Anti-TNF antibodies, compositions, and methods for treatment of active psoriatic arthritis

Abstract

To provide compositions for use in safe and effective treatment of active Psoriatic Arthritis (PsA).SOLUTION: The present invention relates to a composition comprising at least one isolated mammalian anti-TNF antibody having a heavy chain (HC) comprising a specific sequence and a light chain (LC) comprising a specific sequence, and at least one pharmaceutically acceptable carrier or diluent, wherein the composition is administered via IV infusion, and wherein at week 14 of treatment, patients treated with the anti-TNF antibody achieve a mean change from a baseline in one or more criteria selected from a group consisting of HAQ-DI=-0.60±0.53SD, enthesitis=-1.87±1.75SD, dactylitis=-7.8±8.57SD, SF-36 PCS=8.65±7.60SD, and SF-36 MCS=5.33±9.95SD.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a safe and effective treatment for active psoriatic arthritis (PsA). a heavy chain (HC) comprising SEQ ID NO: 36 for use in the effective treatment of Compositions utilizing anti-TNF antibodies having a light chain (LC) comprising SEQ ID NO: 37 and methods. [Background technology]

[0002] TNFα is a soluble homotrimer of 17 kD protein subunits. A membrane-bound 26 kD precursor form also exists.

[0003] Cells other than monocytes or macrophages also produce TNFα. For example, human non-monocytic tumor cells The cell line produces TNFα and CD4+ and CD8+ peripheral blood T lymphocytes, and several cultures Selected T and B cell lines also produce TNFα.

[0004] TNFα induces cartilage and bone degradation, adhesion molecule induction, and procoagulant activity in vascular endothelial cells Increases adhesion of neutrophils and lymphocytes, and promotes adhesion of macrophages, neutrophils and blood vessels Pro-inflammatory effects leading to tissue damage, such as stimulating the release of platelet-activating factor from endothelial cells, Causes use.

[0005] TNFα is involved in infections, immune disorders, neoplastic, autoimmune, and graft-versus-host pathologies. The association of TNFα with cancer and infectious pathologies is related to the catabolic state of the host. Cancer patients often suffer from weight loss, which is usually associated with anorexia.

[0006] The severe wasting associated with cancer and other diseases is known as "cachexia." Symptoms include progressive weight loss, anorexia, and persistent decline in lean body mass in response to tumor growth. Cachexia is a major cause of morbidity and mortality in many cancers. TNFα plays a key role in the prevention of cancer, infectious diseases, and There is evidence that it is involved in cachexia in cardiovascular disease and other catabolic states.

[0007] TNFα contributes to the development of gram-negative sepsis and endotoxin, including fever, fatigue, anorexia, and cachexia. Endotoxin is thought to play a central role in acute shock. It strongly stimulates phage production and secretion of TNFα and other cytokines. Other monocyte-derived cytokines mediate metabolic and neurohormonal responses to endotoxin. Endotoxin administration to human volunteers produces fever, tachycardia, increased metabolic rate, and stress hormones. Circulating TNFα leads to acute illness with flu-like symptoms, including the release of TNFα. , is increased in patients with gram-negative sepsis.

[0008] Therefore, TNFα plays a key role in the treatment of inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections. , malignancies, and / or neurodegenerative diseases, and has been associated with rheumatoid arthritis and crow's feet. It is a useful target for specific biological treatments in diseases such as Crohn's disease. Beneficial effects of the monoclonal antibody cA2 in an open-label study were observed in the suppression of inflammation. and has been reported with successful retreatment after relapse in rheumatoid arthritis and Crohn's disease. The beneficial results of a randomized, double-blind, placebo-controlled trial using cA2 have also It has been reported to suppress inflammation in goat.

[0009] Other investigators have described mAbs specific for recombinant human TNF that had neutralizing activity in vitro. Some of these mAbs map epitopes on human TNF. and used to develop enzyme immunoassays and to aid in the purification of recombinant TNF. However, these studies have been hampered by immunogenicity, low specificity, and / or pharmaceutical incompatibility. and TNF-neutralizing antibodies that can be used for in vivo diagnostic or therapeutic applications in humans. does not provide a basis for generating

[0010] Neutralizing antisera or mAbs against TNF have been shown to inhibit experimental endotoxin production in mammals other than humans. To prevent adverse physiological changes and death after lethal challenge in hemodialysis and bacteremia. This effect has been demonstrated, for example, in rodent lethality assays and primate pathology models. It is shown in the system.

[0011] The putative receptor binding site for hTNF has been disclosed, and amino acids 11-13 and 37 of TNF are The receptor binding locus of TNFα consisting of 42, 49, 57, and 155, 157 is disclosed. There are.

[0012] Non-human mammalian, chimeric, polyclonal (e.g., antisera), and / or monoclonal Monoclonal antibodies (Mabs) and fragments thereof (e.g., proteolytic digestion or fusion protein production thereof) In some cases, drugs are potent drugs that are being investigated in an attempt to treat certain diseases. However, such antibodies or fragments may induce an immune response when administered to humans. Such an immune response may be mediated by immune complexes of antibodies or fragments from the blood circulation. This can lead to clearance, making repeated administration unsuitable for therapy, thereby causing discomfort to the patient. This reduces the therapeutic benefit of the antibody or fragment and limits re-administration of the antibody or fragment. Repeated administration of antibodies or fragments containing the antibody may result in serum sickness and / or anaphylaxis. To avoid these and other problems, chimerization and hybridization are used, as is well known in the art. Many approaches have been taken to reduce the immunogenicity of such antibodies and portions thereof, including silencing. However, these and other approaches still suffer from some immunogenicity, low affinity, have low binding activity, or are difficult to culture, scale up, produce and / or yield. Such antibodies or fragments may result in problems in the treatment of They may not be ideally suited for production or use as therapeutic proteins. Summary of the Invention [Problem to be solved by the invention]

[0013] Therefore, an anti-TNF antibody or fragment that overcomes another of these problems, as well as There is a need to provide improvements to known antibodies or fragments thereof. [Means for solving the problem]

[0014] The present invention provides a method for the safe and effective treatment of active psoriatic arthritis using SEQ ID NO: 1. 36 and a light chain (LC) comprising SEQ ID NO: 37. An isolated mammalian anti-TNF antibody is provided, said anti-TNF antibody being administered intravenously. IV) administered via infusion, and at 14 weeks of treatment, patients treated with anti-TNF antibodies Health Assessment Questionnaire Disability Index scores core, HAQ-DI)=-0.60±0.53 standard deviation (SD), Enthesitis = -1.87 ± 1.75 SD, dactylitis = -7.8 ± 8.57 SD, 36-item shot 36-item Short-Form Health Survey Physical Summary Score Summary score (SF-36PCS) = 8.65 ± 7.60 SD, and 36-item show 36-item Short-Form Health Survey Mental Component Summary Score l Component Summary score, SF-36MCS) = 5.33 ± 9.95 SD Achieve a mean change from baseline in one or more criteria selected from .

[0015] The present invention provides a method for the safe and effective treatment of active psoriatic arthritis using SEQ ID NO: 1. 36 and a light chain (LC) comprising SEQ ID NO: 37. An isolated mammalian anti-TNF antibody is provided, said anti-TNF antibody being administered at 0 and 4 weeks, and then every 8 weeks (q8w) at a dose of 2 mg / kg intravenously over 30 ± 10 minutes (IV) Patients treated with anti-TNF antibodies administered via infusion at week 14 of treatment: HAQ-DI=-0.60±0.53SD, Enthesitis=-1.87±1.75SD, Finger inflammation = -7.8 ± 8.57 SD, SF-36PCS = 8.65 ± 7.60 SD, and SF- One or more criteria selected from the group consisting of 36MCS = 5.33 ± 9.95 SD Achieve a mean change from baseline in

[0016] The present invention provides a compound of SEQ ID NO: 1 for use in the safe and effective treatment of active psoriatic arthritis. At least one having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37 and at least one pharmaceutically acceptable carrier or diluent. and a composition comprising an agent and a medicament for treating atopic dermatitis, the composition being administered via IV infusion, and the composition being administered within 14 weeks of treatment. Patients treated with anti-TNF antibodies had an HAQ-DI of -0.60 ± 0.53 SD in the eyes and tendons. Enthesitis = -1.87 ± 1.75 SD, dactylitis = -7.8 ± 8.57 SD, SF-36PC S = 8.65 ± 7.60 SD, and SF-36MCS = 5.33 ± 9.95 SD. Achieve a mean change from baseline in one or more criteria selected from the group do.

[0017] The present invention provides a compound of SEQ ID NO: 1 for use in the safe and effective treatment of active psoriatic arthritis. At least one having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37 and at least one pharmaceutically acceptable carrier or diluent. and a composition comprising the agent, wherein the composition is administered at weeks 0 and 4, and then every 8 weeks thereafter ( q8w) at a dose of 2 mg / kg administered via IV infusion over 30 ± 10 minutes. At 14 weeks of treatment, patients treated with anti-TNF antibodies had a HAQ-DI of -0.60±0. .53SD, enthesitis=-1.87±1.75SD, dactylitis=-7.8±8.57SD, SF-36PCS = 8.65 ± 7.60 SD, and SF-36MCS = 5.33 ± 9.9 Average change from baseline in one or more criteria selected from the group consisting of 5 SD Achieve an even change.

[0018] The present invention provides a compound of SEQ ID NO: 1 for use in the safe and effective treatment of active psoriatic arthritis. At least one having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37 and at least one pharmaceutically acceptable carrier or diluent. and a composition comprising the agent, wherein the composition is administered with or without MTX. The composition was administered via IV infusion and treated with anti-TNF antibody at week 14 of treatment. Patients had a HAQ-DI of -0.60 ± 0.53 SD and an enthesitis score of -1.87 ± 1.75. SD, dactylitis=-7.8±8.57SD, SF-36PCS=8.65±7.60SD, and and SF-36MCS=5.33±9.95SD Achieve mean change from baseline in the above criteria.

[0019] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). wherein the composition is administered via IV infusion, and at 14 weeks of treatment, Patients treated with TNF antibody had HAQ-DI = -0.60 ± 0.53 SD and enthesitis =-1.87±1.75SD, dactylitis=-7.8±8.57SD, SF-36PCS=8. 65±7.60SD, and SF-36MCS=5.33±9.95SD. Achieve a mean change from baseline in one or more criteria of your choice.

[0020] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). the composition is administered at weeks 0 and 4, and then every 8 weeks (q8w) thereafter. administered via IV infusion at a dose of 2 mg / kg over 30 ± 10 minutes, At week 14, patients treated with anti-TNF antibodies had a HAQ-DI of -0.60 ± 0.53 S D, enthesitis = -1.87±1.75SD, dactylitis = -7.8±8.57SD, SF-3 6PCS=8.65±7.60SD, and SF-36MCS=5.33±9.95SD The mean change from baseline in one or more criteria selected from the group consisting of: Achieve.

[0021] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). The composition may be administered with or without MTX, and The composition was administered over 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter. The anti-TNF antibody was administered via IV infusion at a dose of 2 mg / kg at week 14 of treatment. Patients treated with HAQ-DI = -0.60 ± 0.53 SD, enthesitis = -1.8 7±1.75SD, dactylitis=-7.8±8.57SD, SF-36PCS=8.65±7. SF-36MCS=5.33±9.95SD, and SF-36MCS=5.60SD. Achieve a mean change from baseline in one or more criteria.

[0022] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). wherein the composition is administered via IV infusion, and at 14 weeks of treatment, Patients treated with TNF antibody had HAQ-DI = -0.60 ± 0.53 SD and enthesitis =-1.87±1.75SD, dactylitis=-7.8±8.57SD, SF-36PCS=8. 65±7.60SD, and SF-36MCS=5.33±9.95SD. The method involves achieving a mean change from baseline in one or more criteria selected. before, simultaneously with, or after the foregoing, (a) adding a detectable label or reporter, a TNF antagonist, an anti-TNF antibody, Anti-rheumatic drugs, muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, Sedatives, local anesthetics, neuromuscular blockers, antibacterials, antipsoriatics, corticosteroids, anabolites steroids, erythropoietin, immunizing agents, immunoglobulins, immunosuppressants, growth hormones Months, hormone replacement drugs, radioactive drugs, antidepressants, antipsychotics, stimulants, asthma medications, beta agonists, inhaled steroids, epinephrine or similar drugs, cytokines, or cytokines an effective amount of at least one compound selected from at least one of the following: The method further comprises administering at least one composition comprising the protein.

[0023] The present invention provides a compound of SEQ ID NO: 1 for use in the safe and effective treatment of active psoriatic arthritis. At least one having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37 and wherein the anti-TNF antibody is administered intravenously (IV) infusion. At 24 weeks of treatment, patients treated with anti-TNF antibodies had a modified total venous Total modified van der Heijde-Sharp score , vdH-S) = -0.36 ± 0.144 Standard error (SE) Achieve average change from baseline.

[0024] The present invention provides a method for the safe and effective treatment of active psoriatic arthritis using SEQ ID NO: 1. 36 and a light chain (LC) comprising SEQ ID NO: 37. An isolated mammalian anti-TNF antibody is provided, said anti-TNF antibody being administered at 0 and 4 weeks, and then every 8 weeks (q8w) at a dose of 2 mg / kg intravenously over 30 ± 10 minutes (IV) Infusion administered via anti-TNF antibody at 24 weeks of treatment, patients Achieving a mean change from baseline in vdH-S = -0.36 ± 0.144 SE do.

[0025] The present invention provides a method for the safe and effective treatment of active psoriatic arthritis using SEQ ID NO: 1. 36 and a light chain (LC) comprising SEQ ID NO: 37. An isolated mammalian anti-TNF antibody is provided, said anti-TNF antibody being administered in combination with MTX or M TX was administered without anti-TNF antibody at weeks 0 and 4, then every 8 weeks thereafter (q8w ) administered via intravenous (IV) infusion at a dose of 2 mg / kg over 30 ± 10 minutes At 24 weeks of treatment, patients treated with anti-TNF antibodies had a vdH-S of -0.36± Achieving a mean change from baseline in SE of 0.144.

[0026] The present invention provides a compound of SEQ ID NO: 1 for use in the safe and effective treatment of active psoriatic arthritis. At least one having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37 and at least one pharmaceutically acceptable carrier or diluent. and a composition comprising an agent, wherein the composition is administered via IV infusion and is administered for 24 weeks of treatment. In the eyes, patients treated with anti-TNF antibodies had a vdH-S of -0.36 ± 0.144 SE. Achieve a mean change from baseline in

[0027] The present invention provides a compound of SEQ ID NO: 1 for use in the safe and effective treatment of active psoriatic arthritis. At least one having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37 and at least one pharmaceutically acceptable carrier or diluent. and a composition comprising the agent, wherein the composition is administered at weeks 0 and 4, and then every 8 weeks thereafter ( q8w) at a dose of 2 mg / kg administered via IV infusion over 30 ± 10 minutes. ,At 24 weeks of treatment, patients treated with anti-TNF antibodies had vdH-S=-0.36±0. Achieve a mean change from baseline in 144 SEs.

[0028] The present invention provides a compound of SEQ ID NO: 1 for use in the safe and effective treatment of active psoriatic arthritis. At least one having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37 and at least one pharmaceutically acceptable carrier or diluent. and a composition comprising the agent, wherein the composition is administered at weeks 0 and 4, and then every 8 weeks thereafter ( q8w) at a dose of 2 mg / kg administered via IV infusion over 30 ± 10 minutes. ,At 24 weeks of treatment, patients treated with anti-TNF antibodies had vdH-S=-0.36±0. Achieve a mean change from baseline in 144 SEs.

[0029] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). the composition is administered via IV infusion, and at 24 weeks of treatment, Patients treated with TNF antibody had a baseline of vdH-S = -0.36 ± 0.144 SE. Achieve average change from baseline.

[0030] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). the composition is administered at weeks 0 and 4, and then every 8 weeks (q8w) thereafter. administered via IV infusion at a dose of 2 mg / kg over 30 ± 10 minutes, At week 24, patients treated with anti-TNF antibodies had a vdH-S of -0.36 ± 0.144S The mean change from baseline in E is achieved.

[0031] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). The composition may be administered with or without MTX, and the composition was administered for 30 ± 10 minutes at 0 and 4 weeks, then every 8 weeks (q8w) thereafter. g / kg via IV infusion and treated with anti-TNF antibody at week 24 of treatment. Patients who underwent a 2-month follow-up study showed a mean change from baseline in vdH-S=-0.36±0.144SE. Achieve an even change.

[0032] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). The composition may be administered with or without MTX, and the composition was administered for 30 ± 10 minutes at 0 and 4 weeks, then every 8 weeks (q8w) thereafter. g / kg via IV infusion and treated with anti-TNF antibody at week 24 of treatment. Patients who underwent a 2-month follow-up study showed a mean change from baseline in vdH-S=-0.36±0.144SE. To achieve this equilibrium change, the method may further comprise, before, simultaneously with, or after the foregoing, (a) adding a detectable label or reporter Anti-inflammatory drugs, TNF antagonists, anti-rheumatic drugs, muscle relaxants, narcotics, non-steroidal anti-inflammatory drugs (NSAIDS) D), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blocking agents, antibacterial agents, antipsoriatic agents, corticosteroids Corticosteroids, anabolic steroids, erythropoietin, immunizing agents, immunoglobulins , immunosuppressants, growth hormones, hormone replacement drugs, radioactive pharmaceuticals, antidepressants, antipsychotics, Stimulants, asthma medications, beta-agonists, inhaled steroids, epinephrine or similar drugs, and administering an effective amount of at least one of a steroid hormone, a steroid hormone, or a cytokine antagonist. and further administering at least one composition comprising at least one compound or protein. include.

[0033] The present invention provides a method for the safe and effective treatment or prevention of active psoriatic arthritis. At least one antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37.

[0013] Another isolated mammalian anti-TNF antibody is provided, wherein the anti-TNF antibody is administered via IV infusion. and elicit a clinical response selected from the group consisting of the responses in the table below.

[0034] [Table 1]

[0035] The present invention provides a method for the safe and effective treatment or prevention of active psoriatic arthritis. At least one antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37. and an isolated mammalian anti-TNF antibody, the anti-TNF antibody being administered by IV infusion. Administered via the IVF-based anticoagulant, more than 65% of patients receiving treatment achieved an ACR20 response at week 14 of treatment. Achieve.

[0036] The present invention provides a method for the safe and effective treatment or prevention of active psoriatic arthritis. At least one antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37. and a mammalian anti-TNF antibody, the anti-TNF antibody being at 0 and 4 weeks of age. eye, then every 8 weeks (q8w) thereafter at a dose of 2 mg / kg over 30 ± 10 minutes , administered via IV infusion, with over 65% of patients receiving treatment experiencing a ≥ 14 week ACR20 will be achieved.

[0037] The present invention provides a method for the safe and effective treatment or prevention of active psoriatic arthritis. At least one antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37. and a mammalian anti-TNF antibody, the anti-TNF antibody being at 0 and 4 weeks of age. eye, then every 8 weeks (q8w) thereafter at a dose of 2 mg / kg over 30 ± 10 minutes , administered via IV infusion, with over 65% of patients receiving treatment experiencing a ≥ 14 week ACR20 was achieved in 65% or more of the patients, and a treatment difference of 50% or more (compared to placebo) was observed in 65% or more of the patients. (improvement compared to baseline) and achieve ACR20 at 14 weeks of treatment.

[0038] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). the composition is administered at weeks 0 and 4, and then every 8 weeks (q8w) thereafter. Administered via IV infusion at a dose of 2 mg / kg over 30 ± 10 minutes. More than 65% of patients receiving the treatment achieve ACR20 at 14 weeks of treatment.

[0039] The present invention provides a method for treating a TNF-associated condition, the TNF-associated condition being an active psoriatic arthritis, and the method comprises administering to a subject a heavy chain (HC) comprising SEQ ID NO: 36 and a human psoriatic arthritis comprising SEQ ID NO: 37. and administering a composition comprising a safe and effective amount of an isolated mammalian anti-TNF antibody having a light chain (LC). the composition is administered at weeks 0 and 4, and then every 8 weeks (q8w) thereafter. Administered via IV infusion at a dose of 2 mg / kg over 30 ± 10 minutes. More than 65% of patients receiving the drug achieved ACR20 at 14 weeks of treatment, Patients with a treatment difference (improvement compared to placebo) of 50% or more achieved AC at week 14 of treatment. Achieve R20. [Brief explanation of the drawings]

[0040] [Figure 1] Figure 1 shows a graphical representation of an assay for the ability of TNV mAbs in hybridoma cell supernatants to inhibit TNFα binding to recombinant TNF receptors. Various amounts of hybridoma cell supernatant containing known amounts of TNV mAb were preincubated with a fixed concentration (5 ng / mL) of 125I-labeled TNFα. The mixture was transferred to a 96-well Optiplate precoated with the recombinant TNF receptor / IgG fusion protein p55-sf2. The amount of TNFα bound to the p55 receptor in the presence of mAb was determined after washing away unbound material and counting using a gamma counter. Eight TNV ​​mAb samples were tested in these experiments; however, for brevity, three of the mAbs shown by DNA sequence analysis to be identical to one of the other TNV mAbs (see section 5.2.2) are not shown here. Each sample was tested in duplicate. Results shown are representative of two independent experiments. [Figure 2A]The DNA sequence of the TNV mAb heavy chain variable region is shown. 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. A dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single-letter code) beginning 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. Gaps in the germline DNA sequence (CDR3) are due to sequences that are unknown or not present in the germline gene. The TNV mAb heavy chain uses the J6 binding region. [Figure 2B] The DNA sequence of the TNV mAb heavy chain variable region is shown. 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. A dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single-letter code) beginning 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. Gaps in the germline DNA sequence (CDR3) are due to sequences that are unknown or not present in the germline gene. The TNV mAb heavy chain uses the J6 binding region. [Figure 3]The DNA sequence of the TNV mAb light chain variable region is shown. The germline gene shown is a representative member of the Vg / 38K family of human kappa germline variable region genes. Dotted lines in the TNV mAb gene sequence indicate nucleotides that are identical to those in the germline sequence. The first 16 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single-letter 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 due to sequences that are unknown or not present in the germline gene. The TNV mAb light chain uses the J3 joining region. [Figure 4] The deduced amino acid sequences of the TNV mAb heavy chain variable regions are shown. The amino acid sequences (single-letter code) shown were deduced from DNA sequences determined from both uncloned and cloned PCR products. The amino acid sequences are 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 relates to both TNV148 and TNV148B. "TNV" indicates that the sequence shown relates to all TNV mAbs unless a different sequence is indicated. A dashed line in the germline sequence (CDR3) indicates that the sequence is unknown or not present in the germline gene. [Figure 5]The deduced amino acid sequence of the TNV mAb light chain variable region is shown. The amino acid sequence (single-letter code) shown was deduced from DNA sequences determined from both uncloned and cloned PCR products. The amino acid sequence is divided into the 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. 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] Figure 1 shows 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 by black boxes. The immunoglobulin enhancer in the JC intron is indicated by a gray box. Relevant restriction sites are indicated. The plasmids are shown oriented so that transcription of the Ab gene proceeds clockwise. 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 alternative heavy chain variable region sequences by replacing the BsiWI / BstBI restriction fragment. The variable region coding sequence of p1776 can be replaced with alternative variable region sequences by replacing the SalI / AflII restriction fragment. [Figure 7]A graphical representation of the growth curve analysis of five rTNV148B-producing cell lines is shown. Cultures were initiated on day 0 by seeding cells in I5Q+MHX medium in T75 flasks to a viable cell density of 1.0 × 10 cells / ml in a volume of 30 ml. The cell cultures used in these studies were continuous cultures due to the transfection and subcloning procedures. Cells in the T-flasks were then thoroughly resuspended, and 0.3 ml aliquots of the culture were removed. Growth curve studies were terminated when the cell count dropped below 1.5 × 10 cells / ml. The number of viable cells in the aliquots was determined by trypan blue exclusion, and the remaining aliquot was saved for later mAb concentration determination. A human IgG ELISA was performed on all sample aliquots at the same time. [Figure 8] A graphical representation of a comparison of cell growth rates in the presence of various MHX selection concentrations is shown. Cell subclones C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for an additional two days. Both cell cultures were then split into three cultures containing either no MHX, 0.2x MHX, or 1x MHX. One day later, new T75 flasks were seeded with the cultures at a starting density of 1x10 cells / ml, and cells were counted at 24-hour intervals for one week. The doubling time for the first five days was calculated using the formula in SOP PD32.025 and is shown above the bars. [Figure 9] A graphical representation of the stability of mAb production over time from two rTNV148B-producing cell lines is shown. After transfection and subcloning, subclones of cells from the 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 the culture every 4–6 days to maintain new viable cultures while simultaneously exhausting the previous cultures. Aliquots of exhausted cell supernatants were collected immediately after the cultures were exhausted and stored until the 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 Tg197 arthritis mouse model mice in response to an anti-TNF antibody of the present invention compared to the control in Example 4. Approximately 4-week-old Tg197 research mice were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus dose of Dulbecco's PBS (D-PBS) or an anti-TNF antibody of the present invention (TNV14, TNV148, or TNV196) at either 1 mg / kg or 10 mg / kg. 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 from weeks 3 to 7. Animals treated with 10 mg / kg TNV148 also achieved significant weight gain by week 7 of the study. [Figure 11A] The progression of disease severity based on the arthritis index shown in Example 4 is shown. The arthritis index of the 10 mg / kg cA2-treated group was lower than that of the D-PBS control group starting at week 3 and 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-treated group. When each was compared with similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there were no significant differences between the 10 mg / kg treatment groups. When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI than 1 mg / kg cA2 at weeks 3, 4, and 7. 1 mg / kg TNV148 was also significantly lower than the 1 mg / kg TNV14-treated group at weeks 3 and 4. While TNV196 showed a significant reduction in AI (when compared to the D-PBS treatment group) by week 6 of the study, TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 11B]The progression of disease severity based on the arthritis index shown in Example 4 is shown. The arthritis index of the 10 mg / kg cA2-treated group was lower than that of the D-PBS control group starting at week 3 and 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-treated group. When each was compared with similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there were no significant differences between the 10 mg / kg treatment groups. When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI than 1 mg / kg cA2 at weeks 3, 4, and 7. 1 mg / kg TNV148 was also significantly lower than the 1 mg / kg TNV14-treated group at weeks 3 and 4. While TNV196 showed a significant reduction in AI (when compared to the D-PBS treatment group) by week 6 of the study, TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 11C] The progression of disease severity based on the arthritis index shown in Example 4 is shown. The arthritis index of the 10 mg / kg cA2-treated group was lower than that of the D-PBS control group starting at week 3 and 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-treated group. When each was compared with similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148, and 196), there were no significant differences between the 10 mg / kg treatment groups. When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI than 1 mg / kg cA2 at weeks 3, 4, and 7. 1 mg / kg TNV148 was also significantly lower than the 1 mg / kg TNV14-treated group at weeks 3 and 4. While TNV196 showed a significant reduction in AI (when compared to the D-PBS treatment group) by week 6 of the study, TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 12]Figure 1 shows the weight change of Tg197, an arthritic mouse model, in response to an anti-TNF antibody of the present invention compared to the control in Example 5. Approximately 4-week-old Tg197 research mice were assigned to one of eight treatment groups based on body weight and treated with an intraperitoneal bolus of either a control (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 based on the arthritic index in Example 5. The arthritic index for 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. [Figure 13B]1 is a graph depicting the progression of disease severity based on the arthritic index in Example 5. The arthritic index for 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. [Figure 13C]1 is a graph depicting the progression of disease severity based on the arthritic index in Example 5. The arthritic index for 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. [Figure 14] Figure 1 shows the weight change of Tg197 arthritic mouse model mice in response to anti-TNF antibodies of the present invention compared to the control in Example 6. Tg197 study mice, approximately 4 weeks old, 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 a 10 mg / kg cA2 control group. [Figure 15]The progression of disease severity based on the arthritis index shown in Example 6 is shown. 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 experiment, 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 Tg197, a mouse model of arthritis, in response to an anti-TNF antibody of the present invention, compared to the control in Example 7. To compare the efficacy of a single intraperitoneal administration of TNV148 (derived from hybridoma cells) and rTNV148B (derived from transfected cells), approximately 4-week-old Tg197 research 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]Figure 1 depicts the progression of disease severity based on the arthritis index as shown in Example 7. The arthritis index for the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 4 and throughout the remainder of the study (week 8). Both the TNV148-treated group and the 1 mg / kg cA2-treated group showed a significant decrease in AI at week 4. While 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, this study showed slightly higher AIs from groups treated with both versions of the TNV antibody. The group treated with 1 mg / kg cA2 (except at week 6) did not have a significant increase when compared with the 10 mg / kg cA2 group, and the group treated with TNV148 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] FIG. 1 shows a diagram of the study design for a trial of Simponi (golimumab) administered intravenously in subjects with active psoriatic arthritis (PsA). DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3, and / or an isolated recombinant and / or Synthetic anti-TNF human, primate, rodent, mammalian, chimeric, humanized or CDR-grafted and at least one anti-TN Fc antibody, F antibody or anti-idiotypic antibody. The present invention provides diagnostic and therapeutic compositions, methods, and encoding nucleic acid molecules. and the production and use of such nucleic acids and antibodies, including devices, and anti-idiotypic antibodies. Methods further include, but are not limited to:

[0042] As used herein, "anti-tumor necrosis factor alpha antibody," "anti-TNF antibody," "anti-TNF "antibody portion" or "anti-TNF antibody fragment" and / or "anti-TNF antibody variant" etc. At least one complementary sequence of the heavy or light chain that can be incorporated into the antibody of the present invention. constant drive (CDR) or ligand-binding portion thereof, heavy or light chain variable region, heavy or light chain or light chain constant region, framework region, or any part thereof, or TNF receptor immunoglobulins, such as, but not limited to, at least a portion of a protein or binding protein. Any protein- or peptide-containing molecule, including molecules that contain at least a portion of a purine molecule. Such antibodies optionally further affect specific ligands and Without limitation, such antibodies may be used in vitro, in situ, and / or in vivo. Modulating or decreasing at least one TNF activity or binding or TNF receptor activity or binding , increase, antagonize, agonize, reduce, mitigate, block, inhibit, suppress, and / or interfere. By way of example, a preferred anti-TNF antibody, specified portion or variant of the invention may comprise at least one The antibody can bind to TNF or a specified part, variant or domain thereof. Suitable anti-TNF antibodies, specified portions or variants may also optionally be expressed in RNA, DNA, or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TN TNF activity or TNF production and / or synthesis, including, but not limited to, TNF activity, The term "antibody" also refers to a molecule that can affect at least one of the functions of the antibody. The term "antibody" is intended to encompass antibodies, digest fragments, specified portions, and variants thereof, including include antibody mimetics, or portions of antibodies that mimic the structure and / or function of an antibody. or specific fragments or portions thereof, including single chain antibodies and fragments thereof. 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')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion) digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., , by pepsin digestion, partial reduction and reassembly), Fv or scFv (e.g., molecular biology and fragments (e.g., by biological techniques) that bind to TNF or portions thereof. Antibody fragments capable of binding to the nucleotides of the present invention are encompassed by the present invention (see, e.g., Colligan, supra). (See Immunology).

