Anti-TNF antibodies, compositions, and methods for the treatment of active ankylosing spondylitis

Abstract

To provide a composition for use in the safe and effective treatment of active Ankylosing Spondylitis (AS).SOLUTION: The present invention provides 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 a patient treated with the composition achieves an ASDAS inactive disease (<1.3) at 4 weeks of treatment or at 2 weeks of treatment.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides a safe and effective treatment for active ankylosing spondylitis (AS). A heavy chain (HC) comprising SEQ ID NO: 36 and a heavy chain comprising SEQ ID NO: 3 for use in therapy. Compositions and methods using anti-TNF antibodies having light chains (LC) including 7 Regarding. [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 nonmonocytic tumor cells The cell line produced TNFα and CD4+ and CD8+ peripheral blood T lymphocytes, and several cultures The selected T and B cell lines also produce TNFα.

[0004] TNFα induces cartilage and bone degradation, induction of adhesion molecules, and procoagulant activity in vascular endothelial cells , increases adhesion of neutrophils and lymphocytes, and inhibits adhesion of macrophages, neutrophils and blood vessels Proinflammatory 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 conditions, autoimmune conditions, and graft-versus-host conditions. 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". Progressive weight loss, anorexia, and persistent loss of lean body mass in response to malignant tumor growth The cachectic state is responsible for many cancer morbidity and mortality. TNFα promotes the development of cancer, infection, and There is evidence that it is involved in cachexia in sexual pathologies and other catabolic states.

[0007] TNFα is a potent inhibitor of gram-negative sepsis and endotoxin, which can cause symptoms such as fever, fatigue, anorexia, and cachexia. Endotoxin is thought to play a central role in acute shock. Strongly activates phage production and secretion of TNFα and other cytokines. and other monocyte-derived cytokines mediate metabolic and neurohormonal responses to endotoxin Endotoxin administration to human volunteers produces fever, tachycardia, increased metabolic rate, and stress. This leads to acute illness with flu-like symptoms, including the release of TNFα. Circulating IgG is increased in patients with gram-negative sepsis.

[0008] Thus, TNFα plays a key role in the treatment of inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections. It has been linked to rheumatoid arthritis, chronic kidney disease, malignancies, and / or neurodegenerative diseases, and is It is a useful target for certain biological treatments in diseases such as Crohn's disease. Beneficial effects of anti-inflammatory drugs in open-label studies using a human monoclonal antibody (cA2) 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 with cA2 have also It has also been reported to suppress inflammation in utero.

[0009] Other investigators have described mAbs specific for recombinant human TNF that had neutralizing activity in vitro. Several of these mAbs have been shown to map epitopes on human TNF. used to develop enzyme immunoassays and to aid in the purification of recombinant TNF However, these studies have been hampered by issues such as immunogenicity, low specificity, and / or pharmaceutical unsuitability. The properties of the TNF-neutralizing antibodies make them suitable for in vivo diagnostic or therapeutic use in humans. It does not provide a basis for generating the body.

[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. This is shown in the system.

[0011] The putative receptor binding locus for hTNF has been disclosed, which corresponds to amino acids 11-13 and 37 of TNF. 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 (e.g., proteolytic digests or fusion protein production thereof) In some cases, potential drugs that are being investigated in an attempt to treat a particular disease are However, such antibodies or fragments may not 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 result in clearance, making repeated administration unsuitable for treatment, thereby causing patient This reduces the therapeutic benefit to the patient 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, as is well known in the art, is used. Many approaches have been developed to reduce the immunogenicity of such antibodies and portions thereof, including antibody synthesis and humanization. However, these and other approaches still involve some immunogenicity. have low affinity, low binding activity, or are difficult to culture, scale up, produce, and / or may result in antibodies or fragments with problems in low yield. Fragments may not be ideally suited for production or use as therapeutic proteins. There is. 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 relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being administered intravenously (IV), In the present invention, the anti-TNF antibody is administered by injection, and the patient treated with the anti-TNF antibody is At least one isolated feeding achieving ASDAS inactive disease (<1.3) at week 1 Anti-TNF antibodies are provided.

[0015] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being administered at weeks 0 and 4, followed by IV at a dose of 2 mg / kg over 30 ± 10 minutes every 8 weeks (q8w) ) by infusion, and in patients treated with the anti-TNF antibody, At least one isolated animal achieving ASDAS inactive disease (<1.3) at week 2 Mammalian anti-TNF antibodies are provided.

