivity to TNF using a suitable RIA or other assay. For example, as described above, the supernatants are incubated on goat anti-human IgG Fc plates, washed, and then probed with radiolabeled TNF at an appropriate count per well for 1 hour at room temperature. The wells are washed twice with PBS, and bound radiolabeled TNF is quantified using a suitable counter.

[0245] Human IgG1κ anti-TNF-secreting hybridomas can be expanded in cell culture and serially subcloned by limiting dilution. The resulting clonal populations are expanded, cryopreserved in freezing medium (95% FBS, 5% DMSO) and stored in liquid nitrogen.

[0246] Isotype. Determination of the antibody isotype can be accomplished using an EIA in a format similar to that used to screen mouse immune sera for specific titrations. TNF can be coated onto 96-well plates as described above, and 2 μg / mL of purified antibody can be incubated on the plates for 1 hour at room temperature. The plates are washed and incubated with HRP-labeled goat anti-human IgG diluted 1:4000 in 1% BSA-PBS. 1 or HRP-conjugated goat anti-human IgG 3 Probe with 500 mM NaCl for 1 hour at room temperature. Wash plates again and incubate with substrate solution as above.

[0247] Binding kinetics of human anti-human TNF antibodies with human TNF. The binding characteristics of the antibodies can be conveniently evaluated using, for example, TNF capture EIA and BIAcore technology. Graded concentrations of purified human TNF antibodies can be evaluated for binding to EIA plates coated with 2 μg / mL TNF in an assay as described above. The OD can then be expressed as a semi-log plot showing the relative binding efficiency.

[0248] Quantitative binding constants can be obtained, for example, as follows, or by any other known suitable method: A BIAcore CM-5 (carboxymethyl) chip is placed in a BIAcore 2000 unit. HBS buffer (0.01 M Hepes, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v P20 surfactant, pH 7.4) is flowed over the flow cells of the chip at 5 μL / min until a stable baseline is obtained. A solution (100 μL) of 15 mg EDC (N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200 μL water is added to a solution (100 μL) of 2.3 mg NHS (N-hydroxysuccinimide) in 200 μL water. 40 μL of the resulting solution is injected onto the chip. A 6 μL solution of human TNF (15 μg / mL in 10 mM sodium acetate, pH 4.8) is injected over the chip, resulting in an increase of approximately 500 RU. The buffer is changed to TBS / Ca / Mg / BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mM calcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 μg / mL BSA, pH 7.4) and run over the chip overnight to equilibrate it and hydrolyze or cap any unreacted succinates.

[0249] Antibodies are dissolved in running buffer at 33.33, 16.67, 8.33, and 4.17 nM. The flow rate is adjusted to 30 μL / min and the temperature of the instrument is adjusted to 25° C. Two flow cells are used for the kinetic run, one with immobilized TNF (sample) and a second underivatized flow cell (blank). 120 μL of each antibody concentration is injected over the flow cell at 30 μL / min (association phase), followed by a continuous buffer flow for 360 s (dissociation phase). The surface of the chip is regenerated by two sequential injections of 30 μL each of 2 M guanidine thiocyanate (dissociation of tissue necrosis factor α / antibody complexes).

[0250] Data analysis is performed using BIA evaluation 3.0 or CLAMP 2.0, which are known in the art. For each antibody concentration, blank sensograms are subtracted from sample sensograms. Dissociation (k d, sec -1) and meeting (k a ,mol -1 sec -1 ) and perform a global fit for the dissociation constant (K D , mol) (k d / k a ). If the antibody affinity is high enough that the RU of the captured antibody is greater than 100, additional dilutions of the antibody are performed.

[0251] Results and Discussion Generation of anti-human TNF monoclonal antibodies. Several fusions are performed and each fusion generating several dozen antibodies specific for human TNF is seeded onto 15 plates (1440 wells / fusion). Of these, some are found to consist of a combination of human and mouse Ig chains. The remaining hybridomas secrete anti-TNF antibodies consisting of only human heavy and light chains. All of the human hybridomas are expected to be IgG1κ.

[0252] Binding kinetics of human anti-human TNF antibodies. ELISA analysis confirms that purified antibodies from most or all of these hybridomas bind TNF in a concentration-dependent manner. Figures 1 and 2 show the results of the relative binding efficiency of these antibodies. In this case, the binding activity of the antibody to its cognate antigen (epitope) is measured. It should be noted that binding TNF directly to EIA plates can cause denaturation of the protein, and the apparent binding affinity may not reflect binding to the native protein. Fifty percent binding is seen over a wide range of concentrations.

[0253] Quantitative binding constants were obtained using BIAcore analysis of human antibodies, with some human monoclonal antibodies having binding constants of 1x10 -9 ~7x10 -12 K in the range D It is revealed that the affinity is very high.

[0254] Conclusion.

[0255] Several fusions are performed utilizing splenocytes from hybrid mice containing human variable and constant region antibody transgenes that are immunized with human TNF. A set of several fully human TNF-reactive IgG monoclonal antibodies of the IgG1κ isotype was generated. The fully human anti-TNF antibodies are further characterized. Some of the antibodies generated were at 1x10 9 ~9x10 12 The unexpected high affinity of these fully human monoclonal antibodies makes them suitable for therapeutic use in TNF-dependent diseases, pathologies or related conditions.

[0256] Example 3: Generation of human IgG monoclonal antibodies reactive to human TNFα.

[0257] Summary: (CBA / JxC57BL / 6J)F contains human variable and constant region antibody transgenes for both heavy and light chains. 2 Hybrid mice (1-4) were immunized with recombinant human TNFα. One fusion, designated GenTNV, yielded eight fully human IgG1κ monoclonal antibodies that bound to immobilized recombinant human TNFα. Upon identification, eight cell lines were transferred to Molecular Biology for further characterization. Because these Mabs are fully human in sequence, they are expected to be less immunogenic than cA2 (Remicade) in humans.

[0258] Abbreviations: BSA - bovine serum albumin, Co 2 - Carbon dioxide, DMSO - Dimethyl sulfoxide, EIA - Enzyme immunoassay, FBS - Fetal bovine serum, H 2 O 2-hydrogen peroxide, HC-heavy chain, HRP-horseradish peroxidase, ID-interadermal, Ig-immunoglobulin, TNF-tissue necrosis factor alpha, IP-intraperitoneal, IV-intravenous, Mab-monoclonal antibody, OD-optical density, OPD-o-phenylenediamine dihydrochloride, PEG-polyethylene glycol, PSA-penicillin, streptomycin, amphotericin, RT-room temperature, SQ-subcutaneous, TNFα-tumor necrosis factor alpha, v / v-volume per unit volume, w / v-weight per unit volume.

[0259] Introduction. Utilizing transgenic mice containing human heavy and light chain immunoglobulin genes, fully human monoclonal antibodies specific for recombinant human TNFα were generated. It is anticipated that these unique antibodies can be used to therapeutically inhibit inflammatory processes involved in TNFα-mediated diseases, with the benefit of increased serum half-life and reduced immunogenic side effects, cA2 (Remicade).

[0260] As defined herein, the term "half-life" indicates that the plasma concentration of a drug (e.g., a therapeutic anti-TNFα antibody) is halved after one elimination half-life. Thus, with each subsequent half-life, less drug is eliminated. After one half-life, the amount of drug remaining in the body is 50%, after two half-lives, 25%, and so on. The half-life of a drug depends on its clearance and volume of distribution. The elimination half-life is considered to be independent of the amount of drug in the body.

[0261] Materials and Methods.

[0262] Animals. Transgenic mice expressing human immunoglobulins but not mouse IgM or Igκ have been developed by GenPharm International. These mice contain functional human antibody transgenes that undergo V(D)J joining, heavy chain class switching and somatic mutation to generate a repertoire of antigen-specific human immunoglobulins (1). The light chain transgene is derived, in part, from a yeast artificial chromosome clone that contains nearly half of the germline human Vκ locus. In addition to several VH genes, the heavy chain (HC) transgene encodes both human μ and human γ1 (2), and / or γ3 constant regions. Mice from the HCo12 / KCo5 genotype strain were used in the immunization and fusion process to generate the monoclonal antibodies described herein.