[0043] Such fragments may be prepared as known in the art and / or as described herein. Antibodies can be produced by enzymatic cleavage, synthetically, or recombinantly, as described above. Using antibody genes in which these stop codons have been introduced upstream of the natural stop site, various truncations were For example, a combination of genes encoding the F(ab')2 heavy chain portion can be produced. The combination may include a DNA sequence encoding the CH1 domain and / or hinge region of the heavy chain. The various portions of the antibody can be chemically linked by conventional techniques. or can be prepared as a contiguous protein using genetic engineering techniques. It is possible.

[0044] As used herein, the term "human antibody" refers to a human antibody in which substantially all parts of the protein are humanized. minutes (e.g., CDR, framework, C L , C H Domain (e.g., C H 1. C H 2. C H 3), Hinge (V L , V H )) can be effectively detected in humans with only minor sequence changes or mutations. Similarly, antibodies that are non-immunogenic in primates (monkeys, baboons, chimpanzees, etc.) Rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.) and other mammals Species-, subgenus-, genus-, subfamily-, and family-specific antibodies are designated. In addition, chimeric antibodies include any combination of the above. Such changes or mutations may optionally Alternatively, and preferably, the antibody retains immunogenicity in humans or other species compared to the unmodified antibody. Thus, a human antibody is distinct from a chimeric or humanized antibody. Human antibodies contain functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing the gene Furthermore, when a human antibody is a single-chain antibody, it is possible to obtain a sequence that is not found in natural human antibodies. For example, Fv can be composed of a heavy chain variable region and a light chain variable region. It may contain a linker peptide such as 2 to about 8 glycine or other amino acid residues. Such linker peptides are considered to be of human origin.

[0045] Also, monoclonal antibodies having binding specificities for at least two different antigens are preferred. or human or humanized antibodies, bispecific, heterospecific, heterobinding, or similar antibodies. In this case, one of the binding specificities is for at least one TNF-α. One is directed against a protein and the other against any other antigen. Methods for producing bispecific antibodies are known in the art. The synthesis is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains are different. (Milstein and Cuello, Nature, 305: 537 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, These hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules. Only one of these has the correct bispecific structure. Purification of the correct molecule (usually The process of cleaving the product (performed by an affinity chromatography step) is rather laborious and results in poor product yield. Similar procedures are described, for example, in WO 93 / 08829, U.S. Pat. No. 10,668, No. 6,193,967, No. 6,132,992, No. 6,10 No. 6,833, No. 6,060,285, No. 6,037,453, No. 6,010 , No. 902, No. 5,989,530, No. 5,959,084, No. 5,959, No. 083, No. 5,932,448, No. 5,833,985, No. 5,821,3 No. 33, No. 5,807,706, No. 5,643,759, No. 5,601,81 No. 9, No. 5,582,996, No. 5,496,549, No. 4,676,980 No. 91 / 00360, WO 92 / 00373, European Patent No. 030 No. 89, Traunecker et al., EMBO J.10:3655(199 1), Suresh et al., Methods in Enzymology12 1:210 (1986), each of which is incorporated herein by reference in its entirety. will be incorporated into

[0046] Anti-TNF Antibodies (also referred to as TNF Antibodies) Useful in the Methods and Compositions of the Invention are characterized by high affinity binding to TNF, and optionally and preferably low toxicity. Specifically, the variable region, constant region, and frame region may be optionally characterized. Individual components such as the framework may be individually and / or collectively optionally and preferably The antibodies of the present invention, specified fragments, or variants thereof, which have low or no immunogenicity, are also included in the present invention. The antibodies that can be used in the present invention are optionally useful in detecting symptoms. The ability to treat patients for extended periods with affordable palliation and low and / or acceptable toxicity Low or acceptable immunogenicity and / or high affinity, and Other favorable properties can contribute to the therapeutic results obtained. As used herein, significant adverse events are seen in less than about 75% of treated patients, or preferably less than about 50%. HAHA, HACA, or HAMA responses are increased and / or in the patient being treated low titer (less than about 300, preferably about 100, as measured by double antigen enzyme immunoassay) 100) (Elliott et al., Lan cet 344:1125-1127 (1994), which is incorporated herein by reference in its entirety. (It is included).

[0047] Utility: The isolated nucleic acids of the present invention can be used in cells, tissues, organs, or animals (including mammals and humans). and measuring or acting on immune disorders or diseases, cardiovascular disorders or diseases, infectious diseases, , malignant and / or neurological disorders or diseases, and / or a combination of the compounds of the present invention, including, but not limited to, a compound that can be used to diagnose, monitor, regulate, treat, mitigate, or prevent at least one TNF condition. at least one compound that can be used to help prevent or reduce the symptoms of It can be used to generate anti-TNF antibodies or specified variants thereof.

[0048] Such methods include those for modulating, treating, alleviating, preventing, or reducing symptoms, effects, or mechanisms. and administering at least one anti-TNF antibody to a cell, tissue, organ, animal, or patient in need thereof. The effective amount may comprise administering an effective amount of a composition or pharmaceutical composition comprising the compound of formula (I) or (II). When performed and determined using known methods, as described or known in the relevant art, Approximately 0.001 to 500 mg / kg per dose (e.g., bolus), multiple doses, or continuous infusion g dose, or 0.01 to 5000 μg / mL of blood per single, multiple, or continuous administration This may include an amount that achieves a concentration of 100% or any range or value therein. All publications or patents cited in the specification are incorporated herein by reference in their entirety. , representing the state of the art at the time of the invention and / or providing an explanation and enablement of the invention Publications include any scientific publication or patent publication, or any recorded electronic or printed material. This refers to any other information available in any media format, including print formats. Ausubel, et al., ed., Cur rent Protocols in Molecular Biology,John Wiley & Sons, Inc., NY, NY (1987-2001), Sambro ok,et al.,Molecular Cloning:A Laboratory Manual,2nd Edition,Cold Spring Harbor,N Y (1989), Harlow and Lane, antibodies, a Lab. oratory Manual,Cold Spring Harbor,NY(198 9), Colligan,et al.,eds.,Current Protocol s in Immunology,John Wiley&Sons,Inc.,NY( 1994-2001), Colligan et al.,Current Proto cols in Protein Science,John Wiley&Sons, New York, New York, (1997-2001).

[0049] Antibodies of the present invention: all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3, and / or At least one antibody of the present invention comprising all of the light chain variable CDR regions of sequences 4, 5, and 6. The TNF antibody may optionally be administered to a cell line, mixed cell line, or other cell line, as is well known in the art. It may be produced by a cell line, an immortalized cell, or a clonal population of immortalized cells. bel,et al.,ed.,Current Protocols in Mole. cular Biology,John Wiley&Sons,Inc.,NY,NY (1987-2001), Sambrook, et al., Molecular Cl oning:A Laboratory Manual,2nd Edition,Co ld Spring Harbor, NY (1989), Harlow and Lan e,antibodies,a Laboratory Manual,Cold Sp Ring Harbor, NY (1989), Colligan, et al., eds. .,Current Protocols in Immunology,John W. iley & Sons, Inc., NY (1994-2001), Colligan et al. al.,Current Protocols in Protein Science e, John Wiley & Sons, NY, NY, (1997-2001) and each is incorporated herein by reference in its entirety.

[0050] Human antibodies specific for human TNF protein or fragments thereof are isolated and / or Suitable molecules such as TNF proteins or portions thereof (including synthetic molecules such as synthetic peptides) Other specific or general mammalian antibodies may be raised against any suitable immunogenic antigen. Preparation of immunogenic antigens and generation of monoclonal antibodies can be carried out using any suitable technique. It can be done.

[0051] In one approach, hybridomas are cloned from a suitable immortalized cell line (e.g., Sp2 / 0 , Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3 X63Ag8.653, Sp2SA3, Sp2MAI, Sp2SS1, Sp2SA5, U 937, MLA144, ACT IV, MOLT4, DA-1, JURKAT, WEHI , K-562, COS, RAJI, NIH3T3, HL-60, MLA144, NAMA Myeloma cell lines, such as, but not limited to, IWA, NEURO2A, or heteromyeloma Heteromylomas, their fusion products, or any cells or By fusing fusion cells, or any other suitable cell line known in the art. Generated by, for example, www.atcc.org, www.lifetech.co See m. etc. Isolated or cloned spleen, peripheral blood, lymph, tonsils, or antibody-producing cells, such as, but not limited to, other immune or B-cell containing cells; or recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptile Insects, fish, mammals, rodents, horses, sheep, goats, lambs, primates, eukaryotes, geno mitochondrial DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA, or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded or triple-stranded, either endogenous or heterologous nucleic acid, such as hybridized nucleic acid, or combinations thereof. For example, heavy or light chain constant or variable or framework or CDR sequences and any other cells that express the sequence. For example, see Ausubel and Collig, supra. See Immunology chapter 2, the entire contents of which are incorporated herein by reference. It will be incorporated into the specification.

[0052] Antibody-producing cells can also be isolated from the peripheral blood of humans or other suitable animals immunized with an antigen of interest, or Preferably, the host cell may be obtained from the spleen or lymph nodes. , a heterologous or endogenous nucleic acid encoding an antibody of the invention, a specified fragment, or a variant thereof. The fused cells (hybridomas) or recombinant cells can be used to express the desired nucleic acid. isolated using conventional culture conditions or other suitable known methods, and then subjected to limiting dilution or cell sorting or can be cloned by other known methods. The cells can be selected by a suitable assay (eg, ELISA).

[0053] Selecting recombinant antibodies from peptide or protein libraries (e.g., bacteriophages) Display libraries for phages, ribosomes, oligonucleotides, RNA, cDNA, etc. Examples of antibodies include, but are not limited to, Cambridge antibody T echnologies,Cambridgeshire,UK,MorphoSys, Martinsreid / Planegg, DE, Biovation, Aberdee n,Scotland,UK,BioInvent,Lund,Sweden,Dyax Corp., Enzon, Affymax / Biosite, Xoma, Berkel ey, CA, Ixsys. For example, European Patent No. 368,684, International Application PCT / GB 91 / 01134, International Application No. PCT / GB92 / 01755, International Application No. PCT / GB 92 / 002240, International Application No. PCT / GB92 / 00883, International Application No. PCT / G B93 / 00605, U.S. Patent Application Publication 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. 90 / 14 443, International Publication No. 90 / 14424, International Publication No. 90 / 14430, International Application P CT / US94 / 1234, WO 92 / 18619, WO 96 / 077 No. 54, (Scripps), European Patent No. 614 989 (MorphoSys), Country International Publication No. 95 / 16027 (BioInvent), International Publication No. 88 / 06630, WO 90 / 3809 (Dyax), U.S. Pat. No. 4,704,692 (Enzo n), International Application No. PCT / US91 / 02989, International Publication No. 89 / 06283, European Patent No. 371 998, European Patent No. 550 400, (Xoma), European Patent No. 22 No. 9046, International Application No. PCT / US91 / 07149, or probabilistically generated Peptides or proteins - U.S. Patent Nos. 5,723,323, 5,763,192, No. 5814476, No. 5817483, No. 5824514, No. 5976862 No. 86 / 05803, European Patent No. 590 689 (Ixsys, now Applied Molecular Evolution (AME), each of which is referenced (which are incorporated herein in their entirety) or are known in the art, and / or or a transgene capable of generating a repertoire of human antibodies as described herein. It relies on immunization of genetically engineered animals (e.g., SCID mice, Nguyen et al. .,Microbiol.Immunol.41:901-907(1997), San dhu et al.,Crit.Rev.Biotechnol.16:95-118 (1996), Eren et al., Immunol.93:154-161(19 98) (each incorporated by reference in its entirety), and related patents and applications) Other suitable methods for generating or isolating antibodies of the required specificity, including, but not limited to, Such techniques include ribosome display (Hanes et al. al.,Proc.Natl.Acad.Sci.USA,94:4937-4942 (May 1997), Hanes et al., Proc. Natl. Acad.S ci.USA, 95:14130-14135 (Nov. 1998)), single cell antibody production synthesis techniques (e.g., Selected Lymphocyte Antibody Method ("SLAM") (U.S. Patent No. 5,627,055) No. 2, Wen et al., J. Immunol. 17:887-892 (1987) ,Babcook et al.,Proc.Natl.Acad.Sci.USA 9 3:7843-7848(1996)), gel microdroplets (gel micro rodroplet), and flow cytometry (Powell et al., Bi otechnol.8:333-337(1990), One Cell System s, Cambridge, MA, Gray et al., J. Imm. Meth. 18 2:155-163 (1995), Kenny et al., Bio / Technol 13:787-790(1995)), B cell selection (Steenbakkers e t al., Molec. Biol. Reports 19:125-134 (1994 ), Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization in Hybridoma Technol ogy,Borrebaeck,ed.,Elsevier Science Publ. ishers BV,Amsterdam,Netherlands(1988)) These include, but are not limited to:

[0054] Methods for engineering or humanizing non-human or human antibodies can also be used, and Generally, humanized or engineered antibodies are well known in the art, e.g., Such mammals include, but are not limited to, mice, rats, rabbits, non-human primates, or other mammals. These human amino acid residues have one or more amino acid residues from a non-human source. The group is often called an "import" residue and is typically the "import" residue of a known human sequence. " taken from the variable region, constant region, or other domain. Known human Ig sequences are disclosed. For example, see www.ncbi.nlm.nih.gov / entrez / quer y.fcgi, www.atcc.org / phage / hdb.html, www.s ciquest.com / , www.abcam.com / , www.antibody resource.com / onlinecomp.html, www.public. iastate.edu / ~pedro / research_tools.html, w www.mgen.uni-heidelberg.de / SD / IT / IT.html, www.whfreeman.com / immunology / CH05 / kuby05 .htm, www.library.thinkquest.org / 12429 / Im mune / Antibody.html, www.hhmi.org / grants / l ectures / 1996 / vlab / , www.path.cam.ac.uk / ~m rc7 / mikeimages.html, www.antibodyresource .com / , mcb.harvard.edu / BioLinks / Immunolog y.html.www.immunologylink.com / , pathbox.w ustl.edu / ~hcenter / index.html, www.biotech .ufl.edu / ~hcl / , www.pebio.com / pa / 340913 / 3 40913.html、www.nal.usda.gov / awic / pubs / an tibody / 、www.m.ehime-u.ac.jp / ~yasuhito / El isa.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、aximt1.imt. uni-marburg.de / ~rek / AEPStart.html、baserv .uci.kun.nl / ~jraats / links1.html、www.reca b.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.cnu sc.fr:8104 / 、www.biochem.ucl.ac.uk / ~marti n / abs / index.html、antibody.bath.ac.uk / 、ab gen.cvm.tamu.edu / lab / wwwabgen.html、www.u nizh.ch / ~honegger / AHOseminar / Slide01.htm l、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 / ind ex.html, www.cryst.bioc.cam.ac.uk / ~fmolin a / Web-pages / Pept / spottech.html, www.jerin i.de / fr_products.htm, www.patents.ibm.com / ibm.html.Kabat et al.,Sequences of Prot eins of Immunological Interest,USDept. Health (1983), each of which is incorporated herein by reference in its entirety. .

[0055] Such imported sequences may be used to reduce immunogenicity or to improve the performance of the present invention. As known in the art, binding, affinity, on-rate, off-rate, avidity, specificity, affinity, etc. can be used to reduce, enhance or modify the shear life or any other suitable property Generally, some or all of the non-human or human CDR sequences are present in the non-human variable and constant regions. The sequence is maintained while replacing it with human or other amino acids. Alternatively, the antibody may be humanized while retaining high affinity for the antigen and other favorable biological properties. To this end, humanized antibodies can optionally be produced by combining parental and human sequences. The process of analyzing the parent sequence and various conceptual humanized products using a 3D model of the humanized sequence. Three-dimensional immunoglobulin models are commonly available. The selected candidate immunoglobulin sequences are then subjected to a series of potential Computer programs are available to illustrate and display highly accurate three-dimensional conformations. Inspection of these displays can determine the role of the residues in the function of the candidate immunoglobulin sequence. Analysis of the likely binding potential of a candidate immunoglobulin, i.e., its ability to bind to its antigen. In this way, it is possible to analyze residues that affect the affinity of the target antigen. From the consensus sequence and the import sequence, FR residues can be selected and combined. Generally, CDR residues are those that are responsible for antigen binding. Humanization or engineering of the antibodies of the present invention Treatments were performed as described in Winter (Jones et al., Nature 321:522(1 986), Riechmann et al., Nature 332:323(198 8), Verhoeyen et al., Science 239:1534(198 8)), Sims et al., J. Immunol. 151:2296 (1993) , Chothia and Lesk, J. Mol. Biol. 196:901(198 7),Carter et al.,Proc.Natl.Acad.Sci.US A.89:4285(1992), Presta et al., J. Immunol. 151:2623 (1993), U.S. Patent Nos. 5,723,323 and 5,976,862, Same No. 5824514, Same No. 5817483, Same No. 5814476, Same No. 57631 No. 92, No. 5723323, No. 5,766886, No. 5714352, No. No. 6204023, No. 6180370, No. 5693762, No. 5530101 No., No. 5585089, No. 5225539, No. 4816567, International Application P CT / :US98 / 16280, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755, International Publication No. 90 / 14443, International Publication No. 90 / 14424, WO 90 / 14430, and European Patent No. 229246 (each , which is incorporated by reference in its entirety, including the references cited therein. This can be done using any known method, such as, but not limited to, .

[0056] The anti-TNF antibody also optionally comprises any of the antibodies described herein and / or known in the art. Transgenic animals capable of producing a repertoire of human antibodies are known. produced by immunization of an animal (e.g., mouse, rat, hamster, non-human primate, etc.) Cells that produce human anti-TNF antibodies can be isolated from such animals and used in the methods described herein. The cells may be immortalized using any suitable method, such as those described in the literature.

[0057] Transgenic mice capable of generating a repertoire of human antibodies that bind to human antigens The cloning agent can be produced by known methods, including but not limited to However, U.S. Patent Nos. 5,770,428 and 5,569, issued to Lonberg et al. No. 825, No. 5,545,806, No. 5,625,126, No. 5,625,8 Nos. 25, 5,633,425, 5,661,016, and 5,789, No. 650, Jakobovits et al., International Publication No. 98 / 50433, Jakobovi WO 98 / 24893 to ts et al., WO 98 / 2488 to Lonberg et al. No. 4, International Publication No. 97 / 13852, Lonberg et al. No. 94 / 25585, Kucherlapate et al., International Publication No. 96 / 34096, European Patent No. 0463 151(B1) to Kucherlapate et al. European Patent No. 0710 719(A1) to Apate et al., U.S. Patent No. 5 to Surani et al. ,545,807, Bruggemann et al., International Publication No. 90 / 04036, Bru European Patent No. 0438 474(B1) to Ggemann et al., European Patent No. Lonberg et al. No. 0814 259(A2), Lonberg et al., UK Patent No. 2 272 44 No. 0(A), Lonberg et al. Nature 368:856-859(1 994), Taylor et al., Int. Immunol. 6(4)579-5 91 (1994), Green et al, Nature Genetics 7:1 3-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic A cids Research 20(23):6287-6295(1992), Tua illon et al.,Proc Natl Acad Sci USA90(8) 3720-3724(1993), Lonberg et al., Int Rev I mmunol 13(1):65-93(1995), and Fishwald et al. l., Nat Biotechnol 14(7):845-851(1996), this (Each of which is incorporated herein by reference in its entirety.) Generally, these mice at least one human that is functionally rearranged or capable of undergoing functional rearrangement The vector contains at least one transgene containing DNA from a human immunoglobulin locus. The endogenous immunoglobulin loci of such mice are disrupted or deleted to allow the expression of endogenous genes. The ability of the animal to produce antibodies encoded thereby can be eliminated.

[0058] Screening of antibodies for specific binding to similar proteins or fragments can be performed using peptide This can be successfully achieved using a hand-display library. Screening a large sample collection of peptides for individual members with novel functions or structures Antibody screening of peptide display libraries involves screening of the antibodies. The length of the displayed peptide sequence is 3 to 5,000. or more amino acids, frequently 5-100 amino acids long, most often about 8-25 amino acids long. In addition to direct chemical synthesis methods for creating peptide libraries, several Several recombinant DNA methods have also been described. One type involves the use of bacteriophages or cells. Each bacteriophage or The cells contain nucleotide sequences encoding the particular displayed peptide sequences. Such methods are described in WO 91 / 17271, WO 91 / 18980 ... The peptides are described in US Pat. Nos. 91 / 19818 and 93 / 08278. Other systems for generating libraries include both in vitro chemical synthesis and recombinant methods. The surface is as described in WO 92 / 05258, WO 92 / 14843, and WO 96 / See U.S. Patent No. 5,658,754 and U.S. Patent No. 5,645,756. See also U.S. Pat. No. 3,768. Peptide display libraries, vectors, and scripts The screening kit was purchased from Invitrogen (Carlsbad, CA) and Cambri dge Antibody Technologies(Cambridgeshire ,UK). No. 4704692, No. 4939666, No. 4946778, No. 526020 No. 3, No. 5455030, No. 5518889, No. 5534621, No. 56 No. 56730, No. 5763733, No. 5767260, No. 5856456, Nos. 5223409, 5403484, and 557169 assigned to Dyax No. 8, No. 5837500, No. 5427908 assigned to Affymax, No. No. 5580717, Cambridge antibody Technologies No. 5885793 assigned to Genentech, and No. 575037 assigned to Genentech. No. 3, No. 5618920, No. 5595898, No. 557 assigned to Xoma No. 6195, No. 5698435, No. 5693493, No. 5698417, C See Olligan (supra), Ausubel (supra), or Sambrook (supra). Each of the above patents and publications is incorporated herein by reference in its entirety.

[0059] The antibodies of the present invention may also be used in goats, cows, horses, sheep, and other animals that produce such antibodies in their milk. To provide a transgenic animal or mammal, at least one nucleic acid encoding Such animals can also be prepared using one anti-TNF antibody. For example, but not limited to, U.S. Patent Nos. 5,827,627,627 and 5,827,6 .... No. 90, No. 5,849,992, No. 4,873,316, No. 5,849,99 No. 2, No. 5,994,616, No. 5,565,362, No. 5,304,489 Nos. 5,629,593 and 5,729,593, each of which is incorporated herein by reference in its entirety.

[0060] The antibodies of the present invention can be used to detect the specific Transgenic plants and cultured plant cells (e.g., To provide at least Further, the present invention can be prepared using a single anti-TNF antibody-encoding nucleic acid. For example, an inducible promoter may be used to generate a transgene that expresses a recombinant protein. Nicked tobacco leaves have been successfully used to provide large amounts of recombinant proteins. For example, C. ramer et al.,Curr.Top.Microbol.Immunol.2 40:95-118 (1999) and the references cited therein. Transgenic corn may be produced in other recombinant systems or from natural sources. Mammalian proteins at commercial production levels with biological activity equivalent to the purified proteins It has been used to express quality. For example, Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein. Antibodies may also be used in single chain antibodies (scFv), including those derived from tobacco seeds and potato tubers. Large amounts of antibodies have also been produced from transgenic plant seeds containing antibody fragments such as , Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and the references cited therein. Thus, the antibodies of the present invention may also It can also be produced using transgenic plants by known methods. For example, Fischer et al.,Biotechnol.Appl.Biochem .30:99-108(Oct.,1999),Ma et al.,Trends B iotechnol.13:522-7(1995), Ma et al., Plant Physiol.109:341-6(1995), Whitelam et al. , Biochem.Soc.Trans.22:940-944(1994), and its See also the references cited therein, including, but not limited to, the following, generally, regarding plant expression of antibodies: See also, U.S. Pat. No. 6,229,099, each of which is incorporated herein by reference in its entirety.

[0061] The antibodies of the present invention have a wide range of affinities (K D ) and can bind to human TNF In a preferred embodiment, at least one human mAb of the invention optionally comprises a human T For example, human mAbs can bind human TNF with high affinity. -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 of the duties therein Any range or value of K, including but not limited to:D can be combined with .

[0062] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., "Antibody- Antigen Interactions,” In Fundamental Imm. unology, Paul, WE, Ed., Raven Press: New Yo rk,NY(1984);Kuby,Janis Immunology,WHFr eeman and Company: New York, NY (1992); and Honmei (See methods described in the manual.) The measured affinity of a particular antibody-antigen interaction is , may differ when measured under different conditions (e.g., salt concentration, pH). Affinity and other antigen binding parameters (e.g., K D , K. a , K. d ) is preferably measured by , standardized solutions of antibody and antigen, and standardized buffers such as those described herein. This is done using

[0063] Nucleic acid molecule. At least one contiguous amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 a nucleotide sequence encoding at least 70-100% of the amino acids, a specified fragment, a variant or a consensus sequence thereof, or at least one of these sequences Using the information provided herein, including the deposited vectors containing SEQ ID NOs: 1, 2, and 3 and / or the light chain variable CDR regions of SEQ ID NOs: 4, 5, and 6 Nucleic acid molecules of the invention encoding at least one anti-TNF antibody comprising all of the above are described herein. These can be obtained using methods described in the literature or known in the art.

[0064] The nucleic acid molecules of the present invention may be in the form of mRNA, hnRNA, tRNA or any other form. in the form of RNA, or cDNA obtained by cloning or produced synthetically and genomic DNA, or any of these forms of DNA. The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be: It may be the coding strand, also known as the sense strand, or may be called the antisense strand. It may also be a non-coding strand.

[0065] The isolated nucleic acid molecules of the present invention may optionally contain one or more introns. Open Reading Frame (ORF), including, but not limited to, at least one CDR1 of the heavy chain (e.g., SEQ ID NOs: 1 to 3) or light chain (e.g., SEQ ID NOs: 4 to 6), At least one characteristic of at least one CDR, such as CDR2 and / or CDR3 Nucleic acid molecules containing a specified portion, coding sequence of an anti-TNF antibody or variable region (e.g., Nucleic acid molecules containing the above-mentioned nucleic acid molecules (sequence numbers 7 and 8), as well as nucleic acid molecules which are substantially different from the above-mentioned nucleic acid molecules but contain genetic code Due to code degeneracy, at least one of the methods described herein and / or known in the art may be used. The present invention may also include nucleic acid molecules comprising a nucleotide sequence encoding one or more anti-TNF antibodies. Of course, the genetic code is well known in the art. Those skilled in the art will appreciate the ease with which such degenerate nucleic acid variants can be generated that encode the particular anti-TNF antibodies of the present invention. See, for example, Ausubel et al., supra, for such nuclear Non-limiting examples of isolated nucleic acid molecules of the present invention include: HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 Non-limiting examples of nucleic acids encoding the LC CDR3, HC variable region, and LC variable region include: Corresponding sequences include SEQ ID NOs: 10, 11, 12, 13, 14, and 15.