[0016] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the antibody being administered with methotrexate (MT X), sulfasalazine (SSZ) or hydroxychloroquine (hydroxy The anti-TNF antibody is administered intravenously ( IV) administered by infusion, in which patients treated with the anti-TNF antibody meet the criteria for the fourth or final week of treatment. At least one isolated schizophrenia patient achieved ASDAS inactive disease (<1.3) at week 2 of treatment. The present invention provides a mammalian anti-TNF antibody.

[0017] The present invention provides a compound of SEQ ID NO:3 for use in the safe and effective treatment of active ankylosing spondylitis. At least one single antibody having a heavy chain (HC) comprising SEQ ID NO:6 and a light chain (LC) comprising SEQ ID NO:37. an isolated mammalian anti-TNF antibody and at least one pharma- ceutically acceptable carrier or diluent; wherein the composition is administered by IV infusion and a patient is treated with the composition. Patients who achieved ASDAS inactive disease (<1.3) at week 4 of treatment or week 2 of treatment The present invention provides a composition comprising:

[0018] The present invention provides a compound of SEQ ID NO:3 for use in the safe and effective treatment of active ankylosing spondylitis. At least one single antibody having a heavy chain (HC) comprising SEQ ID NO:6 and a light chain (LC) comprising SEQ ID NO:37. an isolated mammalian anti-TNF antibody and at least one pharma- ceutically acceptable carrier or diluent; wherein the composition is administered at weeks 0 and 4, and then every 8 weeks thereafter (q 8 weeks) at a dose of 2 mg / kg administered by IV infusion over 30 ± 10 minutes. Patients treated with the composition showed no ASDAS inactive disorder (< 1.3) A composition is provided that achieves the above.

[0019] The present invention provides a compound of SEQ ID NO:3 for use in the safe and effective treatment of active ankylosing spondylitis. At least one single antibody having a heavy chain (HC) comprising SEQ ID NO:6 and a light chain (LC) comprising SEQ ID NO:37. an isolated mammalian anti-TNF antibody and at least one pharma- ceutically acceptable carrier or diluent; , wherein the composition comprises methotrexate (MTX), sulfasalazine. It is administered with or without SSZ or hydroxychloroquine (HCQ), The composition is administered IV and patients treated with the composition are A composition is provided that achieves an ASDAS inactive disorder (<1.3) at 2 weeks.

[0020] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. administering a composition to a subject, the composition being administered by IV infusion, Patients who received the treatment achieved ASDAS inactive disease (<1.3) at week 4 of treatment or week 2 of treatment. Provide a method for achieving this.

[0021] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. administering the composition at weeks 0 and 4, and then every 8 weeks thereafter (q8w) at a dose of 2 mg / kg administered by IV infusion over 30 ± 10 minutes, Patients treated with the composition have a history of ASDAS inactive disorder at week 4 of treatment or week 2 of treatment. Provide a method to achieve (<1.3).

[0022] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. Administering a composition, the composition being methotrexate (MTX), sulfasara It was administered with or without serotonin-stimulating hormone (SSZ) or hydroxychloroquine (HCQ), The composition was administered at 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter. The composition was administered by IV infusion at a dose of 2 mg / kg over a period of 10 days, and patients treated with the composition , achieving ASDAS inactive disease (<1.3) at week 4 of treatment or week 2 of treatment. to provide.

[0023] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. administering a composition to a subject, the composition being administered by IV infusion, Patients who received the treatment achieved ASDAS inactive disease (<1.3) at week 4 of treatment or week 2 of treatment. and the method comprises the step of adding at least one detectable label or reporter, TNF antagonist, antirheumatic drug, muscle relaxant, narcotic, nonsteroidal anti-inflammatory drug (non-steroid anti-inflammatory drug, NSAID), analgesic, anesthetic, sedative, topical Anesthetics, neuromuscular blocking agents, antibacterial agents, antipsoriatic agents, corticosteroids, anabolic steroids id, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, hormonal Month replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma beta agonists, Inhaled steroids, epinephrine or analogs, cytokines, or cytokine antagonists and at least one compound or protein selected from the group consisting of an agonist and an anti-inflammatory drug. The method further comprises administering another composition.