[0263] Purification of human TNFα. Human TNFα was purified from tissue culture supernatants from C237A cells by affinity chromatography using a column packed with TNFα receptor-Fc fusion protein (p55-sf2) (5) coupled to Sepharose 4B (Pharmacia). The cell supernatant was mixed with one-ninth of its volume of 10x Dulbecco's PBS (D-PBS) and passed through the column at 4 mL / min at 4°C. The column was then washed with PBS and TNFα was eluted with 0.1 M sodium citrate, pH 3.5, and neutralized with 2 M Tris-HCl, pH 8.5. The purified TNFα was buffer exchanged into 10 mM Tris, 0.12 M sodium chloride, pH 7.5, and filtered through a 0.2 um syringe filter.

[0264] Immunization. Female GenPharm mice approximately 16 weeks of age were immunized IP (200 μL) and ID (100 μL at the base of the tail) with a total of 100 μg TNFα (lots JG102298 or JG102098) emulsified in an equal volume of Titermax adjuvant on days 0, 12, and 28. Mice were bled by retro-orbital puncture on days 21 and 35 without anticoagulant. Blood was allowed to clot for 1 hour at room temperature, and serum was collected and titrated using a TNFα solid-phase EIA assay. After injection on day 28, mice were allowed to rest for 7 weeks before undergoing the fusion designated GenTNV. Mice with specific human IgG titers of 1:160 against TNFα were then given a final IV booster injection of 50 μg TNFα diluted in 100 μL physiological saline. After 3 days, mice were euthanized by cervical dislocation and spleens were aseptically removed and immersed in 10 mL of cold phosphate buffered saline (PBS) containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B (PSA). Splenocytes were harvested by aseptically perfusing the spleen with PSA-PBS. Cells were washed once in cold PSA-PBS, counted using a Coulter counter, and resuspended in RPMI 1640 medium containing 25 mM Hepes.

[0265] Cell lines. The Cell Biology Services (CBS) group received the non-secreting mouse myeloma fusion partner 653 from Centocor's Product Development group on 14 May 1997. The cell lines were expanded in RPMI medium (JRH Biosciences) supplemented with 10% (v / v) FBS (Cell Culture Labs), 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine (all from JRH Biosciences), cryopreserved in 95% FBS and 5% DMSO (Sigma) and then stored in vapor phase liquid nitrogen freezers at CBS. The cell bank was sterile (Quality Control Centocor, Malvern) and free of mycoplasma (Bionique Laboratories). Cells were maintained in logarithmic growth culture until confluence. Prior to confluence, they were washed in PBS, counted, and viability determined by trypan blue dye exclusion (>95%).

[0266] Human TNFα was produced by a recombinant cell line, designated C237A, produced at Centocor's Molecular Biology. The cell line was expanded in IMDM medium (JRH Biosciences) supplemented with 5% (v / v) FBS (Cell Culture Labs), 2 mM L-glutamine (all from JRH Biosciences), and 0.5 μg / mL mycophenolic acid, cryopreserved in 95% FBS and 5% DMSO (Sigma), and then stored in a vapor-phase liquid nitrogen freezer at CBS (13). The cell bank was sterile (Quality Control Centocor, Malvern) and free of mycoplasma (Bionique Laboratories).

[0267] Cell fusion. Cell fusion was performed using 653 mouse myeloma cells and live mouse spleen cells at a 1:1 ratio. Briefly, spleen cells and myeloma cells were pelleted together. The pellet was slowly resuspended in 1 mL of 50% (w / v) PEG / PBS solution (PEG molecular weight 1,450 g / mol, Sigma) at 37° C. for 30 seconds. Fusion was stopped by slowly adding 10.5 mL of RPMI medium (without additives) (JRH) (37° C.) for 1 minute. The fused cells were centrifuged at 750 rpm for 5 minutes. The cells were then resuspended in HAT medium (RPMI / Hepes medium containing 10% fetal bovine serum (JRH), 1 mM sodium pyruvate, 2 mM L-glutamine, 10 μg / mL gentamicin, 2.5% Origen culture supplement (Fisher), 50 μM 2-mercaptoethanol, 1% 653-conditioned RPMI medium, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) and then plated at 200 μL / well in five 96-well flat-bottom tissue culture plates. They were then incubated for 7–10 days in 5% CO 2 The plates were placed in a humidified 37° C. incubator containing 10% CO and 95% air.

[0268] Detection of human IgG anti-TNFα antibodies in mouse serum. Mouse sera were screened for human IgG antibodies specific for human TNFα using solid-phase EIA. Briefly, plates were coated overnight with 1 μg / mL TNFα in PBS. After washing with 0.15 M saline containing 0.02% (v / v) Tween 20, wells were blocked with 1% (w / v) BSA in PBS, 200 μL / well for 1 h at room temperature. Plates were either used immediately or frozen at −20° C. for later use. Mouse sera were incubated on human TNFα-coated plates in 2-fold serial dilutions at 50 μL / well for 1 h at room temperature. Plates were washed and then probed with 50 μL / well of Fc-specific (Accurate) HRP-labeled goat anti-human IgG diluted 1:30,000 in 1% BSA-PBS for 1 h at room temperature. The plate was washed again and 100 μL / well of citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0.01% H 2 O 2 and 1 mg / mL OPD) was added over 15 min at room temperature, followed by addition of 25 μL / well of stop solution (4N sulfuric acid) and reading the OD at 490 nm using an automated plate spectrophotometer.

[0269] Detection of fully human immunoglobulins in hybridoma supernatants. Because GenPharm mice can produce both mouse and human immunoglobulin chains, growth positive hybridoma clones were tested for the presence of both human light and heavy chains using two separate EIA assays. Plates were coated as described above, and undiluted hybridoma supernatants were incubated on the plates for 1 hour at 37°C. Plates were washed and probed with either HRP-conjugated goat anti-human kappa (Southern Biotech) antibody diluted 1:10,000 in 1% BSA-HBSS, or HRP-conjugated goat anti-human IgG Fc specific antibody diluted 1:30,000 in 1% BSA-HBSS, for 1 hour at 37°C. Plates were then incubated with substrate solution as described above. Hybridoma clones that did not give a positive signal in both the anti-human kappa and anti-human IgG Fc EIA formats were discarded.

[0270] Isotype. Determination of the antibody isotype was accomplished using an EIA in a format similar to that used to screen mouse immune sera for specific titers. EIA plates were coated with goat anti-human IgG (H+L) at 10:g / mL in sodium carbonate buffer overnight at 4EC and blocked as described above. Undiluted supernatants from 24-well cultures were incubated on the plates for 1 hour at room temperature. Plates were washed and blocked with HRP-labeled goat anti-human IgG diluted 1:4000 in 1% BSA-PBS. 1 , IgG 2 , IgG 3 or IgG 4 (Binding Site) for 1 hour at room temperature. Plates were washed again and incubated with substrate solution as above.

[0271] Results and Discussion. Generation of fully human anti-human TNFα monoclonal antibodies. A single fusion, designated GenTNV, was performed from GenPharm mice immunized with recombinant human TNFα protein. From this fusion, 196 growth positive hybrids were screened. Eight hybridoma cell lines were identified that secreted fully human IgG antibodies reactive with human TNFα. Each of these eight cell lines secreted immunoglobulins of the human IgG1κ isotype and all were subcloned twice by limiting dilution to obtain stable cell lines (>90% homogeneity). The cell line names and their respective C code designations are listed in Table 1. Each of the cell lines was frozen in a 12-vial research cell bank stored in liquid nitrogen.

[0272] Parental cells harvested from wells of 24-well culture dishes for each of the eight cell lines were delivered to the Molecular Biology group on Feb. 18, 1999 for transfection and further characterization.