[0066] As provided herein, nucleic acid molecules of the invention include nucleic acids encoding anti-TNF antibodies. The fragment itself encodes the amino acid sequence of an antibody fragment, a whole antibody or a part thereof. The coding sequence of the antibody, fragment or portion thereof, as well as additional sequences, e.g., at least non-coding sequences, with or without the additional coding sequences mentioned above, such as an intron 5' and 3' sequences, such as splicing and polyadenylation signals (e.g., mR It plays a role in transcription, mRNA processing, including ribosome binding and stability of the mRNA. a small amount of transcriptional activity, along with additional non-coding sequences, including but not limited to transcribed, non-translated sequences that play a role in the transcription of a gene. coding sequence for at least one signal leader or fusion peptide, additional amino acids, For example, including additional coding sequences that encode amino acids that provide additional functionality. Thus, the antibody encoding sequence can be, but is not limited to, an antibody fragment. or a marker such as a sequence encoding a peptide that facilitates purification of the fused antibody containing the nucleotide or portion thereof. It can be fused to the Kerr sequence.

[0067] Polynucleotides that selectively hybridize to the polynucleotides described herein The present invention provides a method for selectively hybridizing to the polynucleotides disclosed herein. The present invention provides an isolated nucleic acid that hybridizes under conditions of hybridization. Polynucleotides may be used to isolate, detect, and / or identify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present invention can be used to quantify and identifying, isolating, or amplifying partial or full-length clones in the accumulated library. In some embodiments, the polynucleotides are isolated or otherwise, a genomic sequence complementary to a cDNA from a human or mammalian nucleic acid library. The sequence is a sequence or cDNA sequence.

[0068] Preferably, the cDNA library contains at least 80% of the full-length sequences, preferably the complete At least 85% or 90% of the full-length sequence, and more preferably at least 95% of the full-length sequence cDNA libraries may be normalized to increase the representation of rare sequences. Stringent binding is used with sequences that have reduced sequence identity to the complementary sequence. Low or moderate hybridization conditions are typical, but not exclusive. Moderate and high stringency conditions optionally allow for the selection of sequences with higher identity. Low stringency conditions are those with approximately 70% sequence identity. and allows selective hybridization of sequences with orthologous or paralogous sequences. It can be used to identify the column.

[0069] Optionally, the polynucleotide of the invention is a polynucleotide described herein. The antibody will encode at least a portion of the antibody encoded by the peptide of the present invention. The polynucleotide may be a selective nucleotide for a polynucleotide encoding an antibody of the present invention. For example, the Ausub el, Colligan, supra, each of which is incorporated herein by reference in its entirety. be absorbed.

[0070] Construction of Nucleic Acids. The isolated nucleic acids of the present invention can be produced by (a) recombinant methods, as known in the art; (b) synthetic techniques; (c) purification techniques; or a combination thereof. Cut.

[0071] The nucleic acid may comprise sequences in addition to the polynucleotides of the present invention. For example, a multiple cloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid. It can also be used to insert translatable sequences, which can aid in the isolation of polynucleotides. and can be used to aid in the isolation of translated polynucleotides of the present invention. The hexahistidine marker sequence provides a convenient means for purifying the proteins of the present invention. The nucleic acids of the invention (excluding coding sequences) are optionally referred to as polynucleotides of the invention. The present invention is directed to a vector, adapter or linker for the cloning and / or expression of a gene.

[0072] Additional sequences may be added to such cloning and / or expression sequences to facilitate cloning and / or expression. or their function in expression can be optimized to aid in the isolation of polynucleotides. This can improve the transfer of polynucleotides into cells. The use of vectors, expression vectors, adapters, and linkers is well known in the art. (See, for example, Ausubel, supra, or Sambrook, supra.)

[0073] Recombinant methods for constructing nucleic acids, including RNA, cDNA, genomic DNA, or any of these. The isolated nucleic acid compositions of the present invention, such as any combination thereof, may be prepared from any number of clones known to those of skill in the art. In some embodiments, the present invention can be obtained from biological sources using enzyme-linked immunosorbent assays. An oligonucleotide that selectively hybridizes to a polynucleotide under stringent conditions. The probes are used to identify desired sequences in a cDNA or genomic DNA library. Isolation of RNA and construction of cDNA and genomic libraries are well within the skill of those in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.) and.)

[0074] Nucleic acid screening and isolation methods. Probes based on the nucleotide sequence are used to screen cDNA or genomic libraries. Probes can be used to identify homologous genes in the same or different organisms. To isolate the target gene, the target gene can be hybridized to genomic DNA or cDNA sequences. Those skilled in the art will appreciate that various degrees of hybridization stringency can be used in the assay. Either the hybridization or the wash medium can be used as stringent. It will be understood that the conditions for hybridization may be more stringent. As the number of probes increases, the degree of complementarity between the probe and the target increases, resulting in duplex formation. The degree of stringency can be controlled by temperature, ionic strength, pH, and formamide. and the presence of a partially denaturing solvent such as acetaldehyde. For example, the stringency of hybridization may be within the range of 0% to 50%. This can be successfully modified by changing the polarity of the reaction solution through manipulation of the formamide concentration. The degree of complementarity (sequence identity) required for detectable binding depends on the hybridization The degree of complementarity varies according to the stringency of the annealing medium and / or the washing medium. Preferably, it is 100%, or 70 to 100%, or any range or value therein. However, slight sequence variations within the probe and primers can affect hybridization and It is understood that this can be compensated for by reducing the stringency of the wash medium and / or the It should be.

[0075] Methods for amplifying RNA or DNA are well known in the art and are described herein. Based on the teachings and guidance provided, one can make use of the present invention without undue experimentation.

[0076] Known methods for DNA or RNA amplification include the polymerase chain reaction (PCR). in reaction, PCR) and related amplification processes (see, e.g., U.S. Pat. No. 5,499,623 to Mullis et al., No. 4,683,195, No. 4,683,202, No. 4,800,159, No. No. 4,965,188 to Tabor et al., No. 4,795,699 to Tabor et al., and No. 4,921, No. 794 to Innis, No. 5,142,033 to Wilson et al., No. 5,122, No. 464 to Innis, No. 5,091,310 to Gyllensten et al., No. 066,584 to Gelfand et al., No. 4,889,818 to Silver et al. No. 4,994,370 of Biswas, No. 4,766,067 of Ringold See US Pat. No. 4,656,134), and templates for double-stranded DNA synthesis. RNA-mediated amplification using antisense RNA against a target sequence as a template (Malek et al. 5,130,238, which is trade name NASBA), (The entire contents of these documents are incorporated herein by reference.) See, for example, Ausubel, supra, or Sambrook, supra.)

[0077] For example, polymerase chain reaction (PCR) techniques can be used to generate genomic DNA or cDNA fragments. Amplifying the sequences of the polynucleotides of the present invention and related genes directly from the library. PCR and other in vitro amplification methods can also be used to, for example, amplify the protein to be expressed. cloning the nucleic acid sequence encoding the desired mRNA; detecting the presence of the desired mRNA in a sample; nucleic acids for use as probes for nucleic acid sequencing, nucleic acid sequencing, or other purposes; It will be useful to generate in vitro amplification methods with sufficient skill to guide the skilled artisan through the process. Examples of techniques include Berger, supra; Sambrook, supra; and Ausubel, supra. Mullis et al., U.S. Pat. No. 4,683,202 (1987), and Innis, et al. al., PCR Protocols A Guide to Methods an d Applications,Eds.,Academic Press Inc,S and Diego, CA (1990). Commercially available kits for genomic PCR amplification are available at These are known in the art, e.g., Advantage-GC Genomic See, for example, the T4 gene 32 Protein (Boehringer Mannheim) was used to improve the yield of long PCR products. The rate can be improved.

[0078] Synthetic Methods for Constructing Nucleic Acids. The isolated nucleic acids of the present invention can be prepared by direct chemical synthesis using known methods. They can also be prepared by chemical synthesis (see, for example, Ausubel et al., supra). Generally, by hybridization with a complementary sequence or by using a single strand as a template. single-stranded oligonucleotides that can be converted into double-stranded DNA by polymerization with the DNA polymerase used Those skilled in the art will understand that chemical synthesis of DNA produces a chain of about 100 or more bases. Although the sequence may be limited, longer sequences can be obtained by ligation of shorter sequences. You will realize that you can.

[0079] Recombinant Expression Cassettes. The present invention further provides recombinant expression cassettes comprising a nucleic acid of the invention. The nucleic acid sequences of the invention, e.g., cDNA or genomic sequences encoding the antibodies of the invention, can be used to and constructing a recombinant expression cassette that can be introduced into at least one desired host cell. Recombinant expression cassettes typically are used to express polynucleotides in the intended host cell. The polynucleotide of the present invention is operably linked to a transcription initiation regulatory sequence that directs transcription of the target gene. Both heterologous and non-heterologous (i.e., endogenous) promoters may be used to produce the vectors of the present invention. The expression of the nucleic acid can be directed.

[0080] In some embodiments, isolated proteins that function as promoters, enhancers, or other elements. The nucleic acid may be used in combination with a polynucleotide of the invention to up- or down-regulate expression of the polynucleotide of the invention. Introducing a non-heterologous form of a nucleotide at the appropriate position (upstream, downstream, or within an intron) For example, by mutation, deletion and / or substitution in vivo or in vitro, The endogenous promoter can be altered.

[0081] Vectors and host cells. The present invention provides vectors, recombinant vectors, and host cells comprising the isolated nucleic acid molecules of the invention. host cells genetically engineered with TA and recombinant techniques well known in the art. The present invention also relates to the production of at least one anti-TNF antibody by, for example, Sambroo et al., supra. See K. et al., supra, and Ausubel et al., supra, each of which is incorporated herein by reference in its entirety. be absorbed.

[0082] The polynucleotide may optionally be a vector containing a selectable marker for propagation in a host. Generally, plasmid vectors are prepared by calcium phosphate precipitation. The vector is introduced into a precipitate such as a globulin or in a complex with a charged lipid. If so, package it in vitro using an appropriate packaging cell line, It can then be transduced into host cells.

[0083] The DNA insert should be operably linked to an appropriate promoter. The target is a transcription start site, a transcription termination site, and, within the transcribed region, a ribosome for translation. The coding portion of the mature transcript expressed by the construct preferably further comprises a nucleotide sequence encoding ... was appropriately positioned at the translation initiation site at the beginning and termination of the mRNA to be translated. Contains a stop codon (e.g., UAA, UGA, or UAG) and is inactivated in mammalian or eukaryotic cells For expression, UAA and UAG are preferred.

[0084] The expression vector preferably, but optionally, contains at least one selectable marker. Such markers include, for example, methotrexate (MTX) for eukaryotic cell culture; Dihydrofolate reductase (DHFR, U.S. Pat. No. 4,399,216, U.S. Pat. No. 4,633,633) No. 4,665, No. 4,656,134, No. 4,956,288, No. 5,149 ,636, 5,179,017, ampicillin, neomycin (G418), Icophenolic acid or glutamine synthetase (GS, U.S. Patent No. 5,122,464 , 5,770,359, 5,827,739) resistance genes, and E. coli and tetracycline or ampicillin resistance for cultivation in other bacteria or prokaryotes These include, but are not limited to, sex genes (the above patents are incorporated herein by reference in their entirety). Appropriate culture media and conditions for the above host cells are well known in the art. Suitable vectors will be readily apparent to those skilled in the art. The vector construct was introduced into the cells by calcium phosphate transfection, DEA E-dextran-mediated transfection, cationic lipid-mediated transfection The cells may be affected by electroporation, transduction, infection or other known methods. Such methods are discussed in Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, Chapters 1, 9, 13, 15, and 16, etc. are.

[0085] At least one antibody of the present invention may be expressed in a modified form, such as a fusion protein. The polypeptide may contain not only a secretion signal but also additional heterologous functional regions. For example, a region of additional amino acids. Adding regions, particularly charged amino acids, to the N-terminus of the antibody to prevent fragmentation during purification or subsequent processing and storage The stability and persistence in host cells can be improved. The antibody or at least one fragment thereof may be added to the antibody to facilitate purification. Such regions can be removed prior to final preparation of the sample. ook, Chapters 17.29-17.42 and 18.1-18.74, Ausub above el, Chapters 16, 17, and 18, and many other standard laboratory manuals. .

[0086] Those skilled in the art will appreciate that there are numerous gene expression vectors available for expressing nucleic acids encoding the proteins of the present invention. He is knowledgeable about the current system.

[0087] Alternatively, nucleic acids of the invention can be expressed in host cells containing endogenous DNA encoding an antibody of the invention. can be expressed in a host cell by switching it on (by engineering) within Such methods are described in U.S. Patent Nos. 5,580,734, 5,641,670, and 5,641,670. As described in US Pat. Nos. 5,733,746 and 5,733,761, are well known in the art and are incorporated herein by reference in their entirety.

[0088] An example of a cell culture useful for the production of antibodies, specified portions or variants thereof is mammalian Mammalian cell lines are often in the form of a monolayer of cells, but suspensions of mammalian cells can also be used. Alternatively, a bioreactor can be used. Many suitable host cell lines have been developed in the art, including COS-1 (e.g. COS-7 (e.g., ATCC CRL1650), 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-A g14, 293 cells, HeLa cells, etc., which are, for example, American Type Culture Collection,Manassas,Va(www Preferred host cells include myeloma and lymphoma cells. Particularly preferred host cells include cells of lymphoid origin, such as tumor cells. 8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells ( ATCC Accession No. CRL-1851. In a particularly preferred embodiment, the recombinant cell is , P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0089] Expression vectors for these cells include an origin of replication, a promoter (e.g., late or early SV4 0 promoter, CMV promoter (U.S. Patent Nos. 5,168,062, 5,385, No. 839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter EF-1α promoter (U.S. Patent No. 5,266,491), at least one human Immunoglobulin promoter, enhancer, and / or ribosome binding site, RNA splicer a sequence site, a polyadenylation site (e.g., the SV40 large T Ag polyaddition site), and These include, but are not limited to, expression control sequences, such as processing information sites, including transcription termination sequences. The sequences may include one or more of the regulatory sequences described in, for example, Ausubel et al., supra; Samb et al., supra See, e.g., rook et al. Other cells useful for producing the nucleic acids or proteins of the invention are known. and / or, for example, American Type Culture Co. Collection cell line and hybridoma catalog (www.atcc.org) or available from other known or commercial sources.

[0090] When eukaryotic host cells are used, polyadenylation or transcription termination sites are typically included in 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 may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague , et al., J. Virol. 45:773-781(1983)). As is known in the art, genetic sequences for controlling replication within a host cell are used to It can be incorporated into the controller.

[0091] Antibody purification. Anti-TNF antibodies were purified using protein A, ammonium sulfate, or ethanol precipitation. precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography hydrophobic interaction chromatography, affinity chromatography, hydroxy Examples of suitable methods include, but are not limited to, leupertite chromatography and lectin chromatography. It can be recovered and purified from recombinant cell culture by any well-known method, including but not limited to. High performance liquid chromatography ("HPLC") can also be used for purification. lligan, Current Protocols in Immunology or Current Protocols in Protein Science,Joh n Wiley & Sons, NY, NY (1997-2001), e.g., Vol. 1, Vol. 4, See Chapters 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety. do.

[0092] Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, as well as products derived from, e.g., Produced by recombinant techniques from eukaryotic hosts, including yeast, higher plants, insect and mammalian cells Depending upon the host employed in a recombinant production procedure, the antibody of the present invention may be glycosylated or It may be sylated or non-glycosylated, but is preferably glycosylated. Such methods are described in Sambrook, supra, Sections 17.37-17.42, and Au, supra. subel, Chapters 10, 12, 13, 16, 18, and 20, Colligan, supra. Many standard laboratory manuals, such as Protein Science, Chapters 12-14 No. 6,299,133, all of which are incorporated herein by reference in their entirety.

[0093] Anti-TNF antibody All of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2, and 3 and / or SEQ ID NOs: 4, 5, and The isolated antibodies of the present invention comprising all six light chain variable CDR regions can be prepared using any suitable polynucleotide. the amino acid sequence of the antibody disclosed herein encoded by the nucleotide, or any Preferably, the human antibody or antigen-binding fragment is an isolated or prepared antibody of human TN F, thereby partially or substantially inhibiting at least one biological activity of the protein. at least one biologically active substance of at least one TNF protein or fragment. Antibodies or specified portions or variants thereof that partially or preferably substantially neutralize the activity The variant binds to the protein or fragment, thereby inhibiting TNF through binding to the TNF receptor. or through other TNF-dependent or -mediated mechanisms. As used herein, the term "neutralizing antibody" refers to a neutralizing antibody that is approximately 20-100% neutralizing, depending on the assay. 20%, preferably at least about 10, 20, 30, 40, 50, 55, 60, 65, 7 0, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 refers to an antibody that can inhibit TNF-dependent activity by 100% or more. The ability of the anti-TNF antibody to inhibit TNF-dependent activity is preferably determined by the methods described herein and / or at least one suitable TNF protein or receptor antigen known in the art. The human antibodies of the present invention can be used in any class (IgG, IgA, IgM ... IgM, IgE, IgD, etc.) or isotype, and may contain kappa or lambda light chains. In one embodiment, the human antibody comprises an IgG heavy chain or a defined fragment, e.g., an Ig The antibody comprises at least one of the following isotypes: IgG1, IgG2, IgG3, or IgG4. Antibodies of this type may be any of the various antibodies described herein and / or known in the art. At least one human light chain (e.g., IgG, IgA) and IgM (e.g., γ1, γ2, Transgenic mice or other transgenic animals containing transgenes (γ3, γ4) In another embodiment, the anti-human TN antibody can be prepared by using a non-human mammal. The F human antibody comprises an IgG1 heavy chain and an IgG1 light chain.

[0094] At least one antibody of the present invention may be directed to at least one TNF protein, subunit, or both. at least one specific epitope specific to the polypeptide, fragment, portion, or any combination thereof; The at least one epitope binds to at least one of the aforementioned proteins. The antigen-binding domain may comprise at least one antibody binding region comprising a portion thereof, and this epitope is preferably is an extracellular, soluble, hydrophilic, external, or cytoplasmic portion of at least one of the aforementioned proteins. At least one identified epitope is comprised of the adjacent amino acids of SEQ ID NO: 9. At least one of at least 1 to 3 amino acids for the entire specified portion of amino acids It can include any combination of amino acid sequences.

[0095] Generally, the human antibodies or antigen-binding fragments of the invention contain at least one human complementarity-determining region. (CDR1, CDR2 and CDR3) or a variant of at least one heavy chain variable region, and At least one human complementarity determining region (CDR1, CDR2, and CDR3) or at least Each antibody comprises an antigen-binding region that includes a variant of one light chain variable region. Alternatively, the antigen-binding portion or variant may comprise a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 and and / or at least one of the light chain CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment has corresponding CDR1, 2, and / or at least one having an amino acid sequence of SEQ ID NO: 1, 2, and / or 3 at least a portion of the heavy chain CDRs (i.e., CDR1, CDR2, and / or CDR3) of In another specific embodiment, the antibody or antigen-binding region may comprise The combination or variant may be a combination of the corresponding CDR1, 2, and / or 3 amino acid sequences (e.g., , SEQ ID NOs: 4, 5, and / or 6). and having an antigen-binding region comprising at least a portion of CDR1, CDR2, and / or CDR3. In a preferred embodiment, the three heavy chain CDRs and The three light chain CDRs are identical to those of mAbs TNV148, TNV14, TN At least one of V15, TNV196, TNV118, TNV32, TNV86 Such antibodies can be prepared by conventional techniques of recombinant DNA technology. to prepare and express a nucleic acid molecule (i.e., one or more) encoding an antibody using or by using any other suitable method. By chemically linking various parts of the body (e.g., CDRs, framework) together It can be prepared more easily.

[0096] Anti-TNF antibodies contain at least one heavy or light chain variable region having a defined amino acid sequence. For example, in a preferred embodiment, the anti-TNF antibody may include at least one of: Optionally, a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and / or At least one of at least one light chain variable region having the amino acid sequence of SEQ ID NO: 8 Antibodies that bind to human TNF and contain defined heavy or light chain variable regions are preferred. methods, such as those known in the art and / or described herein. Display (Katsube, Y., et al., Int J Mol. Med, 1(5):863-868(1998)) or methods employing transgenic animals. For example, functionally rearranged human immunoglobulins can be prepared using any suitable method. Immunoglobulin heavy chain transgene and human immunoglobulin capable of undergoing functional rearrangement. a transgene comprising DNA from a human light chain locus; and a transgenic mouse comprising the Immunization with human TNF or a fragment thereof can be used to induce the production of antibodies. In this manner, antibody-producing cells can be isolated and used as described herein and / or in the art. As is known in the art, hybridomas or other immortalized antibody-producing cells are prepared by the method of Alternatively, the antibody, specified portion or variant can be expressed in a suitable host cell. The encoding nucleic acid or a portion thereof can be used for expression.

[0097] The present invention also provides amino acids within sequences that are substantially the same as the amino acid sequences described herein. The present invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs comprising the amino acid. Such antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs have high affinity (e.g. For example, K D is about 10 -9 M or less) and can bind to human TNF. Amino acid sequences that are substantially the same as the sequences listed include conservative amino acid substitutions, as well as Conservative amino acid substitutions include sequences containing amino acid deletions and / or insertions. Chemical and / or physical properties (e.g., charge, structure, polarity, hydrophobicity) similar to those of amino acids A conservative substitution refers to the substitution of a first amino acid with a second amino acid that has a higher affinity (or affinity / hydrophilicity). Substitutions involve replacing one amino acid with another amino acid within the following group: lysine ( K), arginine (R) and histidine (H); aspartate (D) and glutamine Acid salt (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 (T) thiamin (W), methionine (M), cysteine ​​(C), and glycine (G); F, W, and Y ;C, S, and T.

[0098] Amino Acid Codes: The amino acids that make up the anti-TNF antibodies of the invention are often abbreviated. The amino acid notation can be its one-letter code, its three-letter code, its name, or three nucleotides. The amino acids can be represented by codon(s), and are well known in the art. is well understood in the Biology of The Cell,Third Ed.,Garland P Publishing, Inc., New York, 1994).

[0099] [Table 2]

[0100] The anti-TNF antibodies of the invention may be naturally occurring or humanized, as specified herein. It may involve the substitution, deletion or addition of one or more amino acids by any of the manipulations.

[0101] Of course, the number of amino acid substitutions that one skilled in the art can make is limited to a large number, including those mentioned above. Generally speaking, for any given anti-TNF antibody, fragment or variant, The number of amino acid substitutions, insertions, or deletions in the 0, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, Not exceeding 5, 4, 3, 2, 1, e.g., 1 to 30, or any range or value therein .

[0102] Amino acids within the anti-TNF antibodies of the invention that are essential for function can be identified by site-directed mutagenesis or The specific sequences may be identified by methods known in the art, such as alanine scanning mutagenesis. (See, e.g., Ausubel, supra, Chapters 8 and 15; Cunningham, am and Wells,Science 244:1081-1085(1989) The latter procedure introduces one alanine substitution mutation at each residue in the molecule. The resulting mutant molecule may have, for example, at least one TNF-neutralizing activity. The antibody is tested for biological activity, including but not limited to: Important sites may also be identified by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling. (Smith, et al., J. Mol. Biol. 224:89 9-904 (1992) and de Vos, et al., Science 255:3 06-312(1992)).

[0103] The anti-TNF antibody of the present invention has at least one of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. At least one portion, sequence or combination selected from one to all of the adjacent amino acids These may include, but are not limited to:

[0104] The anti-TNF antibody may further optionally comprise at least one of the flanking sequences of SEQ ID NOs: 7, 8. It may contain at least one polypeptide with 70-100% of the amino acids.

[0105] In one embodiment, an immunoglobulin chain or a portion thereof (e.g., a variable region, a CDR) The amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 is the amino acid sequence of at least one corresponding chain of SEQ ID NO: 7, SEQ ID NO: 8. and approximately 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, 9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any of these For example, the amino acid sequence of the light chain variable region may be the sequence of SEQ ID NO: 8. The amino acid sequence of the heavy chain CDR3 can be compared to SEQ ID NO:7. Preferably, the amino acid sequence has 70 to 100% amino acid identity (i.e., 90, 91, 9 2, 93, 94, 95, 96, 97, 98, 99, 100, or any range therein (or values) can be calculated using suitable computer algorithms, as known in the art. is determined using

[0106] Representative sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 7 and 8. The antibody or specified variant thereof may contain any number of contiguous amino acid residues from an antibody of the invention. The number is selected from the group of integers consisting of 10 to 100% of the number of adjacent residues in the anti-TNF antibody. Optionally, this substring of contiguous amino acids is selected from at least about 10, 20, 30 , 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 1 50, 160, 170, 180, 190, 200, 210, 220, 230, 240, 2 50 or more amino acids in length, or any range or value therein. The number of such subsequences is selected from the group consisting of 1 to 20, such as at least 2, 3, 4, or 5. It can be any integer that is

[0107] As will be appreciated by those skilled in the art, the present invention includes a method for producing a human ovarian tumor comprising administering to a subject a subject a biologically active antibody of the present invention. Biologically active antibodies are natural (non-synthetic), endogenous or related and known antibodies. at least 20%, 30% or 40%, and preferably at least 50%, of the total body mass; 60% or 70%, and most preferably at least 80%, 90% or 95% to 1000 % specific activity. Methods for assaying and quantifying enzyme activity and substrate specificity are well known in the art. It is well known to those who

[0108] In another aspect, the present invention provides a method for preparing a compound according to any one of the methods described herein that is modified by the covalent attachment of an organic moiety. Such modifications may result in improved pharmacokinetic properties (e.g., and / or to generate antibodies or antigen-binding fragments with increased serum half-life in vivo. The organic moiety can be a linear or branched hydrophilic polymeric group, a fatty acid group, or a fatty acid ester. In certain embodiments, the hydrophilic polymer group can be a group having a molecular weight of about 800 to about 1000. about 120,000 daltons, and a polyalkane glycol (e.g., polyethylene glycol) Polypropylene glycol (PEG), polypropylene glycol (PPG), carbohydrate polymers, amino The copolymer may be a fatty acid polymer or polyvinylpyrrolidone, and may also contain a fatty acid group or a fatty acid ester group. can contain from about 8 to about 40 carbon atoms.

[0109] The modified antibodies and antigen-binding fragments of the present invention are covalently bound to the antibody, either directly or indirectly. Each organic moiety attached to an antibody or antigen-binding fragment of the invention may comprise one or more organic moieties. The moieties can independently be a hydrophilic polymeric 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 polymeric group" refers to a polymer that is more hydrophilic in water than in octane. For example, polylysine is more soluble in water than in octane. Therefore, antibodies modified by covalent bonding of polylysine are suitable for use in the present invention. Suitable hydrophilic polymers for modifying the antibodies of the invention include linear or branched For example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycols) polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cell sugars, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, poly arginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. The hydrophilic polymer that modifies the antibody of the present invention may be an individual molecular entity having about 800 to about 1500 carbon atoms. 0,000 daltons. 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 polymeric group may 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 the preferred method. For example, a polymer containing amine groups can be prepared by It can be linked to the carboxylate salt of a fatty acid or a fatty acid ester, Activated carboxylates on esters (e.g., activated with N,N-carbonyldiimidazole) The hydroxyl groups on the polymer can be linked to the hydroxyl groups on the polymer.

[0110] Fatty acids and fatty acid esters suitable for modifying antibodies of the invention may be saturated. Suitable lipids for modifying the antibodies of the invention may contain one or more unsaturated units. Examples of fatty acids include n-dodecanoic acid salts (C 12 , laurate), n-tetradecane Acid acid (C 14 , myristate), n-octadecanoate (C 18 , stearates), n-Eicosanoic acid salt (C 20 , arachidate), n-docosanoate (C 22 , behenic acid ), n-triacontanoate (C 30 ), n-tetracontanoate (C 40 ), cis-Δ 9-octadecanoate (C 18 , oleate), all cis-Δ5,8,11,14- Eicosatetraenoate (C 20 , arachidonate), octanedioic acid, tetradecane Suitable fatty acid esters include octadecanedionic acid, octadecanedionic acid, docosanedioic acid, and the like. Esters include monoesters of dicarboxylic acids containing straight or branched chain lower alkyl groups. A lower alkyl group can contain 1 to about 12, preferably 1 to about 6, carbon atoms.

[0111] The modified human antibodies and antigen-binding fragments can be subjected to a suitable treatment, such as by reacting them with one or more modifying agents. As used herein, the term "modifying agent" refers to a compound that can be prepared using a method similar to that described herein. The term refers to any suitable organic group (e.g., hydrophilic polymers, fatty acids, fatty acid esters) that contains an activating group. An "activating group" means a group that, under appropriate conditions, reacts with a second chemical group, thereby modifying the A chemical moiety or functional group capable of forming a covalent bond between a decorator and a second chemical group. For example, amine-reactive activating groups include tosylate, mesylate, halo (chloro, bromo) , fluoro, iodo), N-hydroxysuccinimidyl esters (NH Examples of activating groups that can react with thiols include maleimide, iodine, and the like. Doacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid The aldehyde functional group can be an amine or hydroxyl group. The azide group can be linked to a hydroxyl-containing molecule, and the azide group can react with a trivalent phosphorus group to form a phosphine group. A sulfamidate or phosphorimide bond can be formed. Suitable methods for introduction are known in the art (see, for example, Herman son, GT, Bioconjugate Techniques, Academy (See, e.g., 1996, IEEE Press: San Diego, CA). The activating group is , directly to an organic group (e.g., a hydrophilic polymer, a fatty acid, a fatty acid ester) or via a linker - moiety, e.g., divalent C1-C 12 A group in which one or more carbon atoms is oxygen, nitrogen, or Suitable linker moieties include: For example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH- , -(CH2)2-NH- and -CH2-O-CH2-CH2-O-CH2-CH2-O A modifying agent containing a linker moiety includes, for example, 1-ethyl-3-(3-dimethyl- In the presence of methylaminopropyl carbodiimide (EDC), mono-Boc-alkyl Diamines (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) ) with a fatty acid to form an amine bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be generated by forming a Boc bond. The product can be removed by FA treatment and coupled to another carboxylate as described. The resulting primary amine can be exposed or reacted with maleic anhydride, cyclizing the resulting product to form an activated maleimide derivative of a fatty acid. (See, e.g., Thompsons, 1999, the entire teachings of which are incorporated herein by reference.) See WO 92 / 16221 to N et al.