[0024] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being administered by intravenous (IV) infusion; At 16 weeks of treatment, patients treated with the anti-TNF antibody had a history of Bath ankylosing spondylitis. Bath Ankylosing Spondylitis Functional Index (BASFI) = - 2.4±2.1 standard deviation (SD), Bath ankylosing spondylitis measurement index Bath Ankylosing Spondylitis Metrology Index (BASMI) = -0.4± 0.6SD, 36-item Short-Form Health Survey P hysical Component Summary, SF-36 PCS)=8.5±7.5SD, 36 items omitted 36-item Short-Form Health Survey Mental Component Summar y, SF-36 MCS) = 6.5 ± 9.1 SD, and ankylosing spondylitis quality of life (Ankylos The group consisting of ing Spondylitis Qualification of Life (ASQoL) = -5.4 ± 5.0 SD achieve a mean change from baseline in one or more criteria selected from the following: At least one isolated mammalian anti-TNF antibody is provided.

[0025] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being administered at weeks 0 and 4, followed by IV at a dose of 2 mg / kg over 30 ± 10 minutes every 8 weeks (q8w) ) infusion, and at 16 weeks of treatment, patients treated with the anti-TNF antibody showed BA SFI=-2.4±2.1SD, BASMI=-0.4±0.6SD, SF-36 PC S = 8.5 ± 7.5 SD, SF-36 MCS = 6.5 ± 9.1 SD, and ASQoL = -5.4±5.0SD based on one or more criteria selected from the group consisting of Provide at least one isolated mammalian anti-TNF antibody that achieves a mean change from the line. Provide.

[0026] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being selected from methotrexate (MTX), With or without sulfasalazine (SSZ) or hydroxychloroquine (HCQ) and the anti-TNF antibody was administered by intravenous (IV) infusion at a dose of 2 mg / kg. At 16 weeks of treatment, patients treated with the anti-TNF antibody had a BASFI of -2.4± 2.1SD, BASMI=-0.4±0.6SD, SF-36 PCS=8.5±7.5 SD, SF-36 MCS = 6.5 ± 9.1 SD, and ASQoL = -5.4 ± 5.0 S D. The mean change from baseline in one or more criteria selected from the group consisting of The present invention provides at least one isolated mammalian anti-TNF antibody that achieves the above-mentioned therapeutic effect.

[0027] The present invention provides a compound of SEQ ID NO:3 for use in the safe and effective treatment of active ankylosing spondylitis. At least one single antibody having a heavy chain (HC) comprising SEQ ID NO:6 and a light chain (LC) comprising SEQ ID NO:37. an isolated mammalian anti-TNF antibody and at least one pharma- ceutically acceptable carrier or diluent; wherein the composition is administered by IV infusion, and at 16 weeks of treatment, Patients treated with the anti-TNF antibody had BASFI = -2.4 ± 2.1 SD, BASMI = -0.4±0.6SD, SF-36 PCS=8.5±7.5SD, SF-36 MCS = 6.5 ± 9.1 SD, and ASQoL = -5.4 ± 5.0 SD. The present invention provides a composition that achieves a mean change from baseline in one or more criteria Provide.

[0028] The present invention provides a compound of SEQ ID NO:3 for use in the safe and effective treatment of active ankylosing spondylitis. At least one single antibody having a heavy chain (HC) comprising SEQ ID NO:6 and a light chain (LC) comprising SEQ ID NO:37. an isolated mammalian anti-TNF antibody and at least one pharma- ceutically acceptable carrier or diluent; wherein the composition is administered at weeks 0 and 4, and then every 8 weeks thereafter (q 8 weeks) at a dose of 2 mg / kg administered by IV infusion over 30 ± 10 minutes. At week 16, patients treated with the anti-TNF antibody had a BASFI of -2.4±2.1S. D, BASMI=-0.4±0.6SD, SF-36 PCS=8.5±7.5SD, S F-36 MCS=6.5±9.1SD, and ASQoL=-5.4±5.0SD Achieve a mean change from baseline in one or more criteria selected from the following group: A composition is provided.