[0273] [Table 2]

[0274] Conclusion. GenTNV fusions were performed utilizing splenocytes from hybrid mice containing human variable and constant region antibody transgenes immunized with recombinant human TNFα prepared at Centocor. Eight fully human TNFα-reactive IgG monoclonal antibodies of the IgG1κ isotype were generated. The parent cell lines were transferred to the Molecular Biology group for further characterization and development. One of these new human antibodies may be useful in anti-inflammation, with the potential benefit of reduced immunogenicity and allergy-type complications compared to Remicade.

[0275] References: Taylor, et al., International Immunology 6:579-591 (1993). Lonberg, et al., Nature 368:856-859 (1994). Neuberger, M. Nature Biotechnology 14:826 (1996). Fishwild, et al., Nature Biotechnology 14:845-851 (1996). Scallon, et al., Cytokine 7:759-770 (1995).

[0276] Example 4: Cloning and preparation of cell lines expressing human anti-TNFα antibodies. Summary. A panel of eight human monoclonal antibodies (mAbs), designated TNV, were found to bind immobilized human TNFα with apparently high avidity. Seven of the eight mAbs were shown to efficiently block human TNFα binding to recombinant TNF receptors. Sequence analysis of the DNA encoding the seven mAbs confirmed that all mAbs possessed human V regions. The DNA sequences also revealed that three pairs of mAbs were identical to each other, such that the original panel of eight mAbs contained only four distinct mAbs, designated TNV14, TNV15, TNV148, and TNV196. Based on analysis of the deduced amino acid sequences of the mAbs and the results of the in vitro TNFα neutralization data, mAbs TNV148 and TNV14 were selected for further studies.

[0277] Because a proline residue at position 75 (framework 3) of the TNV148 heavy chain was not found at that position in database searches of other human antibodies of the same subgroup, site-directed DNA mutagenesis was performed to encode a serine residue at that position to match it to the known germline framework e sequence. The serine-modified mAb was designated TNV148B. PCR-amplified DNA encoding the heavy and light chain variable regions of TNV148B and TNV14 were cloned into a newly prepared expression vector based on the recently cloned heavy and light chain genes of another human mAb (12B75) (U.S. Patent Application No. 60 / 236,827, filed October 7, 2000, entitled IL-12 Antibodies, Compositions, Methods and Uses, published as WO 02 / 12500, which is incorporated herein by reference in its entirety).

[0278] P3X63Ag8.653 (653) cells or Sp2 / 0-Ag14 (Sp2 / 0) mouse myeloma cells were transfected with the respective heavy and light chain expression plasmids and screened for cell lines producing high levels of recombinant TNV148B and TNV14 (rTNV148B and rTNV14) mAbs by two rounds of subcloning. Evaluation of growth curves and stability of mAb production over time showed that 653 transfectant clones C466D and C466C stably produced approximately 125:g / mL of rTNV148B mAb in spent cultures, while Sp2 / 0 transfectant 1.73-12-122 (C467A) stably produced approximately 25:g / mL of rTNV148B mAb in spent cultures. Similar analysis showed that the Sp2 / 0 transfectant clone C476A produced 18 μg / mL of rTNV14 in spent cultures.

[0279] Introduction. A panel of eight mAbs from human TNFα immunized GenPharm / Medarex mice (HCo12 / KCo5 genotype) were previously shown to bind human TNFα and have a fully human IgG1κ isotype. A simple binding assay was used to determine whether exemplary mAbs of the invention may have TNFα neutralizing activity by assessing their ability to block TNFα binding to recombinant TNF receptors. Based on these results, DNA sequence results, and some in vitro characterization of the mAbs, TNV148 was selected as the mAb to be further characterized.

[0280] The DNA sequence encoding the TNV148 mAb was cloned and modified into a gene expression vector encoding the appropriate constant region, introduced into well-characterized 653 and Sp2 / 0 mouse myeloma cells, and the resulting transfected cell lines were screened until a subclone was identified that produced 40-fold more mAb than the original hybridoma cell line.

[0281] Materials and Methods. Reagents and Cells. TRIZOL reagent was purchased from Gibco BRL. Proteinase K was obtained from Sigma Chemical Company. Reverse transcriptase was obtained from Life Sciences, Inc. Taq DNA polymerase was obtained from either Perkin Elmer Cetus or Gibco BRL. Restriction enzymes were purchased from New England Biolabs. QIA quick PCR Purification Kit was obtained from Qiagen. QuikChange Site-Directed Mutagenesis Kit was purchased from Stratagene. Wizard Plasmid Miniprep Kit and RNasin were from Promega. Optiplates were obtained from Packard. 125Iodine was purchased from Amersham. Custom oligonucleotides were purchased from Keystone / Biosource International. The names, identification numbers, and sequences of the oligonucleotides used in this work are shown in Table 2.

[0282] Table 2. Oligonucleotides used to clone, engineer, or sequence TNV mAb genes The amino acids encoded by oligonucleotides 5'14s and HuH-J6 are shown above the sequences. The "M" amino acid residue represents the translation initiation codon. The underlined sequences in oligonucleotides 5'14s and HuH-J6 indicate the BsiWI and BstBI restriction sites, respectively. The diagonal lines in HuH-J6 correspond to the exon / intron boundary. Note that oligonucleotides whose sequences correspond to the minus strand are written in the 3'-5' orientation.

[0283] [Table 3]

[0284] One frozen vial of 653 mouse myeloma cells was obtained. The vial was thawed the same day and expanded in IMDM, 5% FBS, and 2 mM glutamine (media) in a T-flask. These cells were maintained in continuous culture until 2-3 weeks later when transfected with anti-TNF DNA as described herein. Some of the culture was harvested 5 days after thawing, pelleted by centrifugation, resuspended in 95% FBS, 5% DMSO, aliquoted into 30 vials, frozen, and stored for later use. Similarly, one frozen vial of Sp2 / 0 mouse myeloma cells was obtained. The vial was thawed, a new freeze-down was prepared as described above, and the frozen vial was stored in freezer boxes AA and AB at CBC. These cells were thawed and used for all Sp2 / 0 transfections described herein.

[0285] Assay for inhibition of TNF binding to the receptor. Hybridoma cell supernatants containing TNV mAbs were used to determine whether the mAbs bind to the recombinant TNF receptor fusion protein p55-sf2. 125 The ability to block binding of I-labeled TNFα was assayed (Scallon et al. (1995) Cytokine 7:759-770). 50:L of p55-sf2 at 0.5:g / mL in PBS was added to Optiplates to coat the wells during a 1 hour incubation at 37°C. Serial dilutions of eight TNV ​​cell supernatants were prepared in 96-well round-bottom plates using PBS / 0.1% BSA as diluent. Cell supernatants containing anti-IL-18 mAb were included as a negative control, and the same anti-IL-18 supernatants spiked with cA2 (anti-TNF chimeric antibody, Remicade, U.S. Patent No. 5,770,198, incorporated herein by reference in its entirety) were included as a positive control. The final TNFα concentration was 5 ng / mL. 125 I-labeled TNFα (58 Ci / g, D. Shealy) was added to 100 L of cell supernatant. The mixture was preincubated at room temperature for 1 h. The coated Optiplates were washed to remove unbound p55-sf2 and 50 L of 125 The I-TNFα / cell supernatant mixture was transferred to Optiplates. After 2 hours at room temperature, the Optiplates were washed 3 times with PBS-Tween. 100:L Microscint-20 was added and cpm bound was determined using a TopCount gamma counter.

[0286] Amplification of V genes and DNA sequence analysis. For RNA preparation, hybridoma cells were washed once with PBS and then TRIZOL reagent was added. 6 ~1.7X10 7The cells were resuspended in 1 mL of TRIZOL. The tube was shaken vigorously after the addition of 200 μL of chloroform. The sample was centrifuged for 10 min at 4 °C. The aqueous phase was transferred to a new microfuge tube and an equal volume of isopropanol was added. The tube was shaken vigorously and incubated at room temperature for 10 min. The sample was then centrifuged for 10 min at 4 °C. The pellet was washed once with 1 mL of 70% ethanol and briefly dried in a vacuum dryer. The RNA pellet was resuspended in 40 μL of DEPC-treated water. The quality of the RNA preparation was determined by fractionating 0.5 μL in a 1% agarose gel. The RNA was stored in a -80 °C freezer until use.