[0112] The modified antibodies of the present invention can be prepared by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, the organic moiety can be generated by an amine-reactive modifying agent, e.g., PEG. By employing NHS esters of Disulfide bonds (e.g., intrachain disulfide bonds) of antibodies or antigen-binding fragments can be reduced. Modified human antibodies or antigen-binding fragments can also be prepared by cloning the antibody or antigen-binding fragments. The reduced antibody or antigen-binding fragment is then reacted with a thiol-reactive modifying agent to form the antibody or antigen-binding fragment of the present invention. It is possible to produce modified antibodies of the above structure. Modified human antibodies and antigen-binding fragments containing organic moieties can be synthesized by reverse proteolysis (Fisch et al., 2001). et al.,Bioconjugate Chem.,3:147-153(199 2), Werlen et al.,Bioconjugate Chem.,5:41 1-417(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 and Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996) The compound can be prepared using any suitable method, such as the method described above.

[0113] Anti-idiotypic antibodies to anti-TNF antibody compositions. Monoclonal or chimeric anti-TNF In addition to F antibodies, the present invention also provides anti-idiotypes (anti-Ids) specific for such antibodies of the present invention. Anti-Id antibodies generally contain unique determinants associated with the antigen-binding region of another antibody. Anti-Id is an antibody that recognizes the Id antibody from an animal (e.g., mouse) of the same species and genotype as the source of the Id antibody. The antibody can be prepared by immunizing a strain (such as a human flu strain) with the antibody or its CDR-containing region. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody. In response, the immune system produces anti-Id antibodies, which can then induce an immune response in another animal. It can be used as an "immunogen" to generate so-called anti-anti-Id antibodies.

[0114] Anti-TNF antibody compositions. The present invention also relates to compositions comprising any of the antibodies described herein and / or known in the art. As known in the art, at least one non-naturally occurring composition, mixture, or form is provided. one, at least two, at least three, at least four, at least five, at least At least one anti-TNF antibody composition comprising six or more of said anti-TNF antibodies is also provided. Such compositions comprise adjacent amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8. 70-100%, or a specified fragment, domain, or variant thereof At least one or two full-length, C and / or C-terminal amino acid sequences of the anti-TNF antibody to be used a non-naturally occurring composition comprising an N-terminal deletion variant, domain, fragment, or variant as specified; A preferred anti-TNF antibody composition comprises 70 to 100 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. % of an anti-TNF antibody, or a specified fragment, domain, or variant thereof, At least one or two full-length, fragment, or domain CDR- or LBR-containing portions More preferred compositions include 70 to 75 of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or variants thereof. 100% or at least one of the identified fragments, domains or variants thereof The percentages of such compositions may be adjusted as known in the art. or as described herein, by weight, volume, concentration, molality, or liquid or molarity as a dry solution, mixture, suspension, emulsion, or colloid This is due to the following.

[0115] The anti-TNF antibody compositions of the present invention further comprise the following: at least one anti-TNF antibody to a cell, tissue, organ, animal, or patient, and optionally Optionally, at least one TNF antagonist (e.g., TNF antibody or fragment, soluble TNF F receptor or fragments, their fusion proteins, or small molecule TNF antagonists. , but not limited to), antirheumatic drugs (e.g., methotrexate, auranofin , aurothioglucose, azathioprine, etanercept, gold sodium thiomalate , hydroxychloroquine sulfate, leflunomide, sulfasalidine), muscle relaxants, narcotics, non Steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers antibacterials (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, Nem, cephalosporin, fluoroquinolone, macrolide, penicillin, sulfonamide anti-psoriatic drugs, corticosteroids, anabolics Steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones Antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietic drugs (e.g. e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), Gramostim (GM-CSF, Leukine), immunizing agent, immunoglobulin, immunosuppression medicines (e.g., basiliximab, cyclosporine, daclizumab), growth hormone, hormone Supplements, estrogen receptor modulators, mydriatics, cyclomodulators, alkylating agents, antimetabolites , mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanic drugs, antipsychotics, anxiolytics, hypnotics , sympathomimetic drugs, stimulants, donepezil, tacrine, asthma medications, beta-agonists, inhaled steroids ides, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or similar drugs , dornase alfa (Pulmozyme), cytokines or cytokine antagonists and any suitable and effective amount of the composition or pharmaceutical composition, further comprising at least one of Non-limiting examples of such cytokines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19 ... The preferred doses include, but are not limited to, any of the following: It is well known in the art, see, for example, Wells et al., eds., Phar macotherapy Handbook, 2 nd Edition, Appleto n and Lange, Stamford, CT (2000), PDR Pharma. copoeia, Tarascon Pocket Pharmacopoeia 20 00, Deluxe Edition, Tarascon Publishing, Lo See Linda, CA (2000), each of which is incorporated by reference in its entirety. is incorporated herein.

[0116] Such anti-cancer or anti-infective agents may also be associated, bound, or immunoreacted with at least one antibody of the invention. Co-formulation or combination of toxin molecules may also be included. Toxins selectively kill diseased cells or tissues. The pathological cells may be cancer cells or other cells. Such toxins may include, but are not limited to, ricin, dimethicone, At least one of a toxin selected from at least one of a phtheria toxin, a snake toxin, or a bacterial toxin. and a purified or recombinant toxin or toxin fragment containing at least one functional cytotoxic domain. The term toxin also refers to a toxic shock that can be fatal in humans and other mammals. Any naturally occurring, mutated, or recombinant cell that can result in any disease state, including cancer. This includes both endotoxins and exotoxins produced by bacteria or viruses. Enterotoxin-resistant Escherichia coli (ESC) heat-labile enterotoxin (LT), heat-stable enterotoxin (S) T), Shigella cytotoxin, Aeromonas enterotoxin, toxic shock syndrome toxin 1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), streptococcal enterotoxins, etc. Such bacteria include enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., serum strains of type 0157:H7), Staphylococcus species (e.g., Staphylo coccus aureus, Staphylococcus pyogenes), S Shigella species (e.g., Shigella dysenteriae, Shigella la flexneri, Shigella boydii, and Shigella s. onnei), Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella entera itidis), Clostridium species (e.g., Clostridium per fringens, Clostridium dificile, Clostridiu m botulinum), Camphlobacter species (e.g., Camphlob acter jejuni, Camphlobacter fetus), Helioc bacter species (e.g., Heliocbacter pylori), Aeromones as species (e.g., Aeromonas sobria, Aeromonas hydrol phila, Aeromonas caviae), Pleisomonas shig elloides, Yersinia enterocolitica, Vibrio species (e.g., Vibrio cholerae, Vibrio parahemolyti cus), Klebsiella species, Pseudomonas aeruginosa, and strains of Streptococci species, such as, but not limited to, Stein, ed., INTERNAL MEDICINE, 3rd ed., pp. 1-13, Little, Brown and Co., Boston, (1990), Evans et al., eds., Bacterial Infections o f Humans: Epidemiology and Control, 2d.Ed. ,pp239-254,Plenum Medical Book Co.,New Y ork (1991), Mandell et al, Principles and P. practice of Infectious Diseases,3d.Ed.,Ch Urchill Livingstone, New York (1990), Berko w et al, eds., The Merck Manual, 16th edition on, Merck and Co., Rahway, NJ, 1992, Wood e. t al,FEMS Microbiology Immunology,76:121 -134 (1991), Marrack et al, Science, 248:705 -711 (1990), the contents of which are incorporated herein by reference in their entireties. (Incorporated into

[0117] The anti-TNF antibody compounds, compositions or mixtures of the present invention may further comprise a diluent, binder, stabilizer, buffer, or the like. any additives, such as, but not limited to, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. The composition may contain at least one of the following suitable auxiliary agents: Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of and methods for preparing such sterile solutions are well known in the art. See, for example, Gennaro, Ed., Remington's Pharmacy ical Sciences,18 th Edition, Mack Publishing ng Co. (Easton, PA), 1990, but is not limited to this. anti-TNF antibodies, fragments, or antibodies as known in the art or as described herein A pharmaceutically acceptable carrier suitable for the administration method, solubility, and / or stability of the mutant composition is selected. , can be selected on a daily basis.

[0118] Pharmaceutical excipients and additives useful in the present compositions include, but are not limited to, proteins Proteins, peptides, amino acids, lipids and carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and sugars including oligosaccharides, alditols, aldonic acids, esterified sugars, and other derivative sugars; and polysaccharides or sugar polymers), which may be present alone or in combination; Typical proteins, alone or in combination, comprise 1 to 99.99% by weight or volume. Excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (HRA), and Representative examples of cellulose that can also function in buffering capacity include cellulose (rHA), gelatin, and casein. Amino acids / antibody components include alanine, glycine, arginine, betaine, histidine, Glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine , methionine, phenylalanine, aspartame, etc. One of them is glycine.

[0119] Suitable carbohydrate excipients for use in the present invention include, for example, fructose, maltose, Monosaccharides such as saccharides like sucrose, galactose, glucose, D-mannose, and sorbose, lactose, Disaccharides such as sugar, trehalose, cellobiose, raffinose, melezitose, maltodextrin, Polysaccharides such as dextrin, dextran, starch, mannitol, xylitol, Maltitol, lactitol, xylitol, sorbitol (glucitol), myoinosin Preferred carbohydrate excipients for use in the present invention include alditols such as tallow, maltodextrin, thiamin ... are mannitol, trehalose, and raffinose.

[0120] The anti-TNF antibody composition may also include a buffer or pH adjusting agent; typically, the buffer is an organic A salt prepared from an acid or base. Typical buffers include citric acid, ascorbic acid, organic acid salts such as salts of acetic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Suitable anti-inflammatory agents for use in the present compositions include phosphate buffer, thiamin mononitrate, thrombin hydrochloride, and phosphate buffer. Preferred buffers are organic acid salts such as citrate.

[0121] In addition, the anti-TNF antibody composition of the present invention contains polyvinylpyrrolidone, Ficoll (polymer sugars), dextrates (e.g., 2-hydroxypropyl-β-cyclodextrin, etc. (Cyclodextrin), polyethylene glycol, flavoring, antibacterial agent, sweetener, antioxidant agents, antistatic agents, surfactants (e.g., "TWEEN 20" and "TWEEN 80" polysorbates), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol polymeric excipients / additives such as steroids, chelating agents (e.g., EDTA) obtain.

[0122] These and additional compounds suitable for use in anti-TNF antibody, portion or variant compositions according to the invention Additional known pharmaceutical excipients and / or additives are known in the art, e.g. , “Remington: The Science&Practice of Phar macy,” 19 th ed., Williams & Williams, (1995), and "Physician's Desk Reference," 52 nd ed,M listed in Journal of Educational Economics, Montvale, NJ (1998) The disclosures of which are incorporated herein by reference in their entireties. The excipient materials include carbohydrates (e.g., monosaccharides and alditols) and buffers (e.g., chrysanthellin). enoate) or polymeric reagents.

[0123] Formulations. As noted above, the present invention preferably provides a formulation containing saline or a selected salt. Stable formulations that are phosphate buffers, as well as preservative-containing storage solutions and formulations, and pharmaceutical for pharmaceutical or veterinary use comprising at least one anti-TNF antibody in a physiologically acceptable formulation. A suitable multi-use preserved formulation is provided. The preserved formulation comprises at least one known compound in an aqueous diluent. of which at least one of phenol, m-cresol, p-cresol, o-cresol, sol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol ethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), Alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, Benzethonium chloride, sodium dehydroacetate, and thimerosal, or a mixture thereof and optionally containing a preservative selected from the group consisting of: As shown, 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. Such as, but not limited to, 7, 4.8, 4.9, or any range or value therein. Any suitable concentration or mixture may be used, including any range or value therein. Non-limiting examples include preservative-free, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), about 0.1 to 3% benzyl alcohol ( For example, 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% phenanthrene Nor (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0 0.005-1.0% alkylparaben(s) (e.g., 0.00075, 0.000 9, 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%).

[0124] As mentioned above, the present invention provides a pharmaceutical composition comprising a packaging material and, optionally, a buffer and a pharmaceutical composition formulated in an aqueous diluent. and / or a solution of at least one anti-TNF antibody with a preservative. and the packaging material is a solution of 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 includes a packaging material and a label stating that the freeze-dried product can be stored for a long period of time. a first vial containing at least one dried anti-TNF antibody and a formulated buffer or and a second vial containing an aqueous diluent of the preservative, and the packaging material further comprises At least one anti-TNF antibody is reconstituted in an aqueous diluent and administered for at least 24 hours. The label includes instructions to the patient to form a solution that can be maintained.

[0125] The at least one anti-TNF antibody used in accordance with the present invention is any of those described herein or or produced from mammalian cells or transgenic preparations known in the art. The polypeptides may be produced by recombinant means, including by the addition of PEG or purified from other biological sources.

[0126] The range of at least one anti-TNF antibody included in the product of the present invention is a wet / dry system. In this case, the amount is such that upon reconstitution a concentration ranging from approximately 1.0 μg / mL to approximately 1000 mg / mL is obtained. However, lower and higher concentrations are possible and these concentrations are not intended. The delivery vehicle chosen depends on the type of drug, e.g., solution formulations, transdermal patches, pulmonary, transmucosal, or is different from osmotic or micropump methods.

[0127] Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. New preservatives include phenol, m-cresol, p-cresol, o-cresol, and chloroform. Benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butylparaben) Benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and Thimerosal or mixtures thereof. The concentration of preservative used is sufficient to produce an antimicrobial effect. This will vary depending on the preservative selected and is readily determined by one of skill in the art.

[0128] Other excipients, such as isotonicity agents, buffers, antioxidants, preservative enhancers, are optionally included. Preferably, an isotonic agent such as glycerin can be added to the diluent at a known concentration. Preferably, a physiologically tolerable buffer is added to improve p The formulation provides pH control 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 sodium phosphate. Phosphate buffered saline (PBS).

[0129] Other additives, such as Tween 20 (polyoxyethylene (20) sorbitan monolaurate) rate), Tween 40 (Polyoxyethylene (20) sorbitan monopalmitate) , Tween 80 (Polyoxyethylene (20) sorbitan monooleate), Plur onic F68 (polyoxyethylene polyoxypropylene block copolymer), and and PEG (polyethylene glycol), or a pharmaceutically acceptable solubilizer. Poloxamer 20 or 80 or Poloxamer 184 or 188, Pluronic Nonionic surfactants such as ETFE (registered trademark) polyols, other block copolymers, and E Chelating agents such as DTA and EGTA can be optionally added to the formulation or composition to These additives can reduce aggregation. This is particularly useful when a plastic container is used. The presence reduces the tendency of the protein to aggregate.

[0130] The formulations of the present invention comprise at least one anti-TNF antibody and a soluble component selected from the group consisting of phenol, m-cresol, p -cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl para Benzyl (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride from the group consisting of thimerosal, ... and a selected preservative, in an aqueous diluent. The mixture of at least one anti-TNF antibody and a preservative in an aqueous diluent can be used in a variety of ways, including conventional methods. Dissolution and mixing procedures are used to prepare suitable formulations. A quantity of at least one anti-TNF antibody in a solution containing a desired concentration of protein and a preservative is added. The process is combined with the desired preservative in a sufficient amount of buffer solution to provide the desired product. The form will be recognized by one of ordinary skill in the art. For example, the order of addition of the components, the additional addition The use of an antiviral agent, the temperature and pH during preparation of the formulation, all relate to the dosage concentration and administration method used. It is a factor that can be optimized.

[0131] The claimed formulations may be prepared as clear solutions or in the presence of water, preservatives and / or excipients, preferably Preferably, phosphate buffer and / or saline, and selected salts in aqueous diluents. and a second vial containing at least one lyophilized anti-TNF antibody. It can be provided to the patient as a dual vial containing a single vial. Any single solution vial or combination vial requiring reconstitution may be reused multiple times. can satisfy single or multiple patient treatment cycles and therefore are currently available This can provide a more convenient treatment regimen than those previously described.

[0132] The claimed product is useful for administration from immediate to over a 24 hour period. Thus, the products claimed by the present invention provide significant benefits to patients. The formulation may optionally be safely stored at a temperature of about 2 to about 40°C to maintain the biological activity of the protein. The packaging label indicates that the solution is capable of retaining its properties for a long period of time. , 36, 48, 72, or 96 hours or more. If a stored diluent is used, it can be shown that Labels may include use up to 1-12 months, 6 months, 1.5 years, and / or 2 years. .

[0133] Solutions of at least one anti-TNF antibody of the present invention comprise dissolving at least one antibody in an aqueous diluent. The mixture can be prepared by a process comprising mixing in a conventional dissolving and To prepare a suitable diluent, for example, water or a buffer a quantity of at least one antibody in a desired concentration of protein, and optionally storing The mixture is combined in amounts sufficient to provide a suitable agent or buffer. Variations of this process are known to those skilled in the art. For example, the order of addition of components, the effectiveness of using additional additives, etc. All formulation preparation temperatures and pH values ​​should be optimized for the dosage concentration and administration route used. This is a factor that can be used.

[0134] The claimed product is available as a clear solution or in a second vial containing an aqueous diluent. A combination vial containing at least one lyophilized anti-TNF antibody, to be reconstituted with It can be provided to patients as a single solution vial or as a reconstitution-requiring vial. Each combination vial can be reused multiple times for single or multiple patient treatment cycles. This would allow for the patient to meet the needs of the patient and thus provide a more convenient treatment regimen than is currently available. do.

[0135] The claimed product is reconstituted in a second vial containing a clear solution or aqueous diluent. a combination vial comprising at least one lyophilized vial of an anti-TNF antibody, to pharmacies, clinics, or other such institutions and facilities, The clear solution can be provided in volumes of up to 1 liter or even more. from this larger container, a smaller amount of at least one antibody solution may be dispensed at once or Multiple withdrawals are made into smaller vials and distributed to customers and / or can be provided to patients.

[0136] Approved devices containing these single-vial systems include BD Pens, BD A Autojector (registered trademark), Humaject (registered trademark), NovoPen (registered trademark) BD® Pen, AutoPen®, and OptiPe n(R), GenotropinPen(R), Genotronorm Pen (registered trademark), Humatro Pen (registered trademark), Reco-Pen (registered trademark) trademark), Roferon Pen®, Biojector®, ije ct (registered trademark), J-tip Needle-Free Injector (registered trademark) ), Intraject®, Medi-Ject®, and other solution delivery pen-type injector devices for n(Franklin Lakes, NJ, www.bectondickenson. com), Disetronic (Burgdorf, Switzerland, www .disetronic.com), Bioject, Portland, Oregon (www.bioject.com), National Medical Produ cts,Weston Medical(Peterborough,UK,www.w eston-medical.com), Medi-Ject Corp (Minnea Manufactured or developed by (Michigan, MN, www.mediject.com) Approved devices that contain combination vials include HumatroPen (registered trademark) ) and other lyophilized drugs in cartridges for delivery of the reconstituted solution. A pen-type injector system for configuring the device is also provided.

[0137] The products claimed herein include packaging. The packaging may be required by a regulatory agency. In addition to the information about the product, the packaging material of the present invention also provides the conditions under which the product can be used. At least one anti-TNF antibody is reconstituted in an aqueous diluent to form a solution, and the solution is then incubated for 2 to 24 hours. Over the above period, this solution will be used for two vial products, wet and dry. For single-vial solution products, the label should state that this solution is The products claimed herein can be used for a period of time or longer. It is useful for pharmaceutical product applications.

[0138] The formulations of the present invention comprise at least one anti-TNF antibody and a selected buffer, preferably a biocompatible buffer. by a process that includes mixing saline or a phosphate buffer containing the selected salt. The mixture of at least one antibody and a buffer in an aqueous diluent can be prepared by: Conventional dissolution and mixing procedures are used to prepare suitable formulations, e.g. A quantity of at least one antibody in water or buffer is added to the desired concentration of protein and buffer. and a desired buffer in a sufficient amount of water to provide a For example, the order of addition of the components, the amount of additional additives, etc., will be recognized by those skilled in the art. Whether or not it is used, the temperature and pH during preparation of the formulation, and the dosage concentration and administration method used It is a factor that can be optimized.

[0139] The claimed stable or preserved formulations are available as clear solutions or in aqueous diluents containing preservatives or or a lyophilized small amount of lyophilized cereals that are reconstituted in a second vial containing buffer and excipients. may be provided to patients as a combination vial containing at least one vial of an anti-TNF antibody. Any single solution vial or combination vial requiring reconstitution can be reused multiple times. can be used to satisfy single or multiple patient treatment cycles, thus It provides a more convenient treatment regimen than is currently available.

[0140] At least one of the stable or preserved formulations or solutions described herein TNF antibodies can be administered by SC or IM injection, intradermal, or pulmonary route, as is well known in the art. , transmucosal, implant, osmotic pump, cartridge, micropump, or related art The present invention can be applied to a wide variety of patients via a variety of delivery methods, including other means well known in the art and understood by those skilled in the art. The invention can be administered to a patient.

[0141] Therapeutic Uses. The present invention also relates to therapeutic uses of compounds known in the art or described herein. At least one dual integrin antibody of the present invention can be used to target cells, tissues, organs, for modulating or treating at least one TNF-related disorder in a human, animal, or patient A method is also provided.

[0142] The present invention also relates to a method for treating obesity, an immune-related disease, a cardiovascular disease, an infectious disease, a malignant disease, or a neurological disease. a cell, tissue, organ, animal, or patient, including but not limited to at least one of: Also provided are methods for modulating or treating at least one TNF-related disorder in a mammal.

[0143] The present invention also relates to a method for treating rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, and psoriatic arthritis. , ankylosing spondylitis, gastric ulcer, seronegative arthropathy, osteoarthritis, inflammatory bowel disease, ulcerative colitis Enteritis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis / uveitis / optic neuritis inflammation, 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 mu-negative bacterial sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, urinary sepsis, meningitis Bacteremia, trauma / hemorrhage, burns, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis Machi, alcoholic hepatitis, chronic inflammatory diseases, sarcoidosis, Crohn's disease, sickle cell disease Hemoglobinemia, diabetes, nephrosis, atopic diseases, hypersensitivity reactions, allergic rhinitis, hay fever Fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease, thrombocytopenia, any organ or tissue transplant rejection, Kidney transplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection Reaction, bone marrow transplant (BMT) rejection, skin allograft rejection, cartilage transplant rejection, bone transplant Hemoptysis, small intestine transplant rejection, fetal thymus transplant rejection, parathyroid transplant rejection, any Organ or tissue xenograft rejection, allograft rejection, antireceptor hyperreaction, Graves' disease , Raynaud's disease, type B insulin-resistant diabetes, asthma, myasthenia gravis, antibody-mediated cells Disorders, Type III hypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome (polyneuropathy) , organomegaly, endocrinopathy, monoclonal gammopathy, and cutaneous syndrome), Neuropathy, organomegaly, endocrine disorders, monoclonal gammopathy, skin symptoms group, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus Diarrhea, chronic active hepatitis, primary biliary cirrhosis, vitiligo, vasculitis, post-MI open-heart syndrome, Type IV hyperimmunity, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, granulation caused by intracellular organisms tumors, drug hypersensitivity, metabolic / idiopathic Wilson's disease, hemacromatosis, alpha-1-antithrombin Psin deficiency, diabetic retinopathy, Hashimoto's thyroiditis, osteoporosis, primary biliary cirrhosis, thyroid inflammation, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, chronic obstructive pulmonary disease (COPD) ), familial hemophagocytic lymphohistiocytosis, dermatological conditions, psoriasis, alopecia, nephrotic syndrome group, nephritis, glomerulonephritis, acute renal failure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy , anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy (e.g., asthenia, anemia, These include, but are not limited to, diathesis, chronic salicylate intoxication, and at least one of, but not limited to, a cell, tissue, organ, animal, or patient Also provided are methods for modulating or treating at least one immune-related disease in a subject. For example, Merck Manual, 12th to 17th Editions, Merck &Company,Rahway,NJ(1972,1977,1982,1987,1 992,1999),Pharmacotherapy Handbook,Wells et al., eds., Second Edition, Appleton and See Lange, Stamford, and Conn. (1998, 2000). each incorporated by reference in its entirety.

[0144] The present invention provides a method for treating cardiac stun syndrome, myocardial infarction, congestive heart failure, and other conditions. , stroke, ischemic attack, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic arteries Sclerotic diseases, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, syphilis of the cardiovascular system , heart failure, cor pulmonale, primary pulmonary hypertension, arrhythmia, atrial ectopic beats, atrial flutter, atrial fibrillation (chronic persistent or paroxysmal), post-reflow syndrome, cardiopulmonary bypass inflammatory response, chaotic or multifocal atrial tachycardia , regular narrow QRS tachycardia, specific arrhythmia ventricular fibrillation, His bundle 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 aneurysms and peripheral aneurysms , aortic dissection, inflammation of the aorta, occlusion of the abdominal aorta and its branches, peripheral vascular disease, occlusive artery disease Pulse disorders, peripheral atherosclerosis, thromboangiitis obliterans, functional peripheral arterial disease, Ray No phenomenon and diseases, acrocyanosis, erythromelalgia, venous diseases, venous thrombosis, varicose veins, arteriovenous Fistula, lymphedema, lipedema, unstable angina, reperfusion injury, post-pump syndrome syndrome), ischemia-reperfusion injury, etc. Also provided are methods for modulating or treating cardiovascular disease in cells, tissues, organs, animals or patients. Such methods include administering an effective amount of a composition or pharmaceutical comprising at least one anti-TNF antibody. The therapeutic composition may be administered to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. The method may optionally include administering the method to a subject.

[0145] The present invention also relates to acute and chronic infections, including acute or chronic bacterial infections, bacterial, viral and fungal infections. Sexual parasitic or infectious processes, HIV infection / HIV neuropathies, meningitis, hepatitis (A, B or C, etc.) etc.), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli 0157:h7, hemolytic uremia Syndrome / embolic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, haematological flu, toxic shock syndrome, streptococcal myositis, gas gangrene, tuberculosis, mycobacterium tuberculosis Mu-avium intracellulare, Pneumocystis carinii pneumonia, pelvic inflammatory disease , orchitis / epididymitis, Legionnaires' disease, Lyme disease, influenza A, Epstein-Barr -Viruses, virus-associated hemophagocytic syndrome, viral encephalitis / aseptic meningitis, etc. In a cell, tissue, organ, animal or patient, including but not limited to, at least one Also provided are methods for modulating or treating at least one infectious disease using the method.

[0146] The present invention also relates to leukemia, acute leukemia, acute lymphocytic leukemia (ALL), B-cell, T-cell cells or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, multiple myeloma tumor, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, malignant paraneoplastic syndromes Syndrome / hypercalcemia, solid tumors, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer including, but not limited to, at least one of the following: bone resorption, cancer-related bone pain, etc. Modulating or treating at least one malignant disease in a cell, tissue, organ, animal, or patient. Also provided is a method for

[0147] The present invention also relates to the treatment of neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia syndrome, demyelinating diseases, For example, multiple sclerosis and acute transverse myelitis, extrapyramidal and cerebellar disorders, e.g., corticospinal Lesions of the cerebral cortex, disorders of the basal ganglia or cerebellar disorders, hyperkinetic movement disorders, e.g., Huntington's disease Chorea and senile chorea, drug-induced movement disorders, e.g., blocking CNS dopamine receptors Drug-induced hypokinetic movement disorders, e.g., Parkinson's disease, progressive Supranuclear palsy, structural lesions of the cerebellum, spinocerebellar degeneration, e.g., spinal ataxia, Friedrich- Ataxia, cerebellar cortical degeneration, multiple system degeneration (Mencel, Dejerine-Tho mas, Shi-Drager and Machado-Joseph), systemic diseases (Lef Sumo disease, abetalipoproteinemia, ataxia, telangiectasia, and mitochondrial polyneuropathy demyelinating core disorder, e.g., multiple sclerosis; Acute transverse myelitis and motor unit disorders, e.g., neuromuscular atrophy (anterior horn cell degeneration, e.g., For example, amyotrophic lateral sclerosis, infantile spinal muscular atrophy, and juvenile spinal muscular atrophy), Alzheimer's disease, Immer's disease, Down's syndrome in middle-aged people, diffuse Lewy body disease, senile dementia with Lewy bodies Alzheimer's disease, Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakob syndrome disease, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and dementia pugilistica a cell, tissue, organ, animal, or patient, including but not limited to at least one of Also provided are methods for modulating or treating at least one neurological disorder in a subject. The method optionally comprises administering at least one TNF antibody or identified portion or variant to a subject. and administering to a patient in need of such regulation, treatment, or therapy an effective amount of a composition or pharmaceutical composition comprising the compound. The method can include administering to a cell, tissue, organ, animal, or patient. erck Manual,16 th Edition, Merck & Company, R See Ahway, N.J. (1992).