[0029] The present invention provides a compound of SEQ ID NO:3 for use in the safe and effective treatment of active ankylosing spondylitis. At least one single antibody having a heavy chain (HC) comprising SEQ ID NO:6 and a light chain (LC) comprising SEQ ID NO:37. an isolated mammalian anti-TNF antibody and at least one pharma- ceutically acceptable carrier or diluent; , wherein the composition comprises methotrexate (MTX), sulfasalazine. It is administered with or without SSZ or hydroxychloroquine (HCQ), The composition is administered by IV infusion, and at week 16 of treatment, patients treated with the anti-TNF antibody The subjects had BASFI=-2.4±2.1SD, BASMI=-0.4±0.6SD, and SF- 36 PCS = 8.5 ± 7.5 SD, SF-36 MCS = 6.5 ± 9.1 SD, and A SQoL = -5.4 ± 5.0 SD The present invention provides compositions that achieve a mean change from baseline in

[0030] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. administering a composition to a subject, the composition being administered by IV infusion, at week 16 of treatment , Patients treated with the anti-TNF antibody had BASFI=-2.4±2.1SD, BASM I=-0.4±0.6SD, SF-36 PCS=8.5±7.5SD, SF-36 M Selected from the group consisting of CS=6.5±9.1SD and ASQoL=-5.4±5.0SD and achieving a mean change from baseline in one or more measures measured using the method. provide.

[0031] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. administering the composition at weeks 0 and 4, and then every 8 weeks thereafter (q8w) at a dose of 2 mg / kg administered by IV infusion over 30 ± 10 minutes, At 16 weeks of treatment, patients treated with the anti-TNF antibody had a BASFI of -2.4 ± 2. 1SD, BASMI=-0.4±0.6SD, SF-36 PCS=8.5±7.5SD , SF-36 MCS=6.5±9.1SD, and ASQoL=-5.4±5.0SD The mean change from baseline in one or more criteria selected from the group consisting of: To achieve, to provide a method.

[0032] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. Administering a composition, the composition being methotrexate (MTX), sulfasara It was administered with or without serotonin-stimulating hormone (SSZ) or hydroxychloroquine (HCQ), The composition is administered by IV infusion, and at week 16 of treatment, subjects are treated with the anti-TNF antibody. The patients who received the BASFI were -2.4±2.1SD, BASMI were -0.4±0.6SD, and S F-36 PCS=8.5±7.5SD, SF-36 MCS=6.5±9.1SD, and and ASQoL=-5.4±5.0SD. The present invention provides a method for achieving a mean change from baseline in

[0033] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method comprises administering to the patient a safe and effective amount of a heavy chain (H C) and an isolated mammalian anti-TNF antibody having a light chain (LC) comprising SEQ ID NO: 37. administering a composition to a subject, the composition being administered by IV infusion, at week 16 of treatment , Patients treated with the anti-TNF antibody had BASFI=-2.4±2.1SD, BASM I=-0.4±0.6SD, SF-36 PCS=8.5±7.5SD, SF-36 M Selected from the group consisting of CS=6.5±9.1SD and ASQoL=-5.4±5.0SD and achieving a mean change from baseline in one or more criteria determined by the method. before, simultaneously with, or after said administration, at least one detectable label or reporter, TNF antagonists, antirheumatic drugs, muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs) , analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials, antipsoriatics, corticosteroids steroids, anabolic steroids, erythropoietin, immunity, immunoglobulin, Immunosuppressants, growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, Stimulants, asthma medications, beta agonists, inhaled steroids, epinephrine or analogues, an effective amount of at least one compound selected from the group consisting of cytokines, cytokine antagonists, and cytokine agonists; The method further comprises administering at least one composition comprising the compound or protein. Provide.

[0034] The present invention relates to a compound of SEQ ID NO:3 for use in the safe and effective treatment or prevention of ankylosing spondylitis. At least one single antibody having a heavy chain (HC) comprising SEQ ID NO:6 and a light chain (LC) comprising SEQ ID NO:37. an isolated mammalian anti-TNF antibody, the anti-TNF antibody being administered to a subject and having the following structure: and at least one isolated antibody that induces a clinical response selected from the group consisting of responses in The present invention provides a mammalian anti-TNF antibody.

[0035] [Table 1]

[0036] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being administered by intravenous (IV) infusion; At least 65% of treated patients achieve ASAS20 at week 16 of treatment. At least one isolated mammalian anti-TNF antibody is provided.

[0037] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being administered by intravenous (IV) infusion; ≥ 65% of treated patients achieved a treatment difference (improvement compared to placebo) of ≥ 45% ) to achieve ASAS20 at week 16 of treatment NF antibodies are provided.