[0287] To prepare heavy and light chain cDNA, a mixture was prepared containing 3 μL of RNA and 1 μg of either oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light chain) (see Table 1) in a volume of 11.5 μL. This mixture was incubated at 70° C. in a water bath for 10 minutes and then cooled on ice for 10 minutes. A separate mixture was prepared consisting of 2.5 μL of 10× reverse transcriptase buffer, 10 μL of 2.5 mM dNTPs, 1 μL of reverse transcriptase (20 units), and 0.4 μL of ribonuclease inhibitor RNasin (1 unit). 13.5 μL of this mixture was added to the 11.5 μL of cold RNA / oligonucleotide mixture and the reaction was incubated at 42° C. for 40 minutes. The cDNA synthesis reaction was then stored in a −20° C. freezer until use.

[0288] Unpurified heavy and light chain cDNAs were used as templates to PCR amplify the variable region coding sequences. Five oligonucleotide pairs (366 / 354, 367 / 354, 368 / 354, 369 / 354, and 370 / 354, Table 1) were tested simultaneously for their ability to prime amplification of heavy chain DNA. Two oligonucleotide pairs (362 / 208 and 363 / 208) were tested simultaneously for their ability to prime amplification of light chain DNA. PCR reactions were performed using 2 units of PLATINUM™ High Fidelity (HIFI) Taq DNA Polymerase in a total volume of 50 μL. Each reaction contained 2 μL of cDNA reaction, 10 pmoles of each oligonucleotide, 0.2 mM dNTPs, 5 μL of 10X HIFI buffer, and 2 mM magnesium sulfate. The thermal cycler program was 95°C for 5 min, followed by 30 cycles of (94°C for 30 s, 62°C for 30 s, 68°C for 1.5 min), followed by a final incubation at 68°C for 10 min.

[0289] To prepare the PCR products for direct DNA sequencing, they were purified using the QIAquick™ PCR Purification Kit according to the manufacturer's protocol. The DNA was eluted from the spin column using 50 μL of sterile water and then dried to a volume of 10 μL using a vacuum dryer. DNA sequencing reactions were then set up with 1 μL of purified PCR product, 10 μM oligonucleotide primers, 4 μL of BigDye Terminator™ ready reaction mix, and 14 μL of sterile water in a total volume of 20 μL. The heavy chain PCR product generated with the oligonucleotide pair 367 / 354 was sequenced using oligonucleotide primers 159 and 360. The light chain PCR product generated with the oligonucleotide pair 363 / 208 was sequenced using oligonucleotides 34 and 163. The thermal cycler program for sequencing was 25 cycles of (96° C. for 30 s, 50° C. for 15 s, 60° C. for 4 min) followed by overnight at 4° C. Reaction products were fractionated through polyacrylamide gels and detected using an ABI 377 DNA sequencer.

[0290] Site-directed mutagenesis to change the amino acid Pro in TNV148 mAb 75A single nucleotide was changed in the TNV148 heavy chain variable region DNA sequence to replace the nucleotide with a serine residue. Complementary oligonucleotides 399 and 400 (Table 1) were designed and this change was made using QuikChange™ site-directed mutagenesis as described by the manufacturer. The two oligonucleotides were first fractionated on a 15% polyacrylamide gel and the major band was purified. Mutagenesis reactions were prepared using either 10 ng or 50 ng of TNV148 heavy chain plasmid template (p1753), 5 μL of 10X reaction buffer, 1 μL of dNTP mix, 125 ng of primer 399, 125 ng of primer 400 and 1 μL of Pfu DNA polymerase. Sterile water was added to bring the total volume to 50 μL. The reaction mix was then incubated in a thermal cycler programmed to perform 30 seconds at 95°C, followed by 14 cycles of 30 seconds at 95°C, 1 minute at 55°C, 1 minute at 64°C, and 7 minutes at 68°C, followed by 2 minutes at 30°C (1 cycle). These reactions were designed to incorporate the mutagenic oligonucleotide into an otherwise identical newly synthesized plasmid. To remove the original TNV148 plasmid, 1 μL of DpnI endonuclease, which cuts only the original methylated plasmid, was added, after which the samples were incubated at 37°C for 1 hour. 1 μL of the reaction was then used to transform Epicurian Coli XL1-Blue supercompetent E. coli by standard heat shock methods, and transformed bacteria were identified after plating on LB-ampicillin agar plates. Plasmid minipreps were prepared using the Wizard™ kit as described by the manufacturer. After elution of the samples from the Wizard™ column, the plasmid DNA was further purified by precipitating it with ethanol and then resuspending it in 20 μL of sterile water. DNA sequence analysis was then performed to identify plasmid clones with the desired base changes and to confirm that no other base changes were inadvertently introduced into the TNV148 coding sequence.Using the same parameters described in section 4.3, 1 μL of plasmid was subjected to a cycle sequencing reaction prepared with 3 μL of BigDye mix, 1 μL of pUC19 forward primer, and 10 μL of sterile water.

[0291] Construction of Expression Vectors from 12B75 Genes. Several recombinant DNA steps were performed to prepare new human IgG1 and new human Kappa expression vectors from genomic copies of previously cloned 12B75-encoding heavy and light chain genes, respectively (as disclosed in U.S. Patent Application Serial No. 60 / 236,827, filed October 7, 2000, entitled IL-12 Antibodies, Compositions, Methods and Uses, published as WO 02 / 12500, which is incorporated herein by reference in its entirety). The final vectors were designed to allow for simple one-step replacement of existing variable region sequences with any appropriately designed PCR amplified variable region.

[0292] To modify the 12B75 heavy chain gene of plasmid p1560, a 6.85 kb BamHI / HindIII fragment containing the promoter and variable region was transferred from p1560 to pUC19 to generate p1743. The smaller size of this plasmid compared to p1560 allowed the use of QuikChange™ mutagenesis (using oligonucleotides BsiWI-1 and BsiWI-2) to introduce a unique BsiWI cloning site immediately upstream of the translation start site, according to the manufacturer's protocol. The resulting plasmid was called p1747. To introduce a BstBI site at the 3' end of the variable region, a 5' oligonucleotide primer was designed with SalI and BstBI sites. This primer was used together with the pUC reverse primer to amplify a 2.75 kb fragment from p1747. This fragment was then cloned back into the naturally occurring SalI and HindIII sites of the 12B75 variable region, thereby introducing a unique BstB1 site. The resulting intermediate vector, designated p1750, was able to accept variable region fragments with BsiWI and BstBI ends. To prepare a version of the heavy chain vector in which the constant region was also derived from the 12B75 gene, the BamHI-HindIII insert of p1750 was transferred to pBR322 in order to have an EcoRI site downstream of the HindIII site. The resulting plasmid p1768 was then digested with HindIII and EcoRI and ligated to the 5.7 kb HindIII EcoRI fragment from p1744, a subclone obtained by cloning the large BamHI-BamHI fragment from p1560 into pBC. The resulting plasmid p1784 was then used as a vector for TNV Ab cDNA fragments with BsiWI and BstBI ends. Additional work was done to prepare expression vectors p1788 and p1798, which contain the IgG1 constant region from the 12B75 gene and differ from each other by how much of the 12B75 heavy chain JC intron they contain.

[0293] To modify the 12B75 light chain gene of plasmid p1558, a 5.7 kb SalI / AflII fragment containing the 12B75 promoter and variable region was transferred from p1558 into the XhoI / AflII sites of plasmid L28. This new plasmid, p1745, provided a smaller template for the mutagenesis step. A unique SalI restriction site was introduced at the 5' end of the variable region by QuikChange™ mutagenesis using oligonucleotides (C340salI and C340sal2). The resulting intermediate vector, p1746, had unique SalI and AflII restriction sites into which variable region fragments could be cloned. Any variable region fragments cloned into p1746 will preferably be joined to the 3' half of the light chain gene. To prepare a restriction fragment from the 3' half of the 12B75 light chain gene that could be used for this purpose, oligonucleotides BAHN-1 and BAHN-2 were annealed together to form a double-stranded linker containing the restriction sites BsiW1, AflII, HindII, and NotI, and containing ends that could be ligated into the KpnI and SacI sites. This linker was cloned between the KpnI and SacI sites of pBC to yield plasmid p1757. A 7.1 kb fragment containing the 12B75 light chain constant region, generated by digesting p1558 with AflII followed by partial digestion with HindIII, was cloned between the AflII and HindII sites of p1757 to yield p1762. This new plasmid contained unique BsiWI and AflII sites where the BsiWI / AflII fragment containing the promoter and variable region could join and transfer the two halves of the gene.