[0148] Any of the methods of the present invention may be used to treat cells, tissues, or tissues in need of such modulation, treatment, or therapy. administering to the organism, animal, or patient an effective amount of a composition or pharmaceutical agent comprising at least one anti-TNF antibody; Such methods may optionally include administering the composition to treat such immune disorders. and the at least one anti-TNF antibody, particularly The administration of the modified portion or variant thereof may be in combination with at least one TNF antagonist (e.g., a soluble TNF receptor or fragment thereof, a fusion protein thereof, or a small molecule T NF antagonists, etc., but are not limited to, anti-rheumatic drugs (e.g., methotrexate, Sirt, auranofin, aurothioglucose, azathioprine, etanercept, gold Sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine ), muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives , local anesthetics, neuromuscular blocking agents, antibacterial agents (e.g., aminoglycosides, antifungals, anthelmintics, Antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins anti-psoriatic drugs, corticosteroids, cyclosporine ... steroids, anabolic steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid medications, vitamins Calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, Erythropoietin (e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunization drugs, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab) ), growth hormone, hormone replacement drugs, estrogen receptor modulators, mydriatics, cycloplegics , alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanic drugs, antipsychotics Diagnosing drugs, anti-anxiety drugs, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta-agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn , epinephrine or analogues, dornase alfa (Pulmozyme), cytokines or or a cytokine antagonist, Suitable dosages are well known in the art. , Wells et al., eds., Pharmacotherapy Handb. ook,2 nd Edition,Appleton and Lange,Stamf ord, CT (2000), PDR Pharmacopoeia, Tarascon. Pocket Pharmacopoeia 2000, Deluxe Edition , Tarascon Publishing, Loma Linda, CA (2000) See, e.g., U.S. Pat. No. 6,229,399, each of which is incorporated herein by reference in its entirety.

[0149] The compositions, combination therapies, co-administrations, devices, and / or methods of the present invention (at least one of the Suitable TNF antagonists include anti-TNF antibodies (including antibodies, specified portions and variants thereof). NF antibodies, antigen-binding fragments thereof, and receptor molecules that specifically bind to TNF, TNF synthesis, Compounds that block and / or inhibit TNF release or its action on target cells, e.g. thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline) adenosine receptor agonists, and rolipram, A2b adenosine receptor agonists, and A2b adenosine receptor enzymes. compounds that block and / or inhibit TNF receptor signaling, e.g., mitogens; MAP kinase inhibitors, which block and / or inhibit membrane TNF cleavage compounds that block and / or inhibit TNF activity, such as metalloproteinase inhibitors compounds, such as angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril) and compounds that block and / or inhibit TNF production and / or synthesis, e.g., MAP kinase These include, but are not limited to, enzyme inhibitors.

[0150] As used herein, "tumor necrosis factor antibody," "TNF antibody," "TNFα antibody" " or "fragment" and the like are intended to mean a compound that can be produced in vitro, in situ, and / or preferably in vivo by the synthesis of TN For example, a preferred TNF human antibody of the present invention reduces, blocks, inhibits, abrogates or interferes with Fα activity. The body is capable of binding to TNFα and is capable of inhibiting TNFα by inhibiting TNFα antibodies, antigen-binding fragments thereof, and TNFα Suitable TNF antibodies or fragments thereof include those identified as variants or domains thereof that specifically bind to TNF. The fragments are involved in TNF RNA, DNA, or protein synthesis, TNF release, and TNF receptor signaling. Reduce, block, inhibit, or interfere with TNF signaling, membrane TNF cleavage, TNF activity, TNF production and / or synthesis It may also interfere with, prevent, and / or inhibit.

[0151] The chimeric antibody cA2 is a high-affinity neutralizing murine anti-human TNFα IgG1 antibody designated A2. It consists of the antigen-binding variable region of human IgG1 and the kappa immunoglobulin constant region of human Ig The G1 Fc region improves the effector function of the cognate antibody and increases its circulating serum half-life. The avidity and epitope specificity of the chimeric antibody cA2 are In certain embodiments, the variable regions of the murine antibody A2 are derived from the variable regions of the murine antibody A2. A preferred source of nucleic acid encoding the region is the A2 hybridoma cell line.

[0152] Chimeric A2 (cA2) inhibits the cytotoxic effects of both natural and recombinant human TNFα. Binding assays of chimeric antibody cA2 with recombinant human TNFα demonstrated that chimeric antibody cA2 neutralizes TNFα in a chimeric manner. The affinity constant of the antibody cA2 is 1.04 x 10 10 M -1 It was calculated that the competitive inhibition The preferred method for determining the specificity and affinity of a monoclonal antibody is by Har low,et al.,antibodies:A Laboratory Manual l,Cold Spring Harbor Laboratory Press,Co ld Spring Harbor, New York, 1988, Colligan et al., eds.,Current Protocols in Immunol ogy,Greene Publishing Assoc.and Wiley In terscience, New York, (1992-2000), Kozbor e t al., Immunol. Today, 4:72-79 (1983), Ausube. l et al., eds.Current Protocols in Molecule lar Biology,Wiley Interscience,New York( 1987-2000), and Muller, Meth. Enzymol., 92:589 -601 (1983), which references are incorporated herein by reference in their entirety. be incorporated into the book.

[0153] In a specific embodiment, the murine monoclonal antibody A2 is a clone designated c134A. The chimeric antibody cA2 is produced by a cell line called c168A. It is done.

[0154] Further examples of monoclonal anti-TNF antibodies that can be used in the present invention include the It has been described in the art (e.g., U.S. Pat. No. 5,231,024, Moll er, A. et al., Cytokine2(3):162-169(1990), US National Application No. 07 / 943,852 (filed September 11, 1992), Rathjen,et al., International Publication No. 91 / 02078 (published February 21, 1991), Rubin, et al., EPO Patent Publication No. 0 218 868 (published April 22, 1987), Yone, et al., EPO Patent Publication No. 0 288 088 (October 2, 1988) 6th), Liang,et al.,Biochem.Biophys.Res.Com m.137:847-854(1986), Meager, et al., Hybrid oma6:305-311(1987), Fendly et al., Hybrido ma6:359-369 (1987), Bringman, et al., Hybrid oma6:489-507 (1987) and Hirai, et al., J. Immun See S.ol. Meth. 96:57-62 (1987). These references are incorporated herein by reference. (The entire contents of which are incorporated herein by reference).

[0155] TNF Receptor Molecules. Preferred TNF receptor molecules useful in the present invention have a high affinity for TNFα. (See, e.g., Feldmann et al., WO 92 / 07076 (1992) (published April 30), Schall et al., Cell61:361-370 (19 90), and Loetscher et al., Cell 61:351-359(19 90), which are incorporated herein by reference in their entireties. , optionally with low immunogenicity. In particular, 55 kDa (p55 TNF-R ) and the 75 kDa (p75TNF-R) TNF cell surface receptor are useful in the present invention. The extracellular domains (ECDs) of these receptors or functional portions thereof are Truncated forms (e.g. Corcoran et al.,Eur.J.Biochem.22 3:831-840 (1994)) are also useful in the present invention. Truncated forms of the TNF receptor containing 30 kDa and 40 kDa TNFα inhibitors were detected in urine and serum. It has been detected as a cytotoxicity-binding protein (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536(1990)). TNF receptor multimers The molecules and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, are disclosed in the present invention. Further examples of TNF receptor molecules useful in the methods and compositions of the present invention are given below. TNF receptor molecules that can treat patients for a long time with good to excellent symptom relief and low toxicity It is characterized by low immunogenicity and / or high affinity and other undefined characteristics. Gender may contribute to the therapeutic results obtained.

[0156] The TNF receptor multimeric molecules useful in the present invention may be prepared by cleaving the multimeric molecules with polyethylene glycol (PEG) linked via one or more polypeptide linkers or other non-peptide linkers, such as The multimeric molecule comprises all or a functional portion of the ECD of two or more TNF receptors. It can further include a signal peptide of a secretory protein to effect expression of the molecule. These multimeric molecules and methods for their production are described in U.S. application Ser. No. 08 / 437,533 (1 No. 6,299,595, filed May 9, 1995, the contents of which are incorporated herein by reference in their entirety. be absorbed.

[0157] TNF immunoreceptor fusion molecules useful in the methods and compositions of the present invention may comprise one or more immunoreceptors. at least one portion of a globulin molecule and all or a functional portion of one or more TNF receptors These immunoreceptor fusion molecules may be present as monomers or hetero- or homo-multimers. Immunoreceptor fusion molecules can also be assembled using either 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. 21:2 883-2886(1991), Ashkenazi et al., Proc. Nat. l.Acad.Sci.USA88:10535-10539(1991), Peppe l et al., J.Exp.Med.174:1483-1489(1991), K olls et al.,Proc.Natl.Acad.Sci.USA91:215 -219(1994), Butler et al., Cytokine6(6):61 6-623(1994), Baker et al.,Eur.J.Immunol.2 4:2040-2048 (1994), Beutler et al., U.S. Pat. No. 5,447,855 No. 1 and U.S. Application No. 08 / 442,133 (filed May 16, 1995), the references thereto. (Each of the references is incorporated herein by reference in its entirety.) The synthesis method is described in Capon et al., U.S. Pat. No. 5,116,964, Capon et al., U.S. Pat. No. 5,225,538 and Capon et al., Nature 337:525-5 31 (1989), which references are incorporated herein by reference in their entireties. To be incorporated.

[0158] Functional equivalents, derivatives, fragments, or regions of the TNF receptor molecule may be used in the present invention. The TNF receptor molecule is of sufficient size and sequence to be functionally similar to the TNF receptor molecule ( For example, it binds to TNFα with high affinity and has low immunogenicity, and binds to the TNF receptor molecule It refers to a portion, or a portion of the TNF receptor molecule sequence, that encodes the TNF receptor molecule. Functional equivalents of the receptor molecules also function as TNF receptor molecules that can be used in the present invention. Modifications that are functionally similar (e.g., bind TNFα with high affinity and have low immunogenicity) For example, functional equivalents of TNF receptor molecules include "SILE" NT" codon, or one or more amino acid substitutions, deletions, or additions (e.g., one amino acid The amino acid may be substituted for another acidic amino acid, or the same or a different hydrophobic amino acid may be used. one codon that codes for an acid instead of another codon that codes for a hydrophobic amino acid Ausubel et al., Current Protocols ols in Molecular Biology,Greene Publishing ng Assoc.and Wiley-Interscience,New York (1987-2000).

[0159] Cytokines include any known cytokines. See Cytokines.com. Cytokine antagonists include any antibody. , fragments or mimetics, any soluble receptor, fragment or mimetic, any small molecule antagonist , or any combination thereof.

[0160] Therapeutic Treatment. Any of the methods of the present invention may be used to treat a cell in need of such modulation, treatment, or therapy. a safe and effective treatment for a cell, tissue, organ, animal, or patient, comprising at least one anti-TNF antibody; and a method for treating a TNF-mediated disease comprising administering a therapeutically effective amount of a composition or pharmaceutical composition. Such methods may optionally include co-administration or The method may further comprise a combination therapy, wherein the at least one anti-TNF antibody, identified portion or portion thereof Administration of the variant may be in combination with at least one TNF antagonist (e.g., a TNF antibody or fragment, possibly soluble TNF receptors or fragments thereof, fusion proteins thereof, or small molecule TNF antagonists. anti-rheumatic drugs (e.g., methotrexate, auranov, aurothioglucose, azathioprine, etanercept, sodium gold thiomalate cerebrospinal fluid, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxants, narcotics , nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neurotransmitters Neuroblockers, antibacterials (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, chloramphenicol ... Bapenams, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfones amides, tetracyclines, other antibacterial agents), antipsoriatics, corticosteroids, anabolic Steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid medications, vitamins, calcium-related Hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietin ( e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen) , Sargramostim (GM-CSF, Leukine), Immunizing agent, Immunoglobulin, Immunoglobulin immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormone, Hormone replacement drugs, estrogen receptor modulators, mydriatics, cycloplegics, alkylating agents, anti- Metabolic agents, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanic drugs, antipsychotics, anxiolytics, Hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta-agonists, inhalants Steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or or analogs, dornase alfa (Pulmozyme), cytokines or cytokine and / or administering the compound before, simultaneously with, and / or after administering at least one selected from the group consisting of a steroid antagonist, ... Included.

[0161] As used herein, the term "safe" means that it is safe for the anti-TNF antibodies of the invention (e.g. For example, the anti-TNF antibody golimumab) If relevant, adverse events compared with standard of care or another comparator, such as other anti-TNF agents. Acceptable frequency of adverse events (AEs) and serious adverse events (SAEs) Adverse events refer to adverse events that are associated with a favorable risk-benefit ratio, with a moderate to severe adverse event and / or acceptable severity. An untoward medical occurrence in a patient who has received a product. Specifically, safety is and the anti-TNF antibodies of the present invention. When relevant, e.g., infusion reactions, laboratory findings of hepatobiliary abnormalities, infections including TB, and malignancies Refers to an acceptable frequency and / or severity of adverse events, including tumors.

[0162] As used herein, the terms "efficacy" and "effectiveness" refer to the composition, dosage, administration, As used herein in the context of a regimen, treatment, or method, the anti-TNF antibody of the present invention Specific compositions, doses, administration, treatments, or combinations of compounds (e.g., the anti-TNF antibody golimumab) refers to the efficacy of the method. Efficacy is based on changes in the course of the disease in response to the agents of the invention. For example, the anti-TNF antibodies of the invention may be used to measure the severity of the disorder being treated. sufficient to cause an improvement, preferably a sustained improvement, in at least one indicator that reflects The amount and duration of the treatment is determined to be sufficient. To determine the severity of the disease, disorder, or condition, various indicators that reflect the severity of the disease, disorder, or condition can be evaluated. Such indicators may include, for example, disease severity, symptoms, or manifestations of the disorder of interest. The degree of improvement is generally assessed by a physician or other appropriate The decision is made by a trained individual who is well trained and who has evidence of improvement in signs, symptoms, biopsies, or clinical symptoms. The determination can be based on other test results indicative of disease activity, or any other measure of disease activity. For example, the anti-TNF antibodies of the invention may improve the condition of patients with psoriatic arthritis (PsA). Improvement in a patient's condition with respect to PsA can be measured, for example, by a health assessment. Questionnaire Disability Index score (HAQ-DI), enthesitis assessment, dactylitis assessment, 36-item short Form Health Survey Physical Summary Score (SF-36PCS), and / or 36-item Show One or more of the following: the Form Health Survey Mental Component Summary Score (SF-36MCS) The HAQ-DI is a questionnaire that assesses eight functional areas (getting dressed, getting up, eating, etc.). The difficulty a person experiences in completing tasks in daily living (e.g., walking, hygiene, extension, grip strength, and activities of daily living). It is a 20-question instrument that assesses the degree of difficulty. Enthesitis can occur in the left and right lateral epicondyles of the elbow, Localized pressure was applied to the left and right medial elbow epicondyles and tendon insertions, including the left and right Achilles tendon insertions. Dactylitis can be assessed by assessing the presence or absence of pain with touching the hands and feet. The SF-36 can be assessed for its presence and severity in eight scored areas. The SF-36PSA and SF-36MCS are questionnaires consisting of multiple scales. from the SF-36, which allows for comparison of the relative burden of different diseases and the relative benefits of different treatments. This is the summary score that is derived.

[0163] Typically, treatment of a condition will, on average, involve a total of: At least about 0.01 to 500 milligrams per kilogram of patient per dose At least one anti-TNF antibody, preferably at least one per kg of patient per single or multiple dose. at least one anti-TNF antibody composition, each in the range of about 0.1 to 100 milligrams of antibody; Alternatively, effective blood transfusion may be achieved by administering a safe and effective amount or dose of Serum concentrations may include serum concentrations of 0.1 to 5000 μg / ml per single or multiple dose. Suitable dosages are known to medical practitioners and will, of course, vary depending on the particular disease state, It depends on the specific activity of the composition being administered and the particular patient undergoing treatment. In some cases, repeated administration, i.e., specific monitored or metered doses, may be required to achieve the desired therapeutic dose. It may be necessary to provide separate doses, in which case the separate doses should be administered on the desired day. Repeated until dose or effect is achieved.

[0164] Preferred doses are optionally 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9 4, 95, 96, 97, 98, 99, and / or 100-500 mg / kg / dose; or any range, value, or fraction thereof, or per single or multiple dose 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, 1 8.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 2 4, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 6 00, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, Serum concentrations of 3500, 4000, 4500, and / or 5000 μg / ml, or The range may be included to obtain any range, value, or fraction of .

[0165] Alternatively, the dose administered may be determined based on the pharmacokinetic characteristics of the particular drug and its method and route of administration. , the recipient's age, health and weight, the nature and severity of symptoms, the type of concurrent treatment, and the frequency of treatment. The dosage of the active ingredient may vary depending on known factors such as the dosage, the amount of the active ingredient, and the desired effect. It can be about 0.1 to 100 milligrams per kilogram of body weight. Usually, it is about 0.1 to 100 milligrams per kilogram of body weight. A dosage of 0.1 to 50, preferably 0.1 to 10 milligrams per gram or sustained release form is desirable. This is effective in obtaining better results.

[0166] As a non-limiting example, human or animal treatment may be performed using single, infusion, or multiple doses. , 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, At least one of the following days: 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 at least one day, or alternatively or additionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2 3, 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, At least one of weeks 50, 51, or 52, or alternatively or additionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17th, 18th, 19th, or 20th year, or any of these years In combination, 0.1 to 100 mg / kg per day, e.g., 0.5, 0.9, 1.0 ,1.1,1.5,2,3,4,5,6,7,8,9,10,11,12,13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2 8, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg , may be provided as a single or periodic dose of at least one antibody of the invention.

[0167] Dosage forms (compositions) suitable for internal administration generally contain about 0.1 milligrams per unit or container. These pharmaceutical compositions contain between about 1000 and about 500 milligrams of the active ingredient. The component is typically present in an amount of about 0.5 to 99.999% by weight, based on the total weight of the composition. .

[0168] For parenteral administration, the antibody is provided in conjunction with or separately from a pharmaceutically acceptable parenteral vehicle. It can be formulated as a solution, suspension, emulsion, or lyophilized powder, which is delivered to the patient. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1 ~10% human serum albumin. Uses non-aqueous vehicles such as liposomes and fixed oils. The vehicle or lyophilized powder may contain additives that maintain isotonicity and chemical stability. agents (e.g., sodium chloride, mannitol for isotonicity; buffers for chemical stability) The formulation may be sterilized by known or suitable techniques. will be done.

[0169] Suitable pharmaceutical carriers are described in Remington's, a standard reference text in this field. Published in the latest edition of Pharmaceutical Sciences, A. Osol It has been done.

[0170] Alternative Administration: Administering a pharmaceutically effective amount of at least one anti-TNF antibody according to the present invention. Many known and developed administration methods can be used in accordance with the present invention to Although pulmonary administration is used in the following description, other modes of administration may be used in accordance with the present invention. This may provide favorable results.

[0171] The TNF antibodies of the present invention may be present in a carrier as a solution, emulsion, colloid or suspension. or as a dry powder, by inhalation or by administration of any of the methods described herein or in the art. Any of a variety of devices and methods suitable for administration by other methods known in the art. can be used to deliver.

[0172] Parenteral Formulations and Administration. Parenteral formulations contain sterile water or saline as the common vehicle. Water, polyalkylene glycols such as polyethylene glycol, plant-derived oils, hydrogen Aqueous or oily suspensions for injection may be prepared according to known methods. Injectable preparations can be prepared by using appropriate emulsifying or wetting agents and suspending agents. A non-toxic parenterally administrable diluent such as an aqueous solution, or a sterile injectable solution, or a suspension in a solvent. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, etc. As a usual solvent or suspending medium, sterile, fixed oils can be used. For these purposes, fatty oils or fatty acids, natural or synthetic or semi-synthetic, includes synthetic or semi-synthetic mono- or di- or triglycerides, Any type of fixed oil and fatty acid may be used. Conventional injection means are known in the art, and are described in U.S. Pat. No. 5,851,198. gas-pressurized needleless injection devices such as those described in U.S. Pat. No. 5,839,446; These include, but are not limited to, laser drilling devices such as 10, No. 10 / 109,493, filed Dec. 1, 2004, which is incorporated herein by reference in its entirety.

[0173] Alternative delivery. The present invention further provides for parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, , intracapsular, intracartilaginous, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, Intraosseous, pelvic, pericardial, abdominal, pleural, prostatic, pulmonary, rectal, renal, retinal, spinal Intramedullary, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, intravaginal, rectal, oral, sublingual, nasal The present invention relates to the administration of at least one anti-TNF antibody by intradermal or transdermal means. The anti-TNF antibody compositions may be administered parenterally (subcutaneously, intramuscularly, or intravenously) or by any other route, particularly , for use in the form of a liquid solution or suspension, in particular creams and suppositories. In semi-solid form, including but not limited to tablets, for use in vaginal or rectal administration. for buccal or sublingual administration, such as, but not limited to, tablets or capsules; or powders, nasal drops or aerosols, or certain medications, but these Intranasally, or by modifying the skin structure or in a transdermal patch, in a form not limited to Chemical enhancers such as dimethyl sulfoxide to either increase drug concentration Using the method (Junginger, et al., "Drug Permeation Enhancement”;Hsieh,DS,Eds.,pp.59-90(Ma Dekker, Inc. New York 1994, incorporated herein by reference in its entirety. (Incorporated into the International Patent Publication) or application of protein and peptide-containing preparations to the skin (International Patent Publication) (Publication No. 98 / 53847) or by creating a transient delivery pathway, such as electroporation. or to increase the mobility of charged drugs through the skin, such as by iontophoresis application of an electric field to induce iontophoresis, or application of ultrasound, such as sonophoresis (U.S. Patent No. 4,309, 989 and 4,767,402) and gels, ointments, and lotions. transdermally, such as, but not limited to, injections, suspensions, or patch delivery systems; (The above publications and patents are incorporated herein by reference in their entirety. (It is included).

[0174] Pulmonary / Intranasal Administration. For pulmonary administration, preferably at least one anti-TNF antibody is administered. The composition is delivered in a particle size effective to reach the lower airways or sinuses of the lung. At least one anti-TNF antibody is described in the art for administering therapeutic agents by inhalation. The drug can be delivered by any of a variety of inhalation or intranasal devices known in the art. These devices are capable of depositing aerosolized formulations into the sinus cavities or alveoli. Examples of devices include metered dose inhalers, nebulizers, dry powder generators, and sprayers. Other devices suitable for pulmonary or intranasal administration are also known in the art. All such devices contain a formulation suitable for administration to dispense the antibody in an aerosol. Such aerosols may be solutions (both aqueous and non-aqueous) or solids. Ventolin® Metered-Dose Inhaler Metered dose inhalers, such as metered dose inhalers, typically use a propellant gas and require actuation during inspiration (e.g., (See, for example, WO 94 / 16970 and WO 98 / 35888.) Urbuhaler™ (Astra), Rotahaler® (Gla xo), Diskus® (Glaxo), Spiros® Inhaler (Du ra), a device marketed by Inhale Therapeutics, and Dry powder inhalers, such as the Spinhaler® powder inhaler (Fisons) uses breath actuation of mixed powders (U.S. Pat. No. 4,668,218 (Astra), European Patent No. 237507 (Astra), International Publication No. 97 / 25086 (Glaxo), WO 94 / 08552 (Dura), U.S. Pat. No. 5,458,135 (Inhal e), WO 94 / 06498 (Fisons), incorporated herein by reference in its entirety. AERx™ (Aradigm), Ultravent® Nebulizer (Mallinckrodt) and Acorn II® nebulizer Nebulizers such as the Iser (Marquest Medical Products) U.S. Patent No. 5,404,871 (Aradigm), International Publication No. 97 / 22376 (above) The above references (which are incorporated herein by reference in their entireties) describe the preparation of aerosols from solutions. Metered dose inhalers and dry powder inhalers generate small particle aerosols. These examples of commercially available inhalation devices are intended to represent specific devices suitable for practicing the present invention. are intended as illustrative only and are not intended as limiting the scope of the invention. Preferably, the composition comprising at least one anti-TNF antibody is administered as a dry powder inhaler. The method for administering at least one antibody of the present invention is delivered by an inhaler or a nebulizer. The device has several desirable features. For example, delivery by inhalation device is effective. The inhalation device is optionally For good respirability, small dry particles, e.g., less than about 10 μm, preferably about 1-5 μm, are used. can be delivered.

[0175] Administration of the TNF antibody composition by spray. The spray containing the TNF antibody composition protein is by passing a suspension or solution of at least one anti-TNF antibody through a nozzle under pressure. The nozzle size and configuration, the applied pressure and the liquid supply The velocity can be selected to achieve the desired output and particle size. An electrospray can be produced by an electric field in conjunction with a nozzle or nozzle feed. In particular, particles of at least one anti-TNF antibody composition protein delivered by a nebulizer. is less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. It has a particle size in the range of m.

[0176] A preparation of at least one anti-TNF antibody composition protein suitable for use with a nebulizer. The agent is typically present in aqueous solution at concentrations of, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 1 00mg / mL or mg / gm, 1mL or mg / g About 0.1 mg to about 100 mg per m solution, or any range or value therein The antibody composition protein is contained in a concentration of at least one anti-TNF antibody composition protein. The formulation may contain excipients, buffers, isotonicity agents, preservatives, surfactants, and preferably pharmaceutical agents such as zinc. The formulation may include additional components of the antibody composition, such as a buffer, a reducing agent, bulk protein or carbohydrate. It may also contain an excipient or agent for stabilizing the composition protein. Bulk proteins useful in formulations include albumin, protamine, etc. Typical carbohydrates useful in formulating antibody composition proteins include sucrose, mannose, and the like. Examples of sugars that can be used include ethanol, lactose, trehalose, and glucose. The agent also prevents the release of the antibody composition protein caused by atomization of the solution when forming the aerosol. Surfactants that can reduce or prevent surface-induced aggregation may also be included. ethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters A variety of conventional surfactants, such as esters, can be used. The amount is generally about 0.1% of the formulation. Particularly preferred surfactants for purposes of the present invention are: Polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 2 0. For formulations of proteins such as TNF antibodies or specified portions or variants Additional drugs known in the art may also be included in the formulation.

[0177] Administration of TNF antibody composition by nebulizer. The antibody composition protein is administered by jet nebulizer. It can be administered by a nebulizer, such as a lysator or ultrasonic nebulizer. Essentially, a jet nebulizer produces a high-velocity jet of air through an orifice. A compressed air source is used to create a low pressure area as the gas expands beyond the nozzle. It draws a solution of antibody composition protein through a capillary tube connected to a liquid reservoir. The liquid flow from the capillary tube is sheared into unstable filaments and droplets as it exits the tube. A range of configurations, flow rates and baffle types are available for a given jet nebulizer. can be used to achieve desired performance characteristics from an ultrasonic nebulizer. In this technique, high frequency electrical energy is used to generate vibrations, typically using a piezoelectric transducer. This energy is transferred directly or through the coupling fluid to the The antibody composition protein is then transferred to a formulation containing the antibody composition protein by any of the methods described above. Advantageously, the antibody composition protein delivered by the nebulizer is The clay particles are less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm. The particle size ranges from 1 μm to approximately 3 μm.

[0178] At least one anti-TN antibody suitable for use in either a jet or ultrasonic nebulizer Formulations of antibody F typically contain at least about 0.1 mg to about 100 mg per mL of solution. The formulation also contains one or more concentrations of an anti-TNF antibody protein. The formulation may also contain an excipient, a buffer, an isotonicity agent, Agents such as preservatives, surfactants, and preferably zinc may be included. At least one anti-TNF antibody composition such as a reducing agent, bulk protein, or carbohydrate. The composition may also contain an excipient or agent for protein stabilization. Bulk proteins useful in product protein formulations include albumin, protamine, etc. Exemplary carbohydrates useful in formulating at least one anti-TNF antibody include: Examples of sugars include sucrose, mannitol, lactose, trehalose, and glucose. At least one anti-TNF antibody formulation also exhibits a toxic effect caused by atomization of the solution upon aerosol formation. A surfactant capable of reducing or preventing surface-induced aggregation of at least one anti-TNF antibody. Polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene A variety of conventional surfactants can be used, such as ethylene sorbital fatty acid esters. The amount is generally in the range of 0.001 to 4% by weight of the formulation. Preferred surfactants are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, etc. For the preparation of proteins such as antibody proteins, Additional pharmaceutical agents known in the art can also be included in the formulation.