[0294] cDNA cloning and assembly of expression plasmids. All RT-PCR reactions (see above) were treated with Klenow enzyme to further fill in the DNA ends. The heavy chain PCR fragment was digested with restriction enzymes BsiWI and BstBI and then cloned between the BsiWI and BstBI sites of plasmid L28 (L28 was used because the 12B75-based intermediate vector p1750 had not yet been prepared). DNA sequence analysis of the cloned inserts showed that the resulting constructs were correct and that no errors had been introduced during PCR amplification. The identification numbers assigned to these L28 plasmid constructs (TNV14, TNV15, TNV148, TNV148B, and TNV196) are shown in Table 3.

[0295] The BsiWI / BstBI inserts of TNV14, TNV148, and TNV148B heavy chains were transferred from the L28 vector into the newly prepared intermediate vector p1750. The identification numbers assigned to these intermediate plasmids are shown in Table 2. This cloning step and the subsequent steps were not performed for TNV15 and TNV196. The variable regions were then transferred into two different human IgG1 expression vectors. Using the restriction enzymes EcoRI and HindIII, the variable regions were transferred into Centocor's previously used IgG1 vector p104. The resulting expression plasmids encoding IgG1 of the Gm(f+) allotype were designated p1781 (TNV14), p1782 (TNV148), and p1783 (TNV148B) (see Table 2). The variable regions were also cloned upstream of the IgG1 constant region derived from the 12B75 (GenPharm) gene. These expression plasmids encoding IgG1 of the G1m(z) allotype are also listed in Table 3.

[0296] Table 3. Plasmid identification numbers for the various heavy and light chain plasmids. L28 or pBC vectors represent the initial Ab cDNA clones. The inserts of these plasmids were transferred into incomplete 12B75-based vectors to generate intermediate plasmids. One additional transfer step resulted in the final expression plasmids that were either linearized and then introduced into cells or used to purify the mAb gene inserts before transfection of cells. ND = not done.

[0297] [Table 4]

[0298] The light chain PCR products were digested with restriction enzymes SalI and SacII and then cloned between the SalI and SacII sites of plasmid pBC. The two different light chain versions differing in one amino acid were designated p1748 and p1749 (Table 2). DNA sequence analysis confirmed that these constructs had the correct sequence. The SalI / AflII fragments of p1748 and p1749 were then cloned between the SalI and AflII sites of intermediate vector p1746 to generate p1755 and p1756, respectively. These 5' halves of the light chain genes were then joined to the 3' halves of the genes by transferring the BsiWI / AflII fragments from p1755 and p1756 to the newly prepared construct p1762 to generate the final expression plasmids p1775 and p1776, respectively (Table 2).

[0299] Cell transfection, screening and subcloning. A total of 15 mouse myeloma cell transfections were performed with various TNV expression plasmids (see Table 3). These transfections were differentiated by (1) whether the host cells were Sp2 / 0 or 653, (2) whether the heavy chain constant region was encoded with Centocor's previous IgG1 vector or the 12B75 heavy chain constant region, (3) whether the mAb was TNV148B, TNV148, TNV14, or the new HC / LC combination, (4) whether the DNA was a linearized plasmid or a purified Ab gene insert, and (5) whether or not the heavy chain gene contained a complete JC intron sequence. In addition, some of the transfections were repeated to increase the likelihood that a large number of clones could be screened.

[0300] Sp2 / 0 and 653 cells were each transfected with a mixture of heavy and light chain DNA (8-12 g each) by electroporation under standard conditions previously described (Knight DM et al. (1993) Molecular Immunology 30:1443-1453). For transfections no. 1, 2, 3, and 16, the appropriate expression plasmids were linearized by digestion with restriction enzymes prior to transfection. For example, SalI and NotI restriction enzymes were used to linearize the TNV148B heavy chain plasmid p1783 and the light chain plasmid p1776, respectively. For the remaining transfections, the DNA inserts containing only the mAb genes were separated from the plasmid vectors by digesting the heavy chain plasmids with BamHI and the light chain plasmids with BsiWI and NotI. The mAb gene inserts were then purified by agarose gel electrophoresis and Qiex purification resin. Cells transfected with purified gene inserts were co-transfected with 3–5 μg of PstI-linearized pSV2gpt plasmid (p13) as a source of selection marker. After electroporation, cells were seeded in IMDM, 15% FBS, 2 mM glutamine in 96-well tissue culture dishes and incubated at 37 °C for 2 h at 5% CO. 2Plates were incubated at 37°C in a 100-mL incubator. After 2 days, an equal volume of IMDM, 5% FBS, 2 mM glutamine, 2X MHX selection medium (1X MHX = 0.5 μg / mL mycophenolic acid, 2.5 μg / mL hypoxanthine, 50 μg / mL xanthine) was added and the plates were incubated for an additional 2-3 weeks while colonies formed.

[0301] Cell supernatants harvested from wells with colonies were assayed for human IgG by ELISA as described. Briefly, various dilutions of cell supernatants were incubated in 96-well EIA plates coated with polyclonal goat anti-human IgG Fc fragment, followed by detection of bound human IgG using alkaline phosphatase-conjugated goat anti-human IgG (H+L) and an appropriate color substrate. A standard curve using the same purified mAb measured in the cell supernatant as a standard was included on each EIA plate to allow quantification of human IgG in the supernatant. Cells in those colonies that appeared to produce the most human IgG were passaged into 24-well plates for further production determination in spent cultures, followed by identification of the highest producing parental clones.

[0302] The highest producing parent clone was subcloned to identify higher producing subclones and prepare more homogenous cell lines. 96-well tissue culture plates were seeded with one cell per well or four cells per well in IMDM, 5% FBS, 2 mM glutamine, 1xMHX and incubated at 37 °C for 12-20 days in 5% CO until colonies appeared. 2 The cells were incubated at 37°C in an incubator. Cell supernatants were collected from wells containing one colony per well and analyzed by ELISA as described above. Selected colonies were passaged into 24-well plates, and the highest producing subclones were identified by quantifying human IgG levels in their supernatants after exhaustion of the cultures. This process was repeated when the selected first round subclones were subjected to a second round of subcloning. The best subclones from the second round were selected as cell lines for development.

[0303] Characterization of cell subclones. The best subclones from the second round were selected and growth curves were performed to assess mAb production levels and cell growth characteristics. A T75 flask was cultured with 1×10 mAbs in 30 mL of IMDM, 5% FBS, 2 mM glutamine, and 1× MHX (or serum-free medium). 5 Cells were seeded at 1 × 10 cells / mL. Aliquots of 300 μL were removed at 24-h intervals to measure viable cell density. 5 Analysis continued until there were less than 100 cells / mL. Aliquots of harvested cell supernatants were assayed for the concentration of antibody present. ELISA assays were performed using rTNV148B or rTNV14 JG92399 as standards. Samples were incubated for 1 hour on ELISA plates coated with polyclonal goat anti-human IgG Fc and bound mAb was detected with alkaline phosphatase-conjugated goat anti-human IgG (H+L) at a 1:1000 dilution.

[0304] Different growth curve analyses were also performed for the two cell lines to compare their growth rates in the presence of various amounts of MHX selection. Cell lines C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for an additional 2 days. Both cell cultures were then split into three cultures containing either no MHX, 0.2X MHX, or 1X MHX (1X MHX = 0.5:g / mL mycophenolic acid, 2.5:g / mL hypoxanthine, 50:g / mL xanthine). After 1 day, 1×10 cells were added to a new T75 flask. 5 Cultures were seeded at a starting density of 1000000 cells / mL and cells were counted at 24 hour intervals for one week. No aliquots were collected for mAb production. Doubling times were calculated for these samples using the formula provided in SOP PD32.025.