[0179] Administration of TNF antibody compositions by metered dose inhaler. Metered dose inhalers (MDIs) contain a propellant, a small amount of At least one anti-TNF antibody and any excipients or other additives are mixed together with a liquefied compressed gas. The particles are contained in a canister as a liquid. By operating the throttle valve, the particles are preferably separated into particles of less than about 10 μm. Preferably, the size ranges from about 1 μm to about 5 μm, and most preferably from about 2 μm to 3 μm. The mixture is released as an aerosol containing particles. Antibody composition proteins produced by various methods known to those skilled in the art, including point aggregation. The formulation can be used to obtain the desired aerosol particle size. The containers are manufactured by 3M or Glaxo and use hydrofluorocarbon propellants. Examples of uses include:

[0180] Formulations of at least one anti-TNF antibody for use in a metered dose inhaler device generally comprise , suspending at least one anti-TNF antibody in a propellant, for example with the aid of a surfactant. The propellant is trichlorofluoromethyl methyl methacrylate. ethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1, 2-Tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227) and other chlorofluoroalkanes. carbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon The propellant can be any conventional material used for this purpose. The surfactant is a hydrofluorocarbon. The surfactant is a fluorocarbon. The surfactant is a hydro ... selected for stabilizing the suspension, protecting the active agent against chemical degradation, etc. Suitable surfactants include sorbitan trioleate, soy lecithin, , oleic acid, etc. In some cases, a solution using a solvent such as ethanol Aerosols are preferred. Additional agents known in the art for formulation of proteins may be added to the formulation. It may be included in the formulation.

[0181] Those skilled in the art will appreciate that the method of the present invention may involve at least one method via a device not described herein. It will be appreciated that this can be achieved by pulmonary administration of the anti-TNF antibody composition.

[0182] Oral Formulations and Administration. Oral formulations may be administered using an agent to artificially increase the permeability of the intestinal wall. Adjuvants (e.g., resorcinol, and polyoxyethylene oleyl ether and simultaneous administration of non-ionic surfactants such as n-hexadecyl polyethylene ether, and Enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl ether) to inhibit enzymatic degradation Co-administration of difluorofluorophosphate (DFF) and trasylol. The active ingredient compounds in the bulk dosage form are sucrose, lactose, cellulose, mannitol, and trehalose. , raffinose, maltitol, dextran, starch, agar, alginate, chitin , chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, at least one of glycerides, such as glycerides, albumin, synthetic or semi-synthetic polymers, and glycerides; These dosage forms may also contain other types of additives, e.g. For example, inert diluents, lubricants (magnesium stearate, parabens, etc.), preservatives (such as (such as benzoyl peroxide, ascorbic acid, α-tocopherol, etc.), antioxidants (such as cysteine, etc.), Disintegrants, binders, thickeners, buffers, sweetening agents, flavoring agents, perfumes, and the like may also be included.

[0183] Tablets and pills can be further processed into enteric coated preparations. Liquid preparations of the present invention include emulsions, syrups, elixirs, suspensions, etc. that are acceptable for medical use. These preparations include suspensions and solution preparations. These preparations are based on the inactive ingredients commonly used in the field. Liposomes may contain a diluent such as water. It has also been described as a drug delivery system (U.S. Pat. No. 4,239,754). More recently, Microspheres of artificial polymers of mixed amino acids (proteinoids) are used to deliver drugs. (U.S. Patent No. 4,925,673). 79,681 and U.S. Pat. No. 5,5871,753. It is known in the art that it can be used to orally deliver biologically active agents. do.

[0184] Mucosal Formulations and Administration. Administer at least one anti-TNF antibody for absorption through mucosal surfaces. The compositions and methods for administering the present invention comprise a plurality of submicron particles, a mucoadhesive macromolecule, and a biological Absorption through mucosal surfaces by achieving mucoadhesion of the active peptide and emulsion particles and an aqueous continuous phase that promotes the formation of a granular polymer (U.S. Pat. No. 5,514,670). Suitable mucosal surfaces for application of the emulsions of the present invention include the cornea, conjunctiva, oral cavity, sublingual, These include nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Preparations, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, Formulations for intranasal administration may be solid and may contain excipients such as cocoa butter. For example, it may contain lactose or may be an aqueous or oily solution nasal spray. For oral administration, sugar, calcium stearate, stearic acid magnesium, pregelatinized starch, etc. (U.S. Patent No. 5,649,299; U ... , No. 849, 695).

[0185] Transdermal Formulations and Administration. For transdermal administration, at least one anti-TNF antibody is incorporated into a liposome. or polymer nanoparticles, microparticles, microcapsules or microspheres (especially those specified Unless otherwise specified, the polymers may be encapsulated in a delivery device such as a polymer (collectively referred to as a microparticle). Polyhydroxy acids such as lactic acid, polyglycolic acid and their copolymers, polyorthoesters Synthetic polymers such as esters, polyanhydrides and polyphosphazenes, as well as collagen, poly Amino acids, natural polymers such as albumin and other proteins, alginate and other polysaccharides Many suitable devices containing microparticles made from sugars and combinations thereof are already available. (U.S. Patent No. 5,814,599).

[0186] Prolonged Administration and Formulations. The compounds of the present invention can be administered in a single dose over an extended period of time, e.g., It may sometimes be desirable to deliver to a subject for periods ranging from one week to one year. For example, the dosage form may contain a compound that has low solubility in body fluids. pharmaceutically acceptable non-toxic salts of the compounds, for example, (a) phosphate, sulfate, citric acid, tartaric acid, Tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfone acid, acid addition salts with polybasic acids such as polygalacturonic acid, (b) zinc, calcium, bismuth , barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc. or, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine. a salt having an organic cation formed from ethylenediamine, or (c) (a) and (b) ) combination, for example, zinc tannate. In addition, the compounds of the present invention, Preferably, a relatively insoluble salt such as those mentioned above is used in a solution suitable for injection, e.g., in sesame oil. The pharmaceutical composition may be formulated into a gel, such as, for example, an aluminum monostearate gel. Particularly preferred salts are zinc salts, zinc tannate, and zinc pamoate. Sustained release depot formulations for injection include, for example, polylactic acid / Encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as polyglycolic acid polymer. The compound or salt preferably contains a compound or salt dispersed therein to be cellularized. Relatively insoluble salts such as those mentioned above are useful for the treatment of cholesterol matrices, particularly for animal use. It can also be formulated in silastic pellets of 100mg / kg. Liposome formulations, such as gas or liquid liposomes, are described in the literature (U.S. Pat. No. 5,770,222, and "Sustained and Controlled Release Drug Delivery Systems”, JR Robinson ed., Marc el Dekker, Inc., NY, 1978).

[0187] Having generally described the invention, the same are offered by way of illustration and not limitation. This will be more readily understood by reference to the following examples, which are not intended to be exhaustive. Deaf.

[0188] Example 1: Cloning and expression of TNF antibodies in mammalian cells. A typical mammalian expression vector contains at least one promoter that mediates the initiation of transcription of mRNA. promoter element, antibody coding sequence, and sequences required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and RNA splicing signals. It contains intervening sequences flanking the donor and acceptor sites for transcription. Transcription may be via early and late promoters from SV40, retroviruses such as RSV, HT LVI, long terminal repeats (LTRS) from HIVI, and sites This can be achieved with 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 practicing the present invention include, for example, pIRES. 1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -) or pcDNA3.1 / Hygro(+ / -)( Invitrogen), PSVL and PMSG (Pharmacia, Uppsala ,Sweden), pRSVcat(ATCC37152), pSV2dhfr(ATC Examples of vectors include pBC12MI (ATCC67109) and pBC12MI (ATCC67146). Mammalian host cells that can be used include human HeLa293, H9, and Jurkat cells. kat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7 and CV1, Zura QC1-3 cells, mouse L cells, and Chinese hamster ovary cells ovary, CHO) cells.

[0189] Alternatively, the gene can be expressed in a stable cell line containing the gene integrated into a chromosome. dhfr, gpt, neomycin or hygromycin can be expressed. Co-transfection with a selectable marker allows for the identification and identification of transfected cells. Allows for separation.

[0190] The transfected gene is also amplified to express large amounts of the encoded antibody. The DHFR (dihydrofolate reductase) marker can be used to identify hundreds or even thousands of genes of interest. This is useful for developing cell lines that contain several thousand copies. The enzyme glutamine synthase (GS) is the key to the synthesis of glutamine. chem.J.227:277-279(1991), Bebbington,et a l., Bio / Technology 10:169-175(1992)). Using a marker, mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are used to produce antibodies. There are many.

[0191] The expression vectors pC1 and pC4 contain the strong promoter (LTR) of the Rous sarcoma virus ( Cullen,et al.,Molec.Cell.Biol.5:438-447( 1985)), as well as the CMV enhancer (Boshart, et al., Cell41 :521-530(1985)). For example, the restriction enzyme cleavage site BamH Multiple cloning sites with I, XbaI, and Asp7l8 allow for cloning of the gene of interest. The vector also contains the 3' introductory sequence of the rat preproinsulin gene. It contains transcription, polyadenylation and termination signals.

[0192] Cloning and expression in CHO cells. The vector pC4 was used to express the TNF antibody. Plasmid pC4 is a clone of plasmid pSV2-dhfr (ATCC accession no. 37 146) is a derivative of the mouse SV40 early promoter. These plasmids contain the DHFR gene. Chinese hamster ovary cells or other cells lacking acid activity can be used to treat the chemotherapy drug methotrexate. Selective medium (e.g., α-MEM, Life Technologies) supplemented with α- , Gaithersburg, MD) to select for the presence of β-glucan. Amplification of the DHFR gene in methotrexate (MTX)-resistant cells is sufficient. It has been documented in recent years (e.g., FWAlt, et al., J. Biol. Chem. m.253:1357-1370 (1978), JL Hamlin and CMa ,Biochem.et Biophys.Acta1097:107-143(199 0) and MJ Page and MA Sydenham, Biotechnol (See Biochemistry 9:64-68 (1991)). Growth at increased MTX concentrations The cells undergo DHFR amplification, resulting in overproduction of the target enzyme, DHFR. If a second gene is linked to the DHFR gene, Using this approach, amplified genes (multiple It has been shown that the technology can develop cell lines with more than 1,000 copies of the gene (possibly 1,000 copies). Subsequently, when the methotrexate is recovered, one of the host cells Cell lines are obtained that contain the amplified gene integrated into one or more chromosomes.

[0193] The plasmid pC4 contains the Rous sarcoma virus long terminal endonucleases (LTDs) for expression of the gene of interest. A strong promoter from the long terminal repeat (LTR) (Cullen, et al., Molec. Cell. Biol. 5: 438-447 (1985)), as well as human cytomegalovirus (CM V) immediate early gene enhancer (Boshart, et al., Cell 41:52 1-530 (1985)). Downstream of the promoter is the gene BamHI, XbaI, and Asp718 restriction enzyme cleavage sites allow for the integration of After these cloning sites, the plasmid contains the It contains a 3' intron and a polyadenylation site. Other highly efficient promoters, e.g. Human β-actin promoter, SV40 early or late promoter, or other retroviruses Long terminal repeats from viruses such as HIV and HTLV-1 can also be used for expression. Clontech's Tet-Off and Tet-On gene expression systems and similar systems TNF can be expressed in a regulated manner in mammalian cells using M.Gossen,and H.Bujard,Proc.Natl.Acad.Sci. USA 89:5547-5551 (1992) for mRNA polyadenylation. Other signals, for example from the human growth hormone or globin genes, can also be used. Stable cell lines with the gene of interest integrated into the chromosome can be generated using gpt, G418 Alternatively, selection can be performed by co-transfection with a selectable marker such as hygromycin. First, more than one selectable marker, e.g., G418, plus methotrexate It is advantageous to use a porcelain sachet.

[0194] After digestion of the plasmid pC4 with restriction enzymes, the plasmid pC4 is purified by procedures known in the art. The vector was then dephosphorylated using calf intestinal phosphatase. It is isolated from a sugar gel.

[0195] The isolated variable and constant region-encoding DNA and the dephosphorylated vector were then ligated to T4 DNA. Ligate the fragments with ligase. Then, transform E. coli HB101 or XL-1 Blue cells. and, using, for example, restriction enzyme analysis, identify the fragment containing the inserted fragment in plasmid pC4. Identify the bacteria.

[0196] For transfection, Chinese hamsters lacking an active DHFR gene were used. CHO cells are used. 5 μg of the expression plasmid pC4 is transfected with lipofectin. The cells are co-transfected with 0.5 μg of the plasmid pSV2-neo using the following procedure. The plasmid pSV2-neo is a vector that binds to a group of antibiotics containing the dominant selectable marker G418. It contains the neo gene from Tn5, which encodes an enzyme that confers resistance to bacteria. The cells were seeded in α-MEM supplemented with 1 μg / ml G418. After 2 days, the cells were trypsinized. and cultured in hybridoma cloning plates (Greiner, Germany). supplemented with 10, 25, or 50 ng / ml methotrexate plus 1 μg / ml G418 After about 10-14 days, single clones were trypsinized and then cultured in α-MEM. Different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 80 100 nM) to seed 6-well Petri dishes or 10 mL flasks. Clones grown at a high concentration of methotrexate were grown at a higher concentration of methotrexate ( Transfer the mixture to a new 6-well plate containing 10 mM NaCl, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM). Repeat the same procedure until you obtain clones that grow at a concentration of 100-200 mM. Expression of the gene product can be determined, for example, by SDS-PAGE and Western blot. or by reverse phase HPLC analysis.

[0197] Example 2: High-affinity human TNF-reactive TNF-α using transgenic mice Generation of IgG monoclonal antibodies. SUMMARY: Transgenic mice containing human heavy and light chain immunoglobulin genes have been developed. and / or to inhibit TNF action for the treatment of one or more TNF-mediated diseases. The heavy chain and Contains human variable and constant region antibody transgenes for both the β and light chains (CBA / JxC57 / B Immunize L6 / J)F2 hybrid mice with human recombinant TNF (Taylor et al. t al., Intl. Immunol. 6:579-591 (1993), Lonbe rg, et al., Nature368:856-859(1994), Neuber Ger, M., Nature Biotech. 14:826 (1996), Fishw. ild,et al.,Nature Biotechnology14:845-85 1 (1996)). Several fusions were synthesized as fully human TNF-reactive IgG monoclonal antibodies. We have generated a panel of one or more fully human anti-TNF antibodies to further characterize. Such antibodies are approximately 1 x 10 9 ~9×10 12 has an affinity constant of The unexpected high affinity of these fully human monoclonal antibodies allowed These make them good candidates for therapeutic use in TNF-related diseases, conditions, or disorders.

[0198] Abbreviations: BSA - bovine serum albumin, Co2 - carbon dioxide, DMSO - dimethyl sulfide oxide, EIA - enzyme immunoassay, FBS - fetal bovine serum, H2O2 - hydrogen peroxide, HRP - horseradish peroxidase, ID - intradermal, Ig - immunoglobulin Phosphorus, 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, v / v - volume per unit volume, w / v - weight per unit volume.

[0199] Materials and Methods Animals. Transgenic mice capable of expressing human antibodies are known in the art. (e.g., GenPharm International, San and others) They express human immunoglobulins but not mouse IgM or Igκ. For example, such transgenic mice exhibit V(D)J joining, heavy chain class switching, and somatic Human sequence introduction, which undergoes cellular mutation to generate a repertoire of human sequence immunoglobulins Gene containing (Lonberg, et al., Nature 368:856-85 9 (1994)). The light chain transgene may, for example, be derived in part from a nearly identical germline human Vκ region. In addition, heavy chain transgenes can be derived from yeast artificial chromosome clones containing half of the human and human γ1 (Fishwild, et al., Nature Biotechn 14:845-851 (1996)) and / or a γ3 constant region. Mice from the appropriate genotype strains can be used in the immunization and fusion process. can be used to generate fully human monoclonal antibodies against TNF.

[0200] Immunization. One or more immunization schedules may be used to generate anti-TNF human hybridomas. Following the exemplary immunization protocol below, the first few fusions Although a combination of these protocols can be used, other similar known protocols can also be used. For 14-20 week old female and / or surgically castrated male transgenic mice Add an equal volume of TITERMAX or complete filler to a final volume of 100-400 μl (e.g., 200 μl). 1-1000 μg of recombinant human TNF emulsified in Roydt's adjuvant was administered IP or ID. Each mouse is also optionally inoculated with 100 μL of physiological saline in each of 2 SQ sites. Mice can also receive 1-10 μg in saline. IP (1-400 μg) and SQ ( 1-400μg x 2) with an equal volume of TITERMAX or complete Freund's adjuvant The patient can be immunized with emulsified TNF. After 12-25 and 25-40 days without anticoagulation, Mice can be bled by retroperitoneal puncture. Blood is then allowed to clot at room temperature for 1 hour. Serum is collected and titrated using a TNF EIA assay by known methods. If the injection does not result in an increase in titer, fusions are performed. At this time, mice are given 100 μL of physiological saline. A final IV booster injection of 1-400 μg of TNF diluted in saline may be given. After 3 days, the mice were euthanized by cervical dislocation, and the spleens were aseptically removed and diluted with 100U 100 μg / mL penicillin, 100 μg / mL streptomycin and 0.25 μg / mL 10 mL of cold phosphate buffered saline (PBS) containing amphotericin B (PSA) The spleen can be immersed in PSA-PBS. Cells were washed once in cold PSA-PBS and isolated using trypan blue dye exclusion. The cells are counted and resuspended in RPMI 1640 medium containing 25 mM Hepes.

[0201] Cell fusion. Cell fusion can be carried out by known methods, for example, 1:1 to 1:1, as known in the art. Fusions can be performed at a ratio of mouse myeloma cells to viable spleen cells of 1:10. The spleen cells and myeloma cells can then be pelleted together. The pellet was then slowly added to 1 mL of 50% (w / v) PEG / PBS solution (PEG content) at 37°C. The solution can then be resuspended in 25 mM NaCl (1,450 mM NaCl, Sigma-Aldrich) for 1 minute. Slowly add 10.5 mL of RPMI 1640 medium (37°C) containing Hepes. Fusion can be stopped by rotating the fused cells at 500-1500 rpm for 5 minutes. The cells were then soaked in HAT medium (25 mM Hepes, 10% fetal clone I serum, 10% fetal bovine serum). Hyclone, 1 mM sodium pyruvate, 4 mM L-glutamine, 10 μL g / mL gentamicin, 2.5% Origen culture supplement (Fisher); 10% 653-adjusted RPMI1640 / Hepes medium, 50 μM 2-mercaptoethanol, containing 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine. After resuspending in RPMI 1640 medium containing 100% ethanol, the cells were plated in 2 wells of a 96-well flat-bottom tissue culture plate. Plate the plates at 100 μL / well. Then incubate in a 5% CO2 / 95% air atmosphere for 7-10 days. Place the plate in a humidified 37 °C incubator with

[0202] Detection of human IgG anti-TNF antibodies in mouse serum. Mouse sera can be screened for human IgG antibodies specific for F. Alternatively, 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, The wells were blocked with 200 μL / well of 1% (w / v) BSA in PBS for 1 hour at room temperature. The plates can be used immediately or frozen at -20°C for later use. Mouse serum dilutions were plated at 50 μL / well on TNF-coated plates at room temperature. After washing the plate, incubate at 1:30 in 1% BSA-PBS for 1 hour. 50 μL / well of Fc-specific HRP-conjugated goat anti-human IgG diluted at 1,000 kJ / ml at room temperature. The plate can be washed again and probed at 100 μL / well for 1 hour. citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0 Add 200 mL of 0.01% H2O2 and 1 mg / mL OPD over 15 minutes at room temperature. Add 5 μL / well of stop solution (4N sulfuric acid) and measure the reaction time using an automated plate spectrophotometer. Read OD at 90 nm.

[0203] Detection of fully human immunoglobulins in hybridoma supernatants using a suitable EIA , growth-positive hybridomas secreting fully human immunoglobulins can be detected. Briefly, 96-well pop-out plates (VWR, 610744) were incubated overnight at 4°C. Coating with 10 μg / mL goat anti-human IgG Fc in sodium carbonate buffer The plate was washed and blocked with 1% BSA-PBS for 1 hour at 37°C, and then immediately Use or freeze at -20°C. Undiluted hybridoma supernatant is plated at 3°C. Incubate for 1 hour at 7°C. Wash the plate and add 1% BSA in PBS. Probe with HRP-conjugated goat anti-human kappa diluted at 1:10,000 for 1 hour at 37°C. The plate is then incubated with substrate solution as described above.

[0204] Determination of fully human anti-TNF reactivity. The hybridomas described above can be used in a suitable RIA or other assay. The antibody can be used to simultaneously assay for responsiveness to TNF. The supernatant was incubated on a goat anti-human IgG Fc plate and washed as described above. Afterwards, plate with radiolabeled TNF at the appropriate count per well for 1 hour at room temperature. The wells are washed twice with PBS and the bound radiolabel is counted using a suitable counter. The recognized TNF is quantified.

[0205] Human IgG1κ anti-TNF-secreting hybridomas were expanded in cell culture and isolated by limiting dilution. The resulting clonal population can then be expanded. The cells are then frozen in freezing medium (95% FBS, 5% DMSO) and stored in liquid nitrogen.

[0206] Isotype. Determination of the antibody isotype is performed by screening mouse immune sera for specific titers. This can be achieved using a similar format EIA to that used to clean TNF can be coated onto 96-well plates as described above, and 1 μg / mL of purified antibody can be incubated on the plate for 1 hour at room temperature. The plate was washed and incubated with HRP-labeled yam diluted 1:4000 in 1% BSA-PBS. Probe with goat anti-human IgG1 or HRP-conjugated goat anti-human IgG3 for 1 hour at room temperature. The plates are washed again and incubated with substrate solution as above.

[0207] Binding kinetics of human anti-human TNF antibodies with human TNF. The binding characteristics of the antibodies can be determined by, for example, The purified product can be suitably evaluated using F capture EIA and BIAcore technology. The concentrations of human TNF antibodies used were graded as described above, with 2 μg / mL TNF in the assay. Binding to NF-coated EIA plates can be assessed. The OD can then be expressed as a semi-logarithmic plot showing the relative binding efficiency.

[0208] The quantitative binding constant can be determined, for example, as follows, or by any other known suitable method: A BIAcore CM-5 (carboxymethyl) chip was used. Place in a re2000 unit. HBS buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.005% v / v P20 surfactant, pH7.4 ) is flowed over the flow cell of the chip at 5 μl / min until a stable baseline is obtained. Dissolve 15 mg of EDC (N-ethyl-N'-(3-dimethylaminopropyl)propionate) in 200 μL of water. A solution (100 μL) of 2.3 mg of benzophenone-4-carbodiimide hydrochloride (p-carbodiimide hydrochloride) in 200 μL of water was Add 100 μL of NHS (N-hydroxysuccinimide) to the solution. Inject 40 μL of the resulting solution onto the chip. Add 6 μL of a solution of human TNF-α (10 mM acetic acid) Injection of 15 μg / mL of sodium chloride, pH 4.8, onto the chip resulted in an increase of approximately 500 RU. The buffer was changed to TBS / Ca / Mg / BSA running buffer (20 mM Tris, 0.1 5M sodium chloride, 2mM calcium chloride, 2mM magnesium acetate, 0.5% Triton Xanthan Gum The solution was then changed to PEG-100, 25 μg / mL BSA, pH 7.4, and run on the chip overnight. It is allowed to equilibrate and any unreacted succinate esters are hydrolyzed or capped.

[0209] Dissolve the antibody in the 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 were used: one underivatized (sample) and the second underivatized (blank) flow cell. Use for kinetic runs. Inject 120 μL of each antibody concentration over the flow cell at 30 μL / min ( The dissociation phase is followed by a continuous flow of buffer for 360 seconds (association phase). Regenerate the surface of the chip by two sequential injections of guanidine anhydride (a cause of tissue necrosis). Dissociation of the α-antibody complex.

[0210] Data analysis was performed using BIA evaluation 3.0 or CLAMP2, which are known in the art. For each antibody concentration, a blank sensogram is taken along with the sample sensogram. Dissociation (k d ,sec -1 ) and meetings (k a ,mol -1 sec -1 ) A global fit was performed on 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, the antibody An additional dilution of 1000 mg / ml is performed.

[0211] Results and Discussion Generation of anti-human TNF monoclonal antibodies. Several fusions were made to target specificity for human TNF. Each fusion, which yields several dozen antibodies, was seeded onto 15 plates (1440 wells / fusion). Some of these are known to consist of a combination of human and mouse Ig chains. The remaining hybridomas secrete anti-TNF antibodies consisting only of human heavy and light chains (se All human hybridomas are expected to be IgG1κ.

[0212] Binding kinetics of human anti-human TNF antibodies. ELISA analysis showed that most of these hybridomas It is confirmed that most or all of the purified antibodies bind to TNF in a concentration-dependent manner. Figures 1-2 show the results of the relative binding efficiencies of these antibodies. The binding activity of TNF to the epitope is measured by directly binding it to the EIA plate. This can cause protein denaturation, and the apparent binding affinity may be reduced to that of the native protein. It should be noted that the 50 percent binding cannot reflect the overall binding. It is found over a range of concentrations.

[0213] Quantitative binding constants were obtained using BIAcore analysis of human antibodies and human monoclonal antibodies. Some of the antibodies were 1 × 10 -9 ~7×10 -12 K in the range D It has very high affinity Reveals that it is sexual.

[0214] Conclusion. Some fusions involve the use of human variable and constant region antibody transgenes that are immunized with human TNF. This is performed using splenocytes from hybrid mice containing the IgG1κ isotype. To generate a set of several fully human TNF-reactive IgG monoclonal antibodies. The human anti-TNF antibodies were further characterized. Some of the antibodies generated were cultured at 1 x 10 9 ~9×10 12 These fully human monoclonal antibodies have an affinity constant of The high affinity makes them suitable for therapeutic use in TNF-dependent diseases, pathologies or related conditions. It becomes something like this.

[0215] Example 3: Generation of human IgG monoclonal antibodies reactive with human TNFα. Summary. Antibody transgenes containing human variable and constant region antibodies of both heavy and light chains (CBA / JxC57BL / 6J)F2 hybrid mice (1-4) were immunized with recombinant human TNFα. One fusion, designated GenTNV, bound to immobilized recombinant human TNFα. Immediately after identification, eight cells were cultured to generate fully human IgG1κ monoclonal antibodies. The strain was submitted to Molecular Biology for further characterization. Since the Mab sequences are completely human, they are the same as those of human cA2 (Remicad It is predicted to be less immunogenic than e).

[0216] Abbreviations: BSA - bovine serum albumin, Co2 - carbon dioxide, DMSO - dimethyl sulfide oxide, EIA - enzyme immunoassay, FBS - fetal bovine serum, H2O2 - hydrogen peroxide, HC - heavy chain, HRP - horseradish peroxidase, ID - interadermal, Ig -Immunoglobulin, TNF-tissue necrosis factor alpha, IP-intraperitoneal, IV-intravenous, Mab-mono 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.

[0217] Introduction. Transgenic mice containing human heavy and light chain immunoglobulin genes have been developed. This was used to generate a fully human monoclonal antibody specific for recombinant human TNFα. 2 (Remicade) has the advantage of increased serum half-life and reduced immunogenic side effects. and can be used to therapeutically inhibit inflammatory processes involved in TNFα-mediated diseases. It is expected that these specific antibodies can be used.

[0218] Materials and Methods. Animal transfection that expresses human immunoglobulins but not mouse IgM or Igκ. The transgenic mice were developed by GenPharm International. These mice undergo V(D)J joining, heavy chain class switching, and somatic mutation. This generates a repertoire of antigen-specific human immunoglobulins (1), functional human antibodies The light chain transgene contains, in part, a clone derived from approximately the germline human Vκ locus. It is derived from a yeast artificial chromosome clone containing half of the heavy chain ( The HC) transgene encodes both human μ and human γ1(2), and / or γ3 constant regions. Immunization and Fusion to Generate Monoclonal Antibodies Described Herein In the process, mice from the HCo12 / KCo5 genotype strain were used.

[0219] Purification of human TNFα. TNFα coupled to Sepharose 4B (Pharmacia). A column packed with a receptor-Fc fusion protein (p55-sf2) (5) was used to Human TNFα was isolated from tissue culture-derived C237A cells by affinity chromatography. The cell supernatant was purified from the supernatant. The cell supernatant was diluted with 1 / 9 of its volume of 10x Dulbecco's PBS ( The mixture was mixed with PBS (D-PBS) and passed through the column at 4 mL / min at 4°C. The column was washed and TNFα was eluted with 0.1 M sodium citrate, pH 3.5, followed by 2 M trichloroethylene. Purified TNFα was neutralized with 10 mM Tris, 0.12 mM Tris HCl, pH 8.5. The solution was buffer exchanged to 1 M sodium chloride, pH 7.5, and passed through a 0.2 μm syringe filter. and filtered.

[0220] Immunization. Female GenPharm mice, approximately 16 weeks old, were immunized with equal doses of IgG1 on days 0, 12, and 28. A total of 100 μg of TNFα (lot JG102) emulsified in Titermax adjuvant 298 or JG102098), IP (200 μL) and ID (100 at the base of the tail) Mice were immunized with 1 μL of IgG1 via retroorbital puncture on days 21 and 35 without anticoagulant. The blood was allowed to clot at room temperature for 1 hour, and serum was collected and analyzed by TNFα solid-phase EIA assay. After the 28th injection, mice were allowed to rest for 7 weeks before the GenTNV The fusion, named , was then performed with a specific human IgG titer of 1:160 against TNFα. Vaccinate mice with 50 μg of TNFα diluted in 100 μL of physiological saline. A final IV booster injection was given. Three days later, the mice were euthanized by cervical dislocation and the spleens were removed. Aseptically remove and add 100 U / mL penicillin and 100 μg / mL streptomycin. and 10 mL of cold phosphoric acid containing 0.25 μg / mL amphotericin B (PSA). The spleen was then immersed in saline-buffered saline (PBS). Splenocytes were collected from the spleen. Cells were washed once in cold PSA-PBS and counted by Coulter count. The cells were counted using a microcentrifuge and resuspended in RPMI 1640 medium containing 25 mM Hepes.