[0305] Additional studies were performed to assess the stability of mAb production over time. Cultures were grown in IMDM, 5% FBS, 2 mM glutamine in 24-well plates, either with or without MHX selection. Once the cultures were confluent, they were split into new cultures and the old cultures were then depleted. At this time, aliquots of supernatant were removed and stored at 4°C. Aliquots were removed over a period of 55-78 days. At the end of this period, the supernatants were tested for the amount of antibody present by anti-human IgG Fc ELISA, as outlined above.

[0306] Results and Discussion. Inhibition of TNF binding to recombinant receptors. A simple binding assay was performed to determine whether the eight TNV ​​mAbs contained in the hybridoma cell supernatants could inhibit TNFα binding to the receptor. The concentration of TNV mAb in each cell supernatant was first determined by standard ELISA analysis of human IgG. The recombinant p55 TNF receptor / IgG fusion protein p55-sf2 was then coated onto an EIA plate and incubated in the presence of various amounts of TNV mAb. 125 I-labeled TNFα was bound to the p55 receptor. As shown in Figure 1, all but one of the eight TNV ​​mAbs (TNV122) efficiently blocked TNFα binding to the p55 receptor. In fact, the TNV mAbs appeared to be more effective at inhibiting TNFα binding than the cA2 positive control mAb spiked into the negative control hybridoma supernatant. These results were interpreted as indicating that the TNV mAbs would likely block the biological activity of TNFα in cell-based assays and in vivo, and therefore, further analysis was required.

[0307] Analysis of DNA sequences. Confirmation that the RNA encodes a human mAb. As a first step in characterizing the seven TNV mAbs (TNV14, TNV15, TNV32, TNV86, TNV118, TNV148, and TNV196) that showed TNFα blocking activity in receptor binding assays, total RNA was isolated from the seven hybridoma cell lines that produce these mAbs. Each RNA sample was then used to prepare human antibody heavy or light chain cDNAs that contained the complete signal sequence, the complete variable region sequence, and a portion of the constant region sequence of each mAb. These cDNA products were then amplified in a PCR reaction, and the PCR-amplified DNA was directly sequenced without first cloning the fragment. The sequenced heavy chain cDNAs were greater than 90% identical to DP-46, one of five human germline genes present in mice (Figure 2). Similarly, the sequenced light chain cDNAs were either 100% identical or 98% identical to one of the human germline genes present in mice (Figure 3). These sequence results confirmed that the RNA molecules that were transcribed into cDNA and sequenced encoded a human antibody heavy chain and a human antibody light chain. It should be noted that because the variable regions were PCR amplified using oligonucleotides that map to the 5' end of the signal sequence coding sequence, the first few amino acids of the signal sequence may not be the actual sequence of the original TNV translation product, but represent the actual sequence of the recombinant TNV ​​mAb.

[0308] Specific neutralizing mAb. Analysis of the cDNA sequences throughout the variable regions of both the heavy and light chains of each mAb revealed that TNV32 was identical to TNV15, TNV118 was identical to TNV14, and TNV86 was identical to TNV148. The results of the receptor binding assay were consistent with the DNA sequence analysis, i.e., both TNV86 and TNV148 were approximately four-fold better than both TNV118 and TNV14 in blocking TNF binding. Therefore, subsequent work was focused only on four unique TNV mAbs, TNV14, TNV15, TNV148, and TNV196.

[0309] Relationships between the four mAbs DNA sequence results revealed that the genes encoding the heavy chains of the four TNV mAbs are all highly homologous to each other and all appear to originate from the same germline gene, DP-46 (Figure 2). In addition, because each of the heavy chain CDR3 sequences are highly similar and of the same length, and because they all use the J6 exon, they clearly arose from a single VDJ gene rearrangement event, followed by somatic changes that make each mAb unique. DNA sequence analysis revealed that there were only two distinct light chain genes in the four mAbs (Figure 3). The light chain variable region coding sequences in TNV14 and TNV15 are identical to each other and to representative germline sequences of the Vg / 38K family of human kappa chains. The TNV148 and TNV196 light chain coding sequences are identical to each other, but differ from the germline sequences at two nucleotide positions (Figure 3).

[0310] The deduced amino acid sequences of the four mAbs revealed the actual relatedness of the mAbs. The four mAbs contain four distinct heavy chains (Figure 4) but only two distinct light chains (Figure 5). The differences between the TNV mAb sequences and the germline sequences were mostly restricted to the CDR domains, but three of the mAb heavy chains also differed from the germline sequence in the framework regions (Figure 4). Compared to the DP-46 germline-encoded Ab framework regions, TNV14 was identical, TNV15 differed by one amino acid, TNV148 differed by two amino acids, and TNV196 differed by three amino acids.

[0311] Cloning of cDNA, site-directed mutagenesis, and assembly of the final expression plasmid. Cloning of cDNA. Based on the DNA sequence of the PCR amplified variable regions, new oligonucleotides were ordered to perform another PCR amplification with the aim of adapting the cloned coding sequence into an expression vector. For the heavy chain, the product of this second PCR was digested with the restriction enzymes BsiWI and BstBI and cloned into the plasmid vector L28 (plasmid identification number shown in Table 2). For the light chain, the product of the second PCR was digested with SalI and AflII and cloned into the plasmid vector pBC. Individual clones were then sequenced to confirm that their sequences were identical to the previous sequences obtained from direct sequencing of the PCR products revealing the most abundant nucleotide at each position of a potentially heterogeneous population of molecules.

[0312] Site-directed mutagenesis to alter TNV148. mAbs TNV148 and TNV196 were consistently observed to be 4-fold more potent than the next best mAb (TNV14) in neutralizing TNFα bioactivity. However, as noted above, the TNV148 and TNV196 heavy chain framework sequences differ from the germline framework sequences. Comparison of the TNV148 heavy chain sequence with other human antibodies showed that many other human mAbs contain an Ile residue at position 28 of framework 1 (counting only the mature sequence), while the Pro residue at position 75 of framework 3 is a rare amino acid at that position.

[0313] A similar comparison of the TNV196 heavy chain suggested that three amino acids that differ from the germline sequence in framework 3 may be rare in human mAbs. These differences could render TNV148 and TNV196 immunogenic when administered to humans. Because TNV148 has only one amino acid residue of interest, which is not believed to be important for TNFα binding, a single nucleotide was changed in the TNV148 heavy chain coding sequence (of plasmid p1753) using site-directed mutagenesis techniques so that the germline Ser residue was encoded instead of the Pro residue at position 75. The resulting plasmid was called p1760 (see Table 2). The resulting gene and mAb were called TNV148B to distinguish it from the original TNV148 gene and mAb (see Figure 5).

[0314] Assembly of the final expression plasmids. New antibody expression vectors were prepared based on the 12B75 heavy and light chain genes previously cloned as genomic fragments. Different TNV ​​expression plasmids were prepared (see Table 2), but in each case the 5' flanking sequences, promoter, and intron enhancer were derived from the respective 12B75 gene. For the light chain expression plasmids, the complete JC intron, constant region coding sequences, and 3' flanking sequences were also derived from the 12B75 light chain gene. For the heavy chain expression plasmids that resulted in the final product cell lines (p1781 and p1783, see below), the human IgG1 constant region coding sequences were derived from the previously used Centocor expression vector (p104). Importantly, the final product cell lines reported here express a TNV mAb of a different allotype (Gm(f+)) than the original hybridoma-derived TNV mAb (G1m(z)). This is because the 12B75 heavy chain gene from GenPharm mouse encodes an Arg residue at the C-terminal end of the CH1 domain, whereas Centocor's IgG1 expression vector p104 encodes a Lys residue at that position. Other heavy chain expression plasmids (e.g., p1786, p1788) in which the JC intron, complete constant region coding sequence, and 3'flank sequence are derived from the 12B75 heavy chain gene have been prepared, but cell lines transfected with these genes were not selected as production cell lines. The vectors were carefully designed to allow for one-step cloning of subsequent PCR amplified V regions that would result in the final expression plasmid.

[0315] The PCR amplified variable region cDNAs were transferred from the L28 or pBC vectors into intermediate 12B75-based vectors that provided the promoter region and part of the JC intron (see Table 2 for plasmid identification numbers). Restriction fragments containing the 5' halves of the antibody genes were then transferred from these intermediate vectors into the final expression vectors that provided the 3' halves of each gene to form the final expression plasmids (see Table 2 for plasmid identification numbers).

[0316] Cell transfection and subcloning. Expression plasmids were either linearized by restriction digestion or the antibody gene insert in each plasmid was purified from the plasmid backbone. Sp2 / 0 and 653 mouse myeloma cells were transfected with heavy and light chain DNA by electroporation. Fifteen different transfections were performed, most of which were unique as defined by the Ab, specific characteristics of the Ab gene, whether the gene was on the linearized whole plasmid or the purified gene insert, and the host cell line (summarized in Table 4). Cell supernatants from clones resistant to mycophenolic acid were assayed by ELISA for the presence of human IgG and quantified using purified rTNV148B as a reference standard curve.

[0317] Highest producing rTNV148B cell line Ten of the highest producing 653 parental lines from rTNV148B transfection 2 (producing 5-10:g / mL in spent 24-well cultures) were subcloned to screen for higher producing cell lines and to prepare a more homogenous cell population. Two of the subclones of parental lines 2.320, 2.320-17, and 2.320-20 produced approximately 50:g / mL in spent 24-well cultures, a 5-fold increase over their parental lines. A second subcloning of subcloned lines 2.320-17 and 2.320-20 resulted in

[0318] The identification numbers of the heavy and light chain plasmids encoding each mAb are shown. For transfections performed with purified mAb gene inserts, plasmid p13 (pSV2gpt) was included as a source of the gpt selection marker. The heavy chain constant region was encoded by either the same human IgG1 expression vector used to encode Remicade ("old") or the constant region contained within the 12B75 (GenPharm / Medarex) heavy chain gene ("new"). H1 / L2 refers to the "new" mAb composed of the TNV14 heavy chain and the TNV148 light chain. Plasmids p1783 and p1801 differ only by the extent to which their heavy chain genes contain a JC intron. The transfection numbers, which define the first digit of the gene name of the cell clone, are shown on the right. The rTNV148B-producing cell lines C466 (A, B, C, D) and C467A described herein were derived from transfection numbers 2 and 1, respectively. The rTNV14-producing cell line, C476A, was derived from transfection no. 3.

[0319] [Table 5]

[0320] ELISA assays on spent 24-well culture supernatants showed that these second round subclones all produced 98-124 μg / mL, which was at least a two-fold increase over the first round subclones. These 653 cell lines were assigned C-code designations, as shown in Table 5.

[0321] Three of the highest producing Sp2 / 0 parental lines from rTNV148B transfection 1 were subcloned. Two rounds of subcloning of parental line 1.73 led to the identification of a clone that produced 25 μg / mL in spent 24-well cultures. This Sp2 / 0 cell line was designated C467A (Table 5).

[0322] Highest producing rTNV14 cell line Three of the highest producing Sp2 / 0 parental lines from rTNV14 transfection 3 were subcloned once. Subclone 3.27-1 was found to be the highest producer in spent 24-well cultures with a production of 19 μg / mL. This cell line was designated C476A (Table 5).

[0323] Table 5. Summary of selected generating cell lines and their C codes. The first digit of the original clone name indicates which transfection the cell line was derived from. All of the C-encoded cell lines reported here were derived from transfections with restriction enzyme linearized heavy and light chain total plasmids.

[0324] [Table 6]

[0325] Characterization of subcloned cell lines To more carefully characterize cell line growth characteristics and determine mAb production levels on a large scale, growth curve analysis was performed using T75 cultures. Results showed that each of the four C466 series of cell lines produced mAb at 1.0 × 10 6 ~1.25×10 6 The results showed that the highest producing Sp2 / 0 subclone, C467A, reached a peak cell density of 2.0 × 10 cells / mL and a maximum mAb accumulation level of 110–140 μg / mL (Figure 7). In contrast, the highest producing Sp2 / 0 subclone, C467A, reached a peak cell density of 2.0 × 10 6 A peak cell density of 100 cells / mL and a maximum mAb accumulation level of 25 μg / mL were reached (FIG. 7). Growth curve analysis was not performed for the rTNV14-producing cell line C476A.

[0326] Further growth curve analysis was performed to compare the growth rates at different MHX selectant concentrations. This comparison was prompted by recent observations that C466 cells cultured in the absence of MHX appeared to grow faster than the same cells cultured in normal amounts of MHX (1X). Because cytotoxic concentrations of compounds such as mycophenolic acid tend to be measured over several orders of magnitude, it was thought possible that by using lower concentrations of MHX, the cell doubling time could be significantly faster without sacrificing the stability of mAb production. Cell lines C466A and C466B were cultured either without MHX, with 0.2X MHX, or with 1X MHX. Viable cell counts were performed at 24-hour intervals for 7 days. The results revealed MHX concentration-dependent cell growth rates (Figure 8). Cell line C466A showed a doubling time of 25.0 hours in 1X MHX, but only 20.7 hours without MHX. Similarly, cell line C466B exhibited a doubling time of 32.4 hours in 1X MHX, but only 22.9 hours without MHX. Importantly, the doubling times of both cell lines in 0.2X MHX were more similar to those observed without MHX than with 1X MHX (Figure 8). This observation indicates that in bioreactors, where doubling time is a critical parameter, enhanced cell performance may be realized by using less MHX. However, while the stability study results (see below) suggest that cell line C466D is capable of stably producing rTNV148B for at least 60 days in the absence of MHX, the stability study also showed higher mAb production levels when cells were cultured in the presence of MHX compared to the absence of MHX.

[0327] Stability studies were performed on cultures either with or without MHX selection to assess mAb production from the various cell lines over a period of approximately 60 days. Not all cell lines maintained high mAb production. After just 2 weeks of culture, production from clone C466A was approximately 45% less than at the beginning of the study. Production from clone C466B also appeared to have dropped significantly. However, clones C466C and C466D maintained fairly stable production, with C466D showing the highest absolute production levels (Figure 9).

[0328] conclusion From an initial panel of eight human mAbs against human TNFα, TNV148B was selected as preferred, similar to TNV14, based on several criteria including protein sequence and TNF neutralization potency. Cell lines producing >100 μg / mL rTNV148B and >19 μg / mL rTNV14 were prepared.

[0329] Example 5: Study of arthritic mice with anti-TNF antibodies and controls using a single bolus injection Tg197 research mice, approximately 4 weeks of age, were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus of Dulbecco's PBS (D-PBS) or an anti-TNF antibody of the invention (TNV14, TNV148, or TNV196) at either 1 mg / kg or 10 mg / kg.

[0330] Results: When body weight was analyzed as a change from pre-dose, animals treated with 10 mg / kg cA2 consistently demonstrated greater weight gain than D-PBS treated animals throughout the study. This weight gain was significant from weeks 3 to 7. Animals treated with 10 mg / kg TNV148 also achieved significant weight gain by week 7 of the study. (See Figure 10).

[0331] 11A-11C depict the progression of disease severity based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to show a significant decrease in AI from week 3 onwards when compared to the D-PBS treated group. There was no significant difference between the 10 mg / kg treatment groups when each was compared to the other at a similar dose (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148 and 196). When comparing 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. TNV148 at 1 mg / kg was also significantly lower than the TNV14 at 1 mg / kg treatment group at weeks 3 and 4. TNV196 showed a significant reduction in AI (when compared to the D-PBS treatment group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study.

[0332] Example 6: Study of arthritic mice using anti-TNF antibodies and controls as multiple bolus doses Tg197 study mice approximately 4 weeks of age were assigned to one of eight treatment groups based on body weight and treated with an intraperitoneal bolus of control article (D-PBS) or 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 efficacy of the test article. Serum samples obtained from animals in groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3, and 4.