[0221] Cell lines. Cell Biology Services (CBS) group On May 14th, Centocor's Product Development group The cell lines received the non-secreting murine myeloma fusion partner 653 from the same company. v) FBS (Cell Culture Labs), 1 mM sodium pyruvate, 0 1mM NEAA, 2mM L-glutamine (all from JRH Biosciences) The cells were expanded in RPMI medium (JRH Biosciences) supplemented with 95% F Cryopreservation in BS and 5% DMSO (Sigma) was followed by vapor-phase liquid nitrogen freezing of CBS. The cell bank was kept sterile (Quality Control Center). cor, Malvern), Mycoplasma (Bionique Laboratori es). Cells were maintained in logarithmic growth culture until confluence. Before confluence, they were washed in PBS, counted, and viability determined by trypan blue dye exclusion (95% super).

[0222] Human TNFα was produced by a recombinant cell line, designated C237A, and was obtained from Centococcus aureus. The cell lines were generated at Molecular Biology, OR. BS (Cell Culture Labs), 2 mM L-glutamine (all JRH Biosciences) and supplemented with 0.5 μg / mL mycophenolic acid. Expanded in MDM medium (JRH Biosciences), containing 95% FBS and 5% DM After cryopreservation in SO (Sigma), the samples were stored in a vapor-phase liquid nitrogen freezer in CBS (13). The cell bank was sterile (Quality Control Centocor, Malvern), Mycoplasma (Bionique Laboratories) There wasn't.

[0223] Cell fusion. Cell fusion was performed using 653 mouse myeloma cells and live mouse spleen cells at a 1:1 ratio. Briefly, splenocytes and myeloma cells were pelleted together for 30 seconds. The pellet was slowly stirred at 37 °C in 1 mL of 50% (w / v) PEG / PBS solution (PE The sample was resuspended in 10.5 mL of R (MW 1,450 g / mol, Sigma) over 1 minute. Fusion was achieved by slowly adding PMI medium (without additives) (JRH) (37 °C). The fused cells were then centrifuged at 750 rpm for 5 minutes. The cells were then resuspended in HAT medium. (10% fetal bovine serum (JRH), 1 mM sodium pyruvate, 2 mM L-glutamine) 10 μg / mL gentamicin, 2.5% Origen culture supplement (Fish er), 50 μM 2-mercaptoethanol, 1% 653-adjusted RPMI medium, 100 μM RPMI containing hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine After resuspending in 2% HEPES medium, the cells were cultured in five 96-well flat-bottom tissue culture plates. The cells were plated at 100 μL / well and then incubated in a 5% CO2 / 95% air atmosphere for 7-10 days. The plate was placed in a humidified 37°C incubator containing 1000 µL of PBS.

[0224] Detection of human IgG anti-TNFα antibodies in mouse serum. Mouse sera were screened for human IgG antibodies specific to NFα. Plates were coated overnight with 1 μg / mL TNFα in 0.02% (v / v) PBS. After washing with 0.15M saline containing Tween 20, the wells were filled with 1% The plates were blocked with 200 μL / well of (w / v) BSA for 1 hour at room temperature. The cells were either used or frozen at -20°C for later use. The serum was incubated with human TNFα coated cells in two-fold serial dilutions at 50 μL / well for 1 hour at room temperature. After washing the plate, the plate was incubated with 1% BSA-P 50 μL / well of Fc specific (Accurate) diluted 1:30,000 in BS The plates were then probed with 100 μl of HRP-conjugated goat anti-human IgG for 1 hour at room temperature. Wash and add 100 μL / well of citrate-phosphate substrate solution (0.1 M citric acid and 0 0.2M sodium phosphate, 0.01% H2O2 and 1mg / mL OPD) for 15 minutes Then, 25 μL / well of reaction stop solution (4N sulfuric acid) was added. The OD was read at 490 nm using a moving plate spectrophotometer.

[0225] Detection of fully human immunoglobulins in hybridoma supernatants. GenPharm Mouse can produce both mouse and human immunoglobulin chains, thus producing two distinct Growth positive hives for the presence of both human light chains and human heavy chains using an EIA assay Plates were coated as described above and undiluted hybridoma clones were tested. The lysoma supernatant was incubated on the plate for 1 hour at 37°C. The plate was washed and HRP-conjugated goat IgG diluted 1:10,000 in 1% BSA-HBSS for 1 hour at 37°C. Anti-human κ (Southern Biotech) antibody or 1% BSA-HBSS Probe with either HRP-conjugated goat anti-human IgG Fc-specific antibody diluted at 1:30,000. The plates were then incubated with substrate solution as described above. The antibody did not give a positive signal in both the human kappa and anti-human IgG Fc EIA formats. The hybridoma clone was discarded.

[0226] Isotype. Determination of the antibody isotype is performed by screening mouse immune sera for specific titers. This was achieved using an EIA similar in format to that used for cleaning. 10 μg / mL goat anti-human IgG (H+L) in sodium carbonate buffer at 37°C overnight. A plate was coated and blocked as described above. Pure supernatant from a 24-well culture was collected. The supernatant was incubated on the plate for 1 hour at room temperature. The plate was washed and resuspended in 1% BSA. -HRP-conjugated goat anti-human IgG1, IgG2, IgG diluted 1:4000 in PBS The plates were probed with IgG3 or IgG4 (Binding Site) for 1 hour at room temperature. The plates were washed again and incubated with substrate solution as described above.

[0227] Results and Discussion. Generation of fully human anti-human TNFα monoclonal antibodies. Recombinant human TNF A fusion virus designated GenTNV was isolated from GenPharm mice immunized with the α protein. From this fusion, 196 growth-positive hybrids were screened. Eight hybridoma cell lines secreting fully human IgG antibodies reactive with human TNFα were isolated. Each of these eight cell lines expresses human immunoglobulins of the IgG1κ isotype. All were subcloned twice by limiting dilution to obtain stable cell lines. (More than 90% homogeneity). The cell line names and their respective C code designations are listed in Table 1. They were frozen in a 12-vial research cell bank stored in liquid nitrogen.

[0228] Parental cells harvested from wells of a 24-well culture dish for each of the eight cell lines were transfected. for transfection and further characterization on February 18, 1999. It was handed over to the Biology group.

[0229] [Table 3]

[0230] Conclusion. GenTNV fusion was immunized with recombinant human TNFα prepared at Centocor. The splenocytes from hybrid mice containing human variable and constant region antibody transgenes were used. Eight fully human TNFα-reactive IgG monoclonal antibodies of the IgG1κ isotype were used. The parent cell line was transferred to Molecul for further characterization and development. One of these new human antibodies was transferred to the Regenerative Biology group. Compared to micade, it has the potential benefit of reducing immunogenicity and allergic complications. and may be useful in anti-inflammatory purposes.

[0231] References

[0232] Taylor,et al.,International Immunology 6:579-591(1993).

[0233] Lonberg,et al.,Nature 368:856-859(1994) .

[0234] Neuberger, M. Nature Biotechnology 14:826 (1996).

[0235] Fishwild,et al.,Nature Biotechnology 14 :845-851(1996).

[0236] Scallon, et al., Cytokine 7:759-770 (1995) .

[0237] Example 4: Cloning and preparation of cell lines expressing human anti-TNFα antibodies. Summary: A panel of eight human monoclonal antibodies (mAbs) representing TNV clearly demonstrated high Of the eight mAbs, the binding activity was observed to be binding to immobilized human TNFα. Seven of these were shown to efficiently block the binding of human TNFα to recombinant TNF receptors. Sequence analysis of the DNA encoding the seven mAbs revealed that all mAbs have human V regions. The DNA sequences were identical to each other in the three pairs of mAbs, and therefore eight pairs were identified. The original panel of mAbs was represented by TNV14, TNV15, TNV148, and TNV196. The predicted amino acid sequences of the mAbs were also shown to be the only four distinct mAbs. Based on the results of sequence analysis and in vitro TNFα neutralization data, mAb TNV148 and TNV14 were selected for further study.

[0238] A database search identified a proline residue at position 75 (framework 3) of the TNV148 heavy chain. It was not found in that position in other human antibodies of the same subgroup, making it a known germline Site-directed DNA mutagenesis was performed to match the sequence of the framework e sequence. A serine residue was encoded at that position. The serine-modified mAb is designated TNV148B. PCR amplification D encoding the heavy and light chain variable regions of TNV148B and TNV14 NA was cloned to the recently cloned heavy and light chain genes of another human mAb (12B75). Based on the IL-12 Antibodies (published as International Publication No. WO 02 / 12500) ies, Compositions, Methods and Uses, 2 No. 60 / 236,827, filed October 7, 2000, which is incorporated herein by reference in its entirety. The resulting DNA fragment was cloned into a newly prepared expression vector.

[0239] 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 expressed at high levels. Recombinant TNV148B and TNV14 (rTNV148B and rTNV14) mAbs Cell lines producing b were screened by two rounds of subcloning. Growth curve and stability evaluation of mAb production were performed using 653 transfectant clone C4 66D and C466C stably maintained approximately 125 μg / ml of rTNV1 in spent cultures. 48B mAb was generated, while Sp2 / 0 transfectant 1.73-12-122 (C467A) was stably expressed in spent cultures at approximately 25 μg / ml of rTNV148B Similar analysis showed that Sp2 / 0 transfectant clones produced mAbs. showed that rTNV14 was produced at 18 μg / ml in spent cultures. did.

[0240] Introduction. Human TNFα-immunized GenPharm / Medarex mice (HCo12 / K A panel of eight mAbs derived from the Co5 genotype binds to human TNFα and is fully human I We previously demonstrated that TN has the gG1κ isotype. The ability of the present invention to block Fα binding to recombinant TNF receptors was assessed. It was determined whether exemplary mAbs may have TNFα neutralizing activity. Based on the results, DNA sequence results, and some in vitro characterization of the mAb, TNV148 was selected as the mAb to be characterized.

[0241] The DNA sequence encoding the TNV148 mAb was cloned and the appropriate constant region was encoded. The well-characterized 653 and Sp2 / 0 mouse myeloma cells, and the resulting transfected cells A subclone was identified that produced 40-fold more mAb than the original hybridoma cell line. Screening was continued until

[0242] 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. Sciences, Inc. Taq DNA polymerase was obtained from Perkin E The restriction enzymes were obtained from either Imner Cetus or Gibco BRL. Purchased from England Biolabs. QIA quick PCR Purification The fication kit was obtained from Qiagen. QuikChange Site- The Directed Mutagenesis Kit was purchased from Stratagene. Wizard plasmid miniprep kit and RNasin were purchased from Promega. Optiplates were obtained from Packard. 125 Iodine is Am Custom oligonucleotides were purchased from Keystone / Bios The oligonucleotides used in this work were purchased from Source International. The names, identification numbers, and sequences of the peptides are shown in Table 2.

[0243] Table 2. TNV mAb genes used to clone, engineer, or sequence oligonucleotides The amino acids encoded by oligonucleotides 5'14s and HuH-J6 were sequenced. The "M" amino acid residue represents the translation initiation codon. Oligonucleotide 5'14 The underlined sequences in s and HuH-J6 indicate BsiWI and BstBI restriction sites, respectively. The diagonal lines in HuH-J6 correspond to the exon / intron boundaries. Note that the corresponding oligonucleotides are written in the 3'-5' orientation.

[0244] [Table 4]

[0245] One frozen vial of 653 mouse myeloma cells was obtained. The vial was thawed the same day and T cells were cultured. The cells were expanded in IMDM, 5% FBS, and 2 mM glutamine (medium) in Lasco. These cells were transfected 2-3 weeks later with anti-TNF DNA as described herein. The cells were maintained in continuous culture until thawing. Some of the cultures were harvested 5 days after thawing. The cells were pelleted by centrifugation, resuspended in 95% FBS, 5% DMSO, and stored for 30 days. The cells were aliquoted into tubes, frozen, and stored for future use. One frozen vial of cells was obtained. The vial was thawed and a new freeze-dried product was prepared as described above. The frozen vials were stored in freezer boxes AA and AB at CBC. These cells were thawed and used for all Sp2 / 0 transfections described herein. Used.

[0246] Assay for inhibition of TNF binding to receptor. Hybridization containing TNV mAb. Using dorma cell supernatant, the mAb was used to detect recombinant TNF receptor fusion protein p55-sf2 to 125 The antibodies were assayed for their ability to block the binding of I-labeled TNFα (Scallion et al. (1995) Cytokine 7:759-770). During incubation, Opt-300 was added to 50 μg / mL of p55-sf2 at 0.5 μg / mL in PBS. The wells were coated with PBS / 0.1% BSA as a diluent. Serial dilutions of eight TNV ​​cell supernatants were prepared in 96-well round-bottom plates using the Cell supernatant containing anti-IL-18 mAb was included as a negative control, and cA2 (anti-T NF chimeric antibody, Remicade, U.S. Patent No. 5,770,198, incorporated herein by reference in its entirety. The same anti-IL-18 supernatant spiked with IL-18 (incorporated herein) was 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 100:1 of cell supernatant. The mixture was incubated at room temperature for 1 The coated Optiplates were washed and the unbound p55-sf2 was removed in the case of 50:1. 125 Opt I-TNFα / cell supernatant mixture After 2 hours at room temperature, the cells were transferred to OptiPla The plates were washed. 100:1 Microscint-20 was added and the TopCounter was Bound cpm was determined using a t gamma counter.

[0247] Amplification of V genes and DNA sequence analysis. For RNA preparation, hybridoma cells were cultured in P After washing once with BS, TRIZOL reagent was added. 6 ~1.7×10 7 The fine The cells were resuspended in 1 ml of TRIZOL. After the addition of 200 μl of chloroform, the tube was shaken. The sample was centrifuged for 10 minutes at 4°C. The aqueous phase was transferred to a new microfuge tube. An equal volume of isopropanol was added. The tube was vigorously shaken and incubated at room temperature for 10 minutes. The samples were then centrifuged for 10 minutes at 4°C. The pellet was then resuspended in 1 ml of 70% ethanol. The RNA pellet was washed once with PBS and briefly dried in a vacuum oven. The RNA was resuspended in treated water. The quality of the RNA preparation was confirmed by fractionating 0.5 μl in a 1% agarose gel. The RNA was stored in a -80°C freezer until use.

[0248] To prepare heavy and light chain cDNA, 3 μl of RNA and 1 μl of ATP were added to a volume of 11.5 μl. 1 μg of either oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light chain) A mixture containing either (see Table 1) was prepared. This mixture was heated in a water bath at 70°C for 1 hour. After incubation for 10 minutes, the mixture was cooled on ice for 10 minutes. 2.5 μl of 10× reverse transcriptase buffer, 10 μl of 2.5 mM dNTPs, 1 μl of reverse transcriptase (20 units), and 0. A separate mixture consisting of 4 μl of the ribonuclease inhibitor RNasin (1 unit) 13.5 μl of this mixture was mixed with 11.5 μl of cold RNA / oligonucleotide The mixture was added and the reaction was incubated at 42°C for 40 minutes. The cDNA synthesis reaction was stored in a −20°C freezer.

[0249] Unpurified heavy and light chain cDNAs were used as templates to clone the variable region coding sequences. The five oligonucleotides were analyzed for their ability to prime the amplification of heavy chain DNA. Otid pairs (366 / 354, 367 / 354, 368 / 354, 369 / 354, and 3 70 / 354 (Table 1) were tested simultaneously for their ability to prime the amplification of light chain DNA. Two oligonucleotide pairs (362 / 208 and 363 / 208) were tested simultaneously. 2 units of PLATINUM™ High Fidelity (HIFI) Ta in a total volume of 50 μl PCR reactions were performed using q DNA polymerase. Each reaction contained 2 μl of cDNA. Reaction A: 10 pmol of each oligonucleotide, 0.2 mM dNTP, 5 μl 10 The thermocycler program contained 2 mM magnesium sulfate and 2 mM HCl. The incubation was carried out at 95°C for 5 min, followed by 94°C for 30 s, 62°C for 30 s, and 68°C for 1.5 min. The reaction was repeated for 30 cycles at 68°C for 10 minutes, followed by a final incubation at 68°C for 10 minutes. It was held.

[0250] Follow the manufacturer's protocol to prepare PCR products for direct DNA sequencing. Therefore, we used the QIAquick™ PCR Purification Kit. They were purified after eluting the DNA from the spin column using 50 μl of sterile water. The mixture was then dried to a volume of 10 μl using a vacuum dryer. 1 μl purified PCR product, 10 μM oligonucleotide primer, 4 μl Big Dye Terminator™ ready reaction mix, and 1 DNA sequencing reactions were set up in 4 μl of sterile water. Oligonucleotide pair 367 / 35 The heavy chain PCR product generated in 4 was amplified using oligonucleotide primers 159 and 360. The light chain PCR product was generated with the oligonucleotide pair 363 / 208. The product was sequenced using oligonucleotides 34 and 163. The program consisted of 25 cycles of 96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 10 seconds. The reaction products were then analyzed by polyacrylamide gel electrophoresis (PEGE) for 4 minutes, followed by overnight at 4°C. The fragments were fractionated and detected using an ABI377 DNA sequencer.

[0251] Site-directed mutagenesis to change the amino acid Pro in TNV148 mAb 7 5 To replace the nucleotide sequence with a serine residue, a single nucleotide fragment of the TNV148 heavy chain variable region DNA sequence was inserted. The complementary oligonucleotides 399 and 400 (Table 1) were designed and prepared. QuikChange™ site-directed mutagenesis method as described by the manufacturer This change was made using the following method. Nucleotides were first fractionated and the major band purified. Either 10 ng or 50 ng 10 μl TNV148 heavy chain plasmid template (p1753), 5 μl 10X reaction buffer , 1 μl dNTP mix, 125 ng primer 399, 125 ng primer Prepare mutagenesis reaction using 400 and 1 μl Pfu DNA polymerase Sterile water was added to bring the total volume to 50 μl. The mixture was then heated at 95°C for 30 seconds, followed by Cycles of 95°C for 30 seconds, 55°C for 1 minute, 64°C for 1 minute, and 68°C for 7 minutes The procedure was repeated 14 times, followed by incubation at 30°C for 2 minutes (1 cycle). The reaction mix was incubated in a programmed thermal cycler. The mutagenic oligonucleotide was then introduced into an otherwise identical newly synthesized plasmid. To remove the original TNV148 plasmid, After adding 1 μl of DpnI endonuclease, which cuts only the purified plasmid, the sample was Incubated at 37°C for 1 hour. 1 μl of the reaction was then used in a standard heat shock. Epicurian Coli XL1-Blue supercompetent by the cloning method E. coli was transformed and plated on LB-ampicillin agar plates. The bacteria were identified using the Wizard™ kit as described by the manufacturer. Plasmid minipreps were prepared. Samples were eluted from Wizard™ columns. Afterwards, the plasmid DNA was further purified by precipitating it with ethanol, and then The clones were then resuspended in 20 μl of sterile water. DNA sequence analysis was then performed to identify clones with the desired base changes. We identified plasmid clones containing the TNV148 base change and determined whether other base changes were inadvertently introduced into the TNV148 coding sequence. Using the same parameters as described in Section 4.3, Mix 1 μl of plasmid, 3 μl of BigDye mix, and 1 μl of pUC19 forward primer. The PCR product was subjected to cycle sequencing reactions prepared with 10 μl of sterile water and 10 μl of primer.

[0252] Construction of an expression vector from the 12B75 gene. Several recombinant DNA steps were performed to from previously cloned genomic copies of the 12B75-encoded heavy and light chain genes. A new human IgG1 expression vector and a new human κ expression vector were prepared (these published as International Publication No. WO 02 / 12500, IL-12 Antibodies ,Compositions,Methods and Uses, 2000 No. 60 / 236,827, filed October 7, 2003, and incorporated by reference in its entirety. The final vector can be prepared by PCR amplification of any appropriately designed variable length vector. The regions were designed to allow for simple one-step replacement of existing variable region sequences.

[0253] To modify the 12B75 heavy chain gene of plasmid p1560, the promoter and variable The 6.85 kb BamHI / HindIII fragment containing the region was cloned from p1560 to pUC 19 to generate p1743. This plasmid is smaller in size compared to p1560. The smid was engineered to incorporate a unique BsiWI cleavage site immediately upstream of the translation start site according to the manufacturer's protocol. Use of QuikChange™ mutagenesis to introduce cloning sites ( The results were obtained using oligonucleotides BsiWI-1 and BsiWI-2. The resulting plasmid was designated p1747. A BstBI site was inserted at the 3' end of the variable region. The 5' oligonucleotide primer was inserted at the SalI and BstBI sites to introduce This primer was used together with the pUC reverse primer to clone p1747. A 2.75 kb fragment was amplified from the 12B75 variable region. The unique Bst The B1 site was introduced. The resulting intermediate vector, designated p1750, It was able to accept variable region fragments with iWI and BstBI ends. To prepare a version of the heavy chain vector derived from the B75 gene, the B75 gene was The mHI-HindIII insert contains an EcoRI site downstream of the HindIII site. The resulting plasmid, p1768, was then transferred to pBR322. , HindIII, and EcoRI, and the large BamHI- p1744, a subclone obtained by cloning a BamHI fragment The resulting 5.7 kb HindIII / EcoRI fragment was then ligated to the 5.7 kb HindIII / EcoRI fragment from The resulting plasmid, p1784, contains TNV Ab carrying BsiWI and BstBI ends. It was used as a vector for cDNA fragments. Additional work was carried out to clone the I gene from the 12B75 gene. It contains the gG1 constant region and depends on the extent to which it contains the 12B75 heavy chain JC intron. This was done to prepare the expression vectors p1788 and p1798, which differ from each other.

[0254] To modify the 12B75 light chain gene of plasmid p1558, the 12B75 promoter was inserted into A 5.7 kb SalI / AflII fragment containing the nucleotide and variable regions was extracted from p1558. The new plasmid p1745 was then transferred to the XhoI / AflII sites of plasmid L28. provided a smaller template for the mutagenesis step. (C340salI and C340sal2) using QuikChange™ Mutagenesis introduced a unique SalI restriction site at the 5' end of the variable region. The resulting intermediate vector, p1746, contains a unique SEQ ID NO: 1 vector into which variable region fragments can be cloned. p1746 contained ll and AflII restriction sites. The region fragment 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 The oligonucleotides BAHN-1 and BAHN-2 were annealed to each other to create the restriction site Bsi W1, containing AflII, HindII, and NotI, and KpnI and SacI sites A double-stranded linker containing the ends that could be ligated was formed. Cloning between the KpnI and SacI sites resulted in plasmid p1757. p1558 was digested with AflII and then partially digested with HindIII. The resulting 7.1 kb fragment containing the 12B75 light chain constant region was inserted into the Afl This new p1762 was cloned between the HindII and HindII sites. The plasmid contains the BsiW promoter and variable region, connecting the two halves of the gene. The I / AflII fragment contained unique BsiWI and AflII sites into which it could be transferred. .

[0255] cDNA cloning and assembly of expression plasmids. To further fill in the DNA ends Additionally, all RT-PCR reactions (see above) were treated with Klenow enzyme. The PCR fragment was digested with restriction enzymes BsiWI and BstBI and then cloned into plasmid L28(1 Since the 2B75-based intermediate vector p1750 had not yet been prepared, L28 was used. The cloned fragment was cloned between the BsiWI and BstBI sites of the vector. DNA sequence analysis of the insert confirmed that the resulting construct was correct and PCR amplified. These L28 plasmid constructs ( TNV14, TNV15, TNV148, TNV148B, and TNV196) The assigned identification numbers are shown in Table 3.

[0256] BsiWI / BstBI inserts in the heavy chains of TNV14, TNV148, and TNV148B The vector was transferred from the L28 vector to a newly prepared intermediate vector, p1750. The identification numbers assigned to these intermediate plasmids are shown in Table 2. The subsequent steps were not performed on TNV15 and TNV196. The vectors were then transferred into two different human IgG1 expression vectors. The vectors were then digested with the restriction enzymes EcoRI and HindI. II to clone the variable regions into Centocor's previously used IgG1 vector p1 The resulting IgG1 encoding the Gm(f+) allotype was transferred into The expression plasmids used were p1781 (TNV14), p1782 (TNV148), and p The variable region was designated 1783 (TNV148B) (see Table 2). Also cloned upstream of the IgG1 constant region derived from the B75 (GenPharm) gene These expression plasmids encoding IgG1 of the G1m(z) allotype were also shown. are listed in 3.

[0257] Table 3. Plasmid identification numbers for the various heavy and light chain plasmids. The L28 vector or pBC vector represents the initial Ab cDNA clone. The insert of this plasmid was inserted into an incomplete 12B75 vector to generate an intermediate plasmid. One additional transfer step allowed the vector to be linearized and then introduced into the cells. or used to purify mAb gene inserts prior to cell transfection This resulted in a final expression plasmid that was either:

[0258] [Table 5]

[0259] The light chain PCR product was digested with restriction enzymes SalI and SacII and then cloned into the plasmid pBC The two fragments differing in one amino acid were cloned between the SalI and SacII sites of The two light chain versions were designated p1748 and p1749 (Table 2). Sequence analysis confirmed that these constructs had the correct sequences. The SalI / AflII fragments of p1748 and p1749 were then ligated into the intermediate vector p1746. Cloning between the SalI and AflII sites gave p1755 and p1 These 5' halves of the light chain gene were then digested with BsiWI / AflII. Transfer the fragments from p1755 and p1756 to the newly prepared construct p1762. The 3' ends of the genes were joined by the ligation of the ligated nucleotides to form the final expression plasmids p1775 and p17 76 were produced (Table 2).

[0260] Cell transfection, screening, and subcloning. A total of 15 mouse Transfection of human myeloma cells was performed with various TNV expression plasmids (Results and (See Table 3 in the Discussion section.) These transfections are: (1) transfection of host cells; (2) the heavy chain constant region is a previous I of Centocor (3) the mAb was encoded with the gG1 vector or the 12B75 heavy chain constant region, or 148B, TNV148, TNV14, or the new HC / LC combination. (4) Whether the DNA is a linearized plasmid or a purified Ab gene insert and (5) the presence or absence of a complete JC intron sequence in the heavy chain gene. In addition, some of the transfections produced multiple clones. This was repeated to increase the likelihood of screening.

[0261] Sp2 / 0 cells and 653 cells were cultured under standard conditions as previously described (Knight DM et al. (1993) Molecular Immunology 30:1 443-1453), and heavy and light chain DNA (8- 12:g) mixture. Transfection numbers 1, 2, 3, For 16 and 17, proper expression was confirmed by digestion with restriction enzymes before transfection. The current plasmid was linearized using the SalI and NotI restriction enzymes, respectively. To linearize the V148B heavy chain plasmid p1783 and the light chain plasmid p1776 For the remaining transfections, the heavy chain plasmid was cleaved with BamHI and the Extract only the mAb gene by digesting the light chain plasmid with BsiWI and NotI. The DNA insert containing the nucleotide sequence was isolated from the plasmid vector. The mAb gene insert was purified by electrophoresis and Qiex purification resin. Cells transfected with gene inserts were cultured for 3-5 min as a source of selectable marker. co-transfected with PstI-linearized pSV2gpt plasmid (p13) After electroporation, the cells were cultured in IMDM, 15% FBS, 2% CO2 in a 96-well tissue culture dish. Cells were seeded in 10 mM glutamine and incubated at 37°C in a 5% CO2 incubator. After 2 days, the cells were incubated in an equal volume of IMDM, 5% FBS, 2 mM glutamine, and 2X MHX. Selection (1X MHX = 0.5 μg / ml mycophenolic acid, 2.5 μg / ml hyaluronan) Add xanthine (50 μg / ml xanthine) and continue for colony formation. The plates were incubated for 2 to 3 weeks.

[0262] Cell supernatants collected from wells with colonies were analyzed by ELISA as described. Briefly, polyclonal goat anti-human IgG was used. Various dilutions of cell supernatant were assayed in a 96-well EIA plate coated with Fc fragments. After incubation, alkaline phosphatase-conjugated goat anti-human IgG (H+L) and appropriate Bound human IgG was detected using an appropriate color substrate. A standard curve using purified mAb as a standard allowed for quantification of human IgG in the supernatant. The antibody that appeared to produce the most human IgG was included on each EIA plate. The cells in these colonies were plated in 24-well plates for further production determination in spent cultures. The cells were passaged into cultures to identify the highest producing parental clones.