[0333] Results: No significant differences were observed when body weight was analyzed as change from pre-treatment. Animals treated with 10 mg / kg cA2 consistently showed higher weight gain than D-PBS treated animals throughout the study. (See FIG. 12).

[0334] 13A-13C depict the progression of disease severity based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and were also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2.

[0335] Example 7: Study of arthritic mice using anti-TNF antibodies and controls as a single intraperitoneal bolus dose Tg197 study mice, approximately 4 weeks of age, were assigned to one of six treatment groups based on sex and weight and treated with a single intraperitoneal bolus of either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148). The study utilized D-PBS and 10 mg / kg cA2 control groups.

[0336] When body weight was analyzed as a change from pre-dose, all treatments achieved similar weight gain. Animals treated with either 3 or 5 mg / kg TNV148 or 5 mg / kg cA2 gained significant weight early in the study (weeks 2 and 3). Only animals treated with TNV148 maintained significant weight gain at later time points. Both 3 and 5 mg / kg TNV148 treated animals showed a significant increase at week 7, with animals treated with 3 mg / kg TNV148 still significantly elevated at week 8 after injection. (See FIG. 14).

[0337] FIG. 15 depicts the progression of disease severity based on the arthritis index. All treatment groups showed some protection at early time points, with 5 mg / kg cA2 and 5 mg / kg TNV148 showing significant reductions in AI at weeks 1-3, and all treatment groups showing significant reductions at week 2. Later in the study, animals treated with 5 mg / kg cA2 showed some protection, with significant reductions at weeks 4, 6, and 7. Both low dose (3 mg / kg) cA2 and TNV148 showed significant reductions at week 6, and all treatment groups showed significant reductions at week 7. None of the treatment groups were able to maintain a significant reduction at the end of the study (week 8). There were no significant differences between any of the treatment groups (except the saline control group) at any time point.

[0338] Example 8: Study in arthritic mice using anti-TNF antibodies and controls as a single intraperitoneal bolus between anti-TNF antibodies and modified anti-TNF antibodies To compare the efficacy of a single intraperitoneal injection of TNV148 (derived from hybridoma cells) and rTNV148B (derived from transfected cells), Tg197 study mice approximately 4 weeks of age were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus injection of Dulbecco's PBS (D-PBS) or 1 mg / kg of antibody (TNV148, rTNV148B).

[0339] When body weight was analyzed as a change from pre-dose, animals treated with 10 mg / kg cA2 consistently showed higher weight gain than D-PBS treated animals throughout the study. This weight gain was significant at week 1 and weeks 3-8. Animals treated with 1 mg / kg TNV148 also achieved significant weight gain at weeks 5, 6, and 8 of the study. (See FIG. 16).

[0340] Figure 17 depicts the progression of disease severity based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 4 and continued throughout the remainder of the study (week 8). Both the TNV148-treated group and the 1 mg / kg cA2-treated group showed a significant reduction in AI at week 4. A previous study (P-099-017) showed that TNV148 was slightly more effective in reducing the arthritis index after a single 1 mg / kg intraperitoneal bolus, but this study showed slightly higher AI from groups treated with both versions of the TNV antibody. The 1 mg / kg cA2 treated group (except at week 6) did not increase significantly when compared to the 10 mg / kg cA2 group, and the TNV148 treated group was significantly higher at weeks 7 and 8, but there were no significant differences in AI between 1 mg / kg cA2, 1 mg / kg TNV148, and 1 mg / kg TNV148B at any time point in the study.

[0341] Example 9: GO-VIVA: A Multicenter, Open-Label Study of Intravenous Golimumab, a Human Anti-TNFα Antibody, in Pediatric Subjects with Active Polyarticular Juvenile Idiopathic Arthritis Despite Methotrexate Therapy Protocol number: CNTO148JIA3003 overview Golimumab is a fully human monoclonal antibody (mAb) that binds to human tumor necrosis factor alpha (TNFα) with high affinity and specificity and neutralizes the biological activity of TNFα. TNFα is...

Claims

1. A pharmaceutical composition for use in a method of treating juvenile idiopathic arthritis (JIA) in pediatric patients, wherein the pharmaceutical composition comprises an anti-TNF antibody, the method comprises administering an intravenous (IV) dose of the anti-TNF antibody to the patient, the IV dose is 80 mg / m2 at week 0, week 4, and then every 8 weeks thereafter, the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and the patient treated with the anti-TNF antibody meets the criteria for inactive disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment. A pharmaceutical composition.

2. More than 10% of the patients meet the criteria for inactive disease after 8 weeks of treatment, more than 20% of the patients meet the criteria for inactive disease after 16 weeks of treatment, and more than 29% of the patients meet the criteria for inactive disease after 28 weeks of treatment. The pharmaceutical composition according to claim 1.

3. The pharmaceutical composition according to claim 1, wherein the pediatric patient is 2 to 17 years old.

4. The pharmaceutical composition according to claim 1, wherein the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA).

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

6. A pharmaceutical composition for use in a method of treating juvenile idiopathic arthritis (JIA) in pediatric patients, wherein the pharmaceutical composition comprises an anti-TNF antibody, the method comprises administering an intravenous (IV) dose of the anti-TNF antibody to the patient, the IV dose is 80 mg / m2 at week 0, week 4, and then every 8 weeks thereafter, the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, the patient treated with the anti-TNF antibody has an improvement from baseline corresponding to a JIA American College of Rheumatology (JIA ACR) response of JIA ACR30, JIA ACR50, JIA ACR70, or JIA ACR90 after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment. A pharmaceutical composition.

7. The pharmaceutical composition according to claim 6, wherein more than 50% of the patients meet the criteria of JIA ACR30 and JIA ACR50 after 4 weeks of treatment.

8. The pharmaceutical composition according to claim 6, wherein more than 50% of the patients meet the criteria of JIA ACR30, JIA ACR50, and JIA ACR70 after 12 weeks of treatment.

9. The pharmaceutical composition according to claim 6, wherein more than 83% of the patients meet the criteria of JIA ACR30, more than 79% of the patients meet the criteria of JIA ACR50, more than 70% of the patients meet the criteria of JIA ACR70, and more than 46% of the patients meet the criteria of JIA ACR90 after 28 weeks of treatment.

10. The pharmaceutical composition according to claim 6, wherein the pediatric patient is 2 to 17 years old.

11. The pharmaceutical composition according to claim 6, wherein the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA).

12. The pharmaceutical composition according to claim 6, wherein the method further comprises administering methotrexate (MTX) to the pediatric patient.

13. A pharmaceutical composition for use in a method of treating juvenile idiopathic arthritis (JIA) in a pediatric patient, wherein the pharmaceutical composition comprises an anti-TNF antibody, wherein the method comprises administering an intravenous (IV) dose of the anti-TNF antibody to the patient, wherein the IV dose is 80 mg / m2 at week 0, week 4, and then every 8 weeks thereafter, wherein the anti-TNF antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 37, and the patient treated with the anti-TNF antibody has a Juvenile Arthritis Disease Activity Score (JADAS) of JADAS10, JADAS27, or JADAS71 minimal disease after 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, or 28 weeks of treatment.

14. The pharmaceutical composition according to claim 13, wherein more than 10% of the patients have a disease of JADAS10, JADAS27, and JADAS71 minimal disease activity after 12 weeks of treatment, 16 weeks of treatment, 20 weeks of treatment, 24 weeks of treatment, and 28 weeks of treatment.

15. The pharmaceutical composition according to claim 13, wherein after 24 weeks of treatment and 28 weeks of treatment, more than 15% of the patients have a disease with minimal disease activity of JADAS10, JADAS27, and JADAS71.

16. The pharmaceutical composition according to claim 13, wherein the pediatric patient is 2 to 17 years old.

17. The pharmaceutical composition according to claim 13, wherein the juvenile idiopathic arthritis (JIA) is polyarticular juvenile idiopathic arthritis (pJIA).

18. The pharmaceutical composition according to claim 13, wherein the method further comprises administering methotrexate (MTX) to the pediatric patient.

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