[0263] The parent clone with the highest yield was subcloned to identify the subclones with higher yields. To prepare a more homogeneous cell line, a 96-well tissue culture plate was filled with IMDM, 5% F BS, 2mM glutamine, 1x MHX, 1 cell per well or 4 per well Seed the cells and incubate in a 5% CO2 incubator for 12-20 days until colonies appear. The wells contained one colony per well and were incubated at 37°C. The cell supernatant was collected from the culture and analyzed by ELISA as described above. After passage into well plates and exhaustion of the cultures, human IgG levels in their supernatants were measured. The highest producing subclones were identified by quantifying the number of clones. This was repeated when the selected first subclones were subjected to a second round of subcloning. The best subclone from the second round was selected as the cell line for development.

[0264] Characterization of cell subclones. The best subclones were selected from the second round and growth curves were performed. The mAb production level and cell growth characteristics were evaluated. 1x1 in MDM, 5% FBS, 2mM glutamine, and 1x MHX (or serum-free medium) 0 5 300 μl aliquots were removed at 24 hour intervals and viable cells were counted. The density was measured and the number of viable cells was 1 × 10 5 Analysis continued until the count was below cells / ml. Aliquots of the collected cell supernatants were assayed for the concentration of antibody present. ELISA assays were performed using rTNV148B or rTNV14JG92399. ELISA plates coated with polyclonal goat anti-human IgG Fc were used. The samples were incubated on a plate for 1 hour, and the bound mAb was then lysed in a 1:1000 dilution of alkaline phosphatase. Detection was performed with phatase-conjugated goat anti-human IgG (H+L).

[0265] To compare growth rates in the presence of various amounts of MHX selection agent, two cell lines were Different growth curve analyses were also performed on the cell lines C466A and C466B. The cells were thawed in IMDM, 5% FBS, and 2 mM glutamine and cultured for another 2 days. Both cell cultures were incubated with no MHX, 0.2X MHX, or 1X MHX (1X MHX = 0.5:g / ml mycophenolic acid, 2.5:g / ml hypoxanthine, 50:g The cultures were split into three cultures containing either 1000mg / ml xanthine or 1000mg / ml ethanol. One day later, new T7 5 flasks, 1 x 10 5 Seed the cultures at a starting density of 24 cells / ml and culture the cells for 1 week. Counts were taken at timed intervals. No aliquots were collected for mAb production. SOP PD The doubling times for these samples were calculated using the formula provided in 32.025.

[0266] Additional studies were performed to assess the stability of mAb production over time. IMDM, 5% FBS in 24-well plates, either with or without Cultures were grown in 2 mM glutamine. When the cultures were confluent, new The culture was split and the old culture was then allowed to drain. At this time, an aliquot of the supernatant was taken. The cells were removed and stored at 4°C. Aliquots were removed over a period of 55 to 78 days. At the end of the period, any antibodies present were assayed by anti-human IgG Fc ELISA as outlined above. The supernatant was tested for the amount of antibody present.

[0267] Results and Discussion. Inhibition of TNF binding to recombinant receptors. Eight TNV ​​mAbs contained in the hybridoma cell supernatant inhibited TNFα binding to the receptor. To determine whether the binding of ATP to ATP can be inhibited, a simple binding assay was performed. Standard ELISA analysis of human IgG revealed that TNV ​​mAbs in each cell supernatant were The concentration of the recombinant p55 TNF receptor / IgG fusion protein p55 was then determined. -sf2 was coated onto EIA plates 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, eight TNFα All but one of the mAbs (TNV122) inhibited the binding of TNFα to the p55 receptor. In fact, TNV mAb spiked into negative control hybridoma supernatants effectively blocked It appeared to be more effective at inhibiting TNFα binding than the cA2 positive control mAb. These results demonstrate that TNV ​​mAbs inhibit the biological activity of TNFα in cell-based assays and in vivo. This indicates that the hypothesis is likely to block the ability to communicate and therefore requires further analysis. Interpreted.

[0268] DNA sequence analysis. Confirmation that the RNA encodes a human mAb. Seven TNV mAbs (TNV 14, TNV15, TNV32, TNV86, TNV118, TNV148, and TNV As a first step in characterizing the seven hybrids that generate these mAbs, Total RNA was isolated from the dorsal cell line. Each RNA sample was then used to assay the complete length of each mAb. a human antibody heavy chain containing a signal sequence, a complete variable region sequence, and a portion of a constant region sequence; Light chain cDNA was prepared. These cDNA products were then amplified by PCR. PCR-amplified DNA was sequenced directly without cloning the fragment into The heavy chain cDNA is a DP- 46 (Fig. 2). Similarly, the sequenced light chain cDNA was It is either 100% or 98% identical to one of the existing human germline genes. These sequence results indicate that the RNA molecules transcribed into cDNA and sequenced are human. It was confirmed that the antibody heavy chain and human antibody light chain were encoded. The sequence was PCR amplified using an oligonucleotide that maps to the 5' end of the However, the first few amino acids of the signal sequence may not be the actual sequence of the original TNV translation product. It should be noted that although it is possible that the sequences represent the actual sequences of the recombinant TNV ​​mAbs.

[0269] Specific neutralizing mAb. Analysis of the cDNA sequences of the entire variable regions of both the heavy and light chains of each mAb revealed that TNV32 is a T TNV15 is identical to TNV15, TNV118 is identical to TNV14, and TNV86 is identical to TNV1 The results of the receptor binding assay were consistent with DNA sequence analysis. Consistent with this finding, both TNV86 and TNV148 were effective in blocking TNF binding. It was approximately four times better than both TNV118 and TNV14. are four specific TNV mAbs, TNV14, TNV15, TNV148, and We focused only on TNV196.

[0270] Relationships between the four mAbs DNA sequencing results showed that the genes encoding the heavy chains of the four TNV mAbs were all highly homologous to each other. It was revealed that they are highly homologous and all appear to derive from the same germline gene, DP-46. In addition, each of the heavy chain CDR3 sequences was highly similar and of the same length. Because they all use the J6 exon, they are clearly single These arise from VDJ gene rearrangement events followed by somatic changes that make each mAb unique. DNA sequence analysis revealed that only two distinct light chain genes were present in the four mAbs. The light chain variable region coding sequences of TNV14 and TNV15 were shown to be identical (Figure 3). The sequences are identical to each other and are 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 The germline sequences differ at two nucleotide positions (Fig. 3).

[0271] The deduced amino acid sequences of the four mAbs revealed the relatedness of the actual mAbs. Abs contain four distinct heavy chains (Figure 4) but only two distinct light chains (Figure 5) The differences between the TNV mAb sequence and the germline sequence are mostly confined to the CDR domains. Although three of the mAb heavy chains also differed from the germline sequence in the framework regions, Compared with the DP-46 germline-encoded Ab framework region, the TN V14 is identical, TNV15 differs by one amino acid, and TNV148 differs by two amino acids. TNV196 differed in three amino acids, while TNV197 differed in four amino acids.

[0272] cDNA cloning, site-directed mutagenesis, and assembly of final expression plasmids cDNA cloning. Based on the DNA sequence of the PCR-amplified variable region, For the purpose of adapting the coding sequence to be expressed into an expression vector, new oligonucleotides was ordered to perform another PCR amplification. For the heavy chain, the product of this second PCR was The plasmid vector L28 (shown in Table 2) was digested with the restriction enzymes BsiWI and BstBI. For the light chain, the second PCR product was cloned into It was digested with SalI and AflII and cloned into the plasmid vector pBC. Individual clones are then sequenced to identify the sequences at each site of a potentially heterogeneous population of molecules. The results obtained from direct sequencing of PCR products reveal the most abundant nucleotide at the position It was confirmed that the sequence was identical to the previous one.

[0273] Site-directed mutagenesis to modify TNV148. mAb TNV148 and TNV 196 was 4.5 times more potent than the next best mAb (TNV14) in neutralizing TNFα bioactivity. However, as mentioned above, TNV148 and The TNV196 and TNV196 heavy chain framework sequences differ from the germline framework sequences. Comparison of the NV148 heavy chain sequence with other human antibodies shows that many other human mAbs share the same framework. 1 contains an Ile residue at position 28 (only the mature sequence is counted), while position 3 of framework 1 The Pro residue at position 75 was shown to be a rare amino acid at that position.

[0274] A similar comparison of the TNV196 heavy chain reveals three regions in framework 3 that differ from the germline sequence. These differences suggest that amino acids may be rare in human mAbs. When administered, TNV148 and TNV196 could be immunogenic. 148 has only one amino acid residue of interest, which is important for TNFα binding. Therefore, site-directed mutagenesis techniques were used to remove the germline Ser residue. The TNV148 heavy chain coding sequence was modified so that the PG residue was coded for in place of the Pro residue at position 75. A single nucleotide in the sequence (of plasmid p1753) was changed. The resulting plasmid was designated p1760 (see Table 2). The mAb was labeled TNV148B to distinguish it from the original TNV148 gene and mAb. called (see Figure 5).

[0275] Assembly of the final expression plasmid. 12B7 previously cloned as a genomic fragment Five new antibody expression vectors based on heavy and light chain genes were prepared. Plasmids were prepared (see Table 2), in each case containing a 5' furan The 12B75 gene contains a transcription sequence, promoter, and intron enhancer. For the light chain expression plasmid, the complete JC intron, constant region coding sequence The 3' flanking sequence was also derived from the light chain gene of 12B75. Regarding the heavy chain expression plasmids that gave rise to p1781 and p1783 (see below), The human IgG1 constant region coding sequence was expressed in the expression vector used before Centocor. Importantly, the final product cell line reported here was derived from the original hybrid (p104). A different allotype (Gm(f+)) from the hybridoma-derived TNV mAb (G1m(z)) ) expresses the TNV mAb 12B75 derived from GenPharm mice. The heavy chain gene encodes an Arg residue at the C-terminal end of the CH1 domain, but This is because the IgG1 expression vector p104 encodes a Lys residue at that position. The JC intron, the complete constant region coding sequence, and the 3' flanking sequence are 12B75 Other heavy chain expression plasmids (e.g., p1786 and p1788) derived from the heavy chain gene The cell lines transfected with these genes were selected as the production cell lines. The vector was then PCR amplified to yield the final expression plasmid. It was carefully designed to allow one-step cloning.

[0276] PCR-amplified variable region cDNA was extracted from L28 or pBC vectors to contain the promoter region and The JC intron was transferred to an intermediate 12B75-based vector (plus (See Table 2 for sequence identification numbers.) Next, a recombinant vector containing the 5' half of the antibody gene is inserted. The restriction fragments were extracted from these intermediate vectors into the final vectors providing the 3' halves of each gene. The final expression vector was then transferred to the final expression plasmid (see Table 2 for plasmid identification numbers). This formed a network of companies.

[0277] Cell transfection and subcloning. Expression plasmids were purified by restriction digestion. Either the antibody gene insert in each plasmid was linearized by the plasmid backbone or Sp2 / 0 and 653 mouse myeloma cells were either purified from electroporated or electroporated. The 15 different genes were transfected with heavy and light chain DNA by troporation. Various transfections were performed, most of which were specific as defined by Abs. and whether the gene is on a linearized whole plasmid or a purified gene insert. These were specific characteristics of the Ab gene and the host cell line, regardless of whether they were present or absent (summarized in Table 4). Cell supernatants from clones resistant to mycophenolic acid were analyzed for the presence of human IgG. Assayed by ELISA using purified rTNV148B as a reference standard curve. was used for quantification.

[0278] Highest producing rTNV148B cell line One of the 653 highest producing parental lines from rTNV148B transfection 2 0 (yielding 5-10 μg / ml in spent 24-well cultures) To screen for higher producing cell lines, a more homogenous cell population was prepared. Two of the subclones of parent strains 2.320, 2.320-17, and 2.320-20 produced approximately 50 μg / ml in spent 24-well cultures, which is higher than their parent strains The subcloned strains 2.320-17 and 2.320 This was the result of a second subcloning of -20.

[0279] The identification numbers of the heavy and light chain plasmids encoding each mAb are shown. Purified mAbs For transfections performed with gene inserts, the source of the gpt selection marker The heavy chain constant region was derived from Remica The same human IgG1 expression vector ("old") used to encode de The constant region contained within the B75 (GenPharm / Medarex) heavy chain gene ("New H1 / L2 is encoded by either the TNV14 heavy chain or the TNV14 The plasmids p1783 and p1801 are the "novel" mAb composed of eight light chains. These heavy chain genes differ only in the extent to which they contain JC introns. The transfection number, which defines the first digit of the loan gene name, is shown on the right. The rTNV148B-producing cell lines C466 (A, B, C, D) and C 467A were derived from transfections no. 2 and 1, respectively. rTNV14 generation Cell line C476A was derived from transfection no. 3.

[0280] [Table 6]

[0281] ELISA assays on spent 24-well culture supernatants were performed using these second subclones. All clones produced 98-124 μg / ml, which is comparable to the initial subclones. These 653 cell lines showed at least a two-fold increase in the expression of 653 cells, as shown in Table 5. C chord notation assigned.

[0282] Of the Sp2 / 0 parental strains, the highest yield was from rTNV148B transfection 1 Two subclonings of the parent strain 1.73 were performed using the spent 24-well plate. This led to the identification of a clone that produced 25 μg / ml in well cultures. The / 0 cell line was designated C467A (Table 5).

[0283] Highest producing rTNV14 cell line Three of the highest yielding Sp2 / 0 parental lines from rTNV14 transfection 3 Subclone 3.27-1 was subcloned once. The production was 19 μg / ml. This cell was found to be the highest producer in the spent 24-well culture. The strain was designated C476A (Table 5).

[0284] 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-encoding cell lines reported herein contain heavy and light chains linearized with restriction enzymes. It was derived from transfection with all plasmids.

[0285] [Table 7]

[0286] Characterization of subcloned cell lines To more carefully characterize cell line growth characteristics and determine mAb production levels at large scale Growth curve analysis was performed using T75 cultures. Results showed that the four C466 series of cell lines Each of the sizes is 1.0 x 10 6 ~1.25×10 6 Peak cell density in cells / ml and 110 A maximum mAb accumulation level of ∼140 μg / ml was achieved (Figure 7). The highest producing Sp2 / 0 subclone, C467A, produced 2.0 × 10 6 Peak cells / ml The cell density and maximum mAb accumulation level of 25 μg / ml were reached (Figure 7). Growth curve analysis , was not performed on the rTNV14-producing cell line C476A.

[0287] Further growth curve analysis was performed to compare growth rates at different concentrations of MHX selection. This comparison shows that C466 cells cultured in the absence of MHX are able to grow with normal amounts of MHX (1X). prompted by recent observations that cells appear to grow faster than the same cells in culture. Cytotoxic concentrations of compounds such as mycophenolic acid tend to be measured over orders of magnitude. Therefore, using a lower concentration of MHX may compromise the stability of mAb production. It was thought that it would be possible to significantly accelerate the cell doubling time without using the C466 cell line. A and C466B were cultured either without MHX, with 0.2X MHX, or with 1X MHX. Viable cell counts were performed every 24 hours for 7 days. The cell line C466A showed a cell growth rate dependent on 1× MHX (Figure 8). With MHX, the doubling time was 25.0 hours, but without MHX, the doubling time was only 20.7 hours. Similarly, cell line C466B had a doubling time of 32.4 hours in 1X MHX. showed a doubling time of only 22.9 hours without MHX. The doubling times of both cell lines in 2X MHX were significantly longer than those observed without MHX compared to 1X MHX. This observation is consistent with the finding that doubling time is an important parameter. Enhanced cell performance by using less MHX in bioreactors However, the stability study results (see below) suggest that The cell line C466D stably expresses rTNV148B for at least 60 days in the absence of MHX. Although stability studies also suggest that MHX can be produced in the absence of MHX, Accordingly, cells also showed higher levels of mAb production when cultured in the presence of MHX.

[0288] To evaluate mAb production from various cell lines over a period of approximately 60 days, MHX Stability studies were performed on cultures either with or without selection. After just 2 weeks of culture, clone C4 The production of 66A was approximately 45% lower than at the start of the study. The production from clone C466B was also However, clones C466C and C466D The production remained fairly stable, with C466D showing the highest absolute production level (FIG. 9).

[0289] conclusion From an initial panel of eight human mAbs against human TNFα, protein sequences and TNFα were identified. Based on several criteria, including neutralization potency, TNV148B as well as TNV14 are preferred. The rTNV148B was selected as a rTNV148B antibody with a concentration of over 100 μg / ml and a rTNV148B antibody with a concentration of over 19 μg / ml. A cell line producing TNV14 was prepared.

[0290] Example 5: Arthritic Mice with Anti-TNF Antibody and Control Using a Single Bolus Injection Research on Approximately 4-week-old Tg197 research mice were assigned to one of nine treatment groups based on sex and weight. Assignment, Dulbecco's PBS (D-PBS), or 1 mg / kg or 10 m g / kg of any of the anti-TNF antibodies of the present invention (TNV14, TNV148, or TN V196) as a single intraperitoneal bolus dose.

[0291] Results: When body weight was analyzed as a change from pre-treatment, treatment with 10 mg / kg cA2 Animals treated with D-PBS consistently gained more weight than animals treated with D-PBS throughout the study. 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).

[0292] Figures 11A-C show the progression of disease severity based on the arthritis index. The arthritis index for the A2-treated group was measured starting at week 3 and throughout the remainder of the study (week 7). ) lower than the D-PBS control group. Animals treated with 1 mg / kg TNV14 and 1 m Animals treated with cA2 at 100 mg / kg had significantly improved A2 levels from week 3 onwards compared to the D-PBS treated group. No significant reduction in I was observed. (c at 10 mg / kg compared with 10 mg / kg TNV14, 148, and 196) A2) There was no significant difference between the 10 mg / kg and 1 mg / kg treatment groups. When the 1 mg / kg dose of TNV148 was administered, the 3, 4, and 7 week TNV148 at 1 mg / kg also showed a significantly lower AI than TNV14 at 1 mg / kg. TNV196 was significantly lower in the AI ​​than the control group at weeks 3 and 4. Although TNV148 showed a significant decrease in the IL-1 receptor agonist activity (when compared with the D-PBS-treated group), This was the only 1 mg / kg treatment group that remained significant at the end of the study.

[0293] Example 6: Study of arthritic mice using anti-TNF antibodies and controls as multiple bolus doses research Approximately 4-week-old Tg197 study mice were assigned to one of eight treatment groups based on body weight. , control (D-PBS), or 3 mg / kg of antibody (TNV14, TNV148) (week 0) All animals were treated with an intraperitoneal bolus injection of 100 mg / kg ... Groups 1-6 were evaluated for efficacy of the test article. Animals in groups 7 and 8 Serum samples obtained from the study were analyzed for the induction of immune responses to TNV14 or TNV148 at 2, 3, and 4 weeks. and pharmacokinetic clearance were evaluated.

[0294] Results: When body weight was analyzed as a change from pre-treatment, no significant difference was observed. Animals treated with cA2 at 200 mg / kg consistently outperformed D-PBS-treated animals throughout the study. They showed high weight gain (see Figure 12).

[0295] Figures 13A-C show the progression of disease severity based on the arthritis index. The arthritis index for the A2-treated group was measured starting at week 2 and throughout the remainder of the study (week 5). ) was significantly lower than the D-PBS control group. Animals placed in the sham group and treated with 3 mg / kg TNV14 showed significantly increased schizophrenia compared to the d-PBS control group. were able to achieve any significant reduction in AI at any time point throughout the study. Animals treated with 3 mg / kg TNV148 showed no significant improvement compared to the d-PBS treated group. When administered at 10 mg / kg, a significant reduction was observed, beginning at week 3 and continuing through week 5. Animals treated with cA2 received lower doses of both cA2 (1 mg / kg) at weeks 4 and 5 of the study. and 3 mg / kg), a significant decrease in AI was observed, and The mean mean age of the 3 mg / kg group was significantly lower than that of the 3 mg / kg group treated with TNV14. There appeared to be no significant difference in the AI ​​for animals treated with 3 mg / kg TNV14. , significantly higher than 10 mg / kg at some time points, while animals treated with TNV148 showed was not significantly different from animals treated with 0 mg / kg cA2.

[0296] Example 7: Arthritic Mice Using Anti-TNF Antibodies and Controls as a Single Intraperitoneal Bolus Research on Approximately 4-week-old Tg197 research mice were assigned to one of six treatment groups based on sex and weight. Assigned to either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148) The study was conducted with D-PBS and 10 mg / kg c The A2 control group was utilized.

[0297] When body weight was analyzed as a change from pre-treatment, all treatments achieved similar weight gain. Treatment with either 3 or 5 mg / kg TNV148 or 5 mg / kg cA2 Animals treated with TNV148 gained significantly more body weight early in the study (weeks 2 and 3). Only animals receiving TN at 3 and 5 mg / kg maintained significant weight gain at later time points. Both V148-treated animals showed significant improvement at week 7, with 3 mg / kg TNV148 Animals treated with α-glucan still had significant increases 8 weeks after injection (see Figure 14).

[0298] Figure 15 depicts the progression of disease severity based on the arthritis index. All treatment groups showed a significant improvement at the initial time point. cA2 at 5 mg / kg and TNV148 at 5 mg / kg showed some protection, while cA2 at 5 mg / kg and TNV148 at 1 to 3 All treatment groups showed a significant reduction in AI at week 1, and all treatment groups showed a significant reduction at week 2. Later in the study, animals treated with 5 mg / kg cA2 showed some protection, with Both cA2 and TNV148 at low doses (3 mg / kg) significantly reduced the At the end of the study, all treatment groups showed a significant reduction in schizophrenia at week 1 and week 7. (Week 8) None of the treatment groups were able to sustain a significant reduction. There were no significant differences between any of them (except the saline control group).

[0299] Example 8: Single intraperitoneal bolus administration of anti-TNF antibody and modified anti-TNF antibody Studies in arthritic mice using anti-TNF antibodies and controls TNV148 (derived from hybridoma cells) and rTNV148B (transfected To compare the efficacy of a single intraperitoneal injection of Tg mice (derived from transfected cells) with that of Tg mice approximately 4 weeks of age. The 197 study mice were assigned to one of nine treatment groups based on sex and weight. Beckmann's PBS (D-PBS) or 1 mg / kg of antibody (TNV148, rTNV 148B) was administered as a single intraperitoneal bolus.

[0300] When body weight was analyzed as a change from pre-treatment, animals treated with 10 mg / kg cA2 showed consistently higher weight gain than D-PBS treated animals throughout the study. The increase was significant at week 1 and weeks 3 to 8. Animals treated with 1 mg / kg TNV148 Subjects also achieved significant weight gain at weeks 5, 6, and 8 of the study (see Figure 16). .

[0301] Figure 17 shows the progression of disease severity based on the arthritis index. The arthritis index for the treated group was D-1 starting at week 4 and throughout the remainder of the study (week 8). The TNV148-treated group and the 1 mg / kg cA2-treated group showed lower levels than the PBS control group. Both groups showed a significant decrease in AI at week 4. 9-017) showed that TNV148 improved arthritis index after a single 1 mg / kg intraperitoneal bolus. This study showed that both versions of TNV The AI ​​from the antibody-treated group was slightly higher. Treatment with 1 mg / kg cA2 The groups treated with cA2 (except for week 6) did not show a significant increase when compared with the 10 mg / kg cA2 group. The TNV148-treated group had significantly higher cA at 7 and 8 weeks, but the 1 mg / kg 2. Between 1 mg / kg TNV148 and 1 mg / kg TNV148B, there is no difference in the There was no significant difference in AI at this time point.

[0302] Example 9: Anti-TNF antibodies for the treatment of active psoriatic arthritis summary Efficacy of intravenously administered anti-TNFα monoclonal antibody in subjects with active psoriatic arthritis (PsA) A multicenter, randomized, double-blind, placebo-controlled study of the monoclonal antibody golimumab

[0303] SIMPONI® (golimumab) binds to the immunoglobulin G1 (IgG1) heavy chain Fully human monoclonal antibody with isotype (G1m[z] allotype) and kappa light chain isotype Golimumab is a clonal antibody. It has a heavy chain (HC) comprising SEQ ID NO: 36 and a heavy chain (HC) comprising SEQ ID NO: 3 The light chain (LC) contains 7. The molecular weight of golimumab is 149,802 to 151,06 Golimumab is a human tumor necrosis factor receptor agonist with high affinity and specificity. It binds to tumor necrosis factor alpha (TNFα) and neutralizes TNFα biological activity.

[0304] Objectives and Hypothesis Main purpose The primary objective of this study was to evaluate the effectiveness of active dry eye treatment by assessing the reduction of signs and symptoms of PsA. Efficacy of IV golimumab 2 mg / kg in subjects with psoriatic arthritis (PsA) The purpose is to evaluate the following.

[0305] Secondary Objectives Secondary objectives are to evaluate IV golimumab's: Associated with improvements in psoriatic skin lesions, physical function, health-related quality of life, and other health outcomes Effectiveness Inhibition of progression of structural damage ·Safety Pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity

[0306] hypothesis To address the primary objective of the study, the statistical hypothesis (alternative hypothesis) was golimumab 2 mg / kg reduced signs and symptoms in subjects with active PsA based on the primary efficacy endpoint. It is statistically superior to placebo in reducing

[0307] The primary endpoint of the study was the American College of Rheumatology (ACR20) score at 14 weeks. This is the proportion of subjects achieving a 20% improvement from baseline in the The endpoints are well accepted by regulatory authorities and the clinical PsA community. was selected because

[0308] Overview of study design This is a comparison of the efficacy of IV golimumab compared with placebo in subjects with active PsA. This is a phase 3, multicenter, randomized, double-blind, placebo-controlled study of efficacy and safety. 0 subjects will be randomized at approximately 90 sites. Subjects will be randomized at 0, 4, 12, and 2 sites. Randomly assigned to receive golimumab 2 mg / kg or placebo IV infusion at week 0 At week 16, all subjects eligible for early withdrawal will be withdrawn as selected by the investigator. One of the following concomitant medications is permitted: Total dose of prednisone 10 mg / day or equivalent), methotrexate (MTX) dose ( Maximum total dose 25 mg / week), or increase in NSAID dose, or NSAID, corticosteroids Steroids (maximum dose of prednisone 10 mg / day or equivalent), MTX (maximum dose of 25 mg / week), SSZ (maximum dose 3g / day), HCQ (maximum dose 400mg / day), or Initiation of flunomide (maximum dose 20 mg / day). Titration to stable doses of these drugs is recommended. To be completed for subjects who are early withdrawal candidates by the Week 4 visit. All subjects receiving a placebo infusion will cross over and begin receiving a golimumab IV infusion.

[0309] Subjects in the golimumab IV treatment group will continue to receive golimumab IV infusion. Database locks (DBL) are scheduled at 24 and 60 weeks. Patients were followed for adverse events (AEs) and serious adverse events (SAEs) for at least 8 weeks after study treatment administration. The end of the study was defined as when the last subject completed the 60-week visit. can be.

[0310] Target population Eligible subjects were 18 years or older with PsA for at least 6 months before the first dose of study medication. Male or female subjects aged 18+ years who meet the CASPAR criteria at screening. Symptoms of active disease (≥5 swollen joints and ≥5 swollen joints) at screening and baseline or more tender joints) and a C-reactive protein (CRP) level ...

Claims

1. A composition comprising at least one isolated mammalian anti-TNF antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37, and at least one pharmaceutically acceptable carrier or diluent, for use in the safe and effective treatment of active psoriatic arthritis in a patient, wherein the composition is administered via IV infusion, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg over 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter, the composition comprising an aqueous vehicle comprising histidine, sorbitol, and polysorbate 80 at a pH of 5.5, and wherein at 14 weeks of treatment the patient has: HAQ-DI=-0.60±0.53 SD; enthesitis=-1.87±1.75 SD; dactylitis=-7.8±8.57 SD; SF-36 A composition that achieves a mean change from baseline in one or more criteria selected from the group consisting of PCS=8.65±7.60 SD, and SF-36 MCS=5.33±9.95 SD.

2. The composition of claim 1, wherein the antibody is administered with or without methotrexate (MTX).

3. A composition comprising at least one isolated mammalian anti-TNF antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37, and at least one pharmaceutically acceptable carrier or diluent, for use in the safe and effective treatment of active psoriatic arthritis in a patient, wherein the composition is administered via IV infusion, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg over 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter, the composition comprising an aqueous vehicle comprising histidine, sorbitol, and polysorbate 80 at a pH of 5.5, and wherein at 24 weeks of treatment, the patient achieves a mean change from baseline in vdH-S=-0.36±0.144 SE.

4. The composition of claim 3, wherein the antibody is administered with or without methotrexate (MTX).

5. A pharmaceutical composition for use in the safe and effective treatment of active psoriatic arthritis in a patient, comprising at least one isolated mammalian anti-TNF antibody having a heavy chain (HC) comprising SEQ ID NO: 36 and a light chain (LC) comprising SEQ ID NO: 37, wherein the pharmaceutical composition is used such that the anti-TNF antibody is administered via intravenous (IV) infusion, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg over 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter, wherein the composition comprises an aqueous vehicle comprising histidine, sorbitol, and polysorbate 80 at a pH of 5.5, wherein 65% or more of patients receiving the treatment achieve an ACR20 at week 14 of treatment.

6. The pharmaceutical composition of claim 5, wherein 65% or more of the patients achieve ACR20 at 14 weeks of treatment with a treatment difference (improvement compared to placebo) of 50% or more.

7. The pharmaceutical composition of claim 5 or 6, wherein the antibody is administered with or without methotrexate (MTX).

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