[0038] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. a mammalian anti-TNF antibody, the anti-TNF antibody being administered at weeks 0 and 4, followed by followed by intravenous administration every 8 weeks (q8w) at a dose of 2 mg / kg administered over 30 ± 10 minutes. Administered by intravenous (IV) infusion, ≥ 65% of patients receiving the treatment achieved a median survival of 16 weeks after treatment At least one isolated mammalian anti-TNF antibody is provided that achieves ASAS20.

[0039] The present invention relates to a compound of SEQ ID NO: 36 for use in the safe and effective treatment of active ankylosing spondylitis. and a light chain (LC) comprising SEQ ID NO: 37. A mammalian anti-TNF antibody, comprising: , containing or not containing sulfasalazine (SSZ) or hydroxychloroquine (HCQ) Administered by intravenous (IV) infusion without any prior therapy, with ≥ 65% of patients receiving the treatment receiving Provide at least one isolated mammalian anti-TNF antibody that achieves ASAS20 at week 6. Provide.

[0040] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method is ankylosing spondylitis, the method comprising: (a) a safe and effective amount of a heavy chain (HC) comprising SEQ ID NO:36 and a light chain comprising SEQ ID NO:37 administering a composition comprising an isolated mammalian anti-TNF antibody having a TNF-α agonist (TNF-α)-binding domain (LC), The composition is administered by IV infusion, and ≥65% of treated patients achieve survival of ≥16 weeks of treatment. The present invention provides a method for achieving ASAS20 in the eye.

[0041] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method is ankylosing spondylitis, the method comprising: (a) a safe and effective amount of a heavy chain (HC) comprising SEQ ID NO:36 and a light chain comprising SEQ ID NO:37 administering a composition comprising an isolated mammalian anti-TNF antibody having a TNF-α agonist (TNF-α)-binding domain (LC), The composition is administered by IV infusion and ≥65% of treated patients have Treatment difference (improvement compared to placebo) in achieving ASAS20 at week 16 of treatment, and methods provide.

[0042] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of Dynamic ankylosing spondylitis, (a) a safe and effective amount of a heavy chain (HC) comprising SEQ ID NO:36 and a light chain comprising SEQ ID NO:37 administering a composition comprising an isolated mammalian anti-TNF antibody having a TNF-α agonist (TNF-α)-binding domain (LC), The composition was administered at 30±10 minutes at weeks 0 and 4, and then every 8 weeks (q8w) thereafter. The drug was administered by IV infusion at a dose of 2 mg / kg administered over a 3-day period. The method provides a method in which ≧65% of subjects achieve ASAS20 at 16 weeks of treatment.

[0043] The present invention relates to a method for treating a TNF-associated condition, the TNF-associated condition being characterized by the inactivation of and the method is ankylosing spondylitis, the method comprising: (a) a safe and effective amount of a heavy chain (HC) comprising SEQ ID NO:36 and a light chain comprising SEQ ID NO:37 administering a composition comprising an isolated mammalian anti-TNF antibody having a TNF-α agonist (TNF-α)-binding domain (LC), The composition contains methotrexate (MTX), sulfasalazine (SSZ) or hydroxypropyl 1,2-dichlorophenyl ether (H2O2). It is administered by IV infusion with or without cyclohexanone (HCQ) to The method provides that ≧65% of treated patients achieve ASAS20 at 16 weeks of treatment. [Brief description of the drawings]

[0044] [Figure 1]Figure 1 shows a graphical representation of an assay of the ability of TNV mAbs in hybridoma cell supernatants to inhibit TNFα binding to recombinant TNF receptors. Various amounts of hybridoma cell supernatants containing known amounts of TNV mAbs were preincubated with a fixed concentration (5 ng / mL) of 125I-labeled TNFα. The mixtures were transferred to 96-well Optiplates precoated with recombinant TNF receptor / IgG fusion protein p55-sf2. The amount of TNFα bound to the p55 receptor in the presence of mAbs was determined after washing away unbound material and counting using a gamma counter. Eight TNV ​​mAb samples were tested in these experiments, but for brevity, three of the mAbs that were shown by DNA sequence analysis to be identical to one of the other TNV mAbs (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] Figure 1 shows the DNA sequence of the TNV mAb heavy chain variable region. The germline gene shown is the DP-46 gene. "TNVs" indicates that the sequence shown is that of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequence define the translation initiation Met codon. The dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides of the TNV sequence (underlined) correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single letter code) starting with the mature mAb is shown only for the germline gene. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. 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]Figure 1 shows the DNA sequence of the TNV mAb heavy chain variable region. The germline gene shown is the DP-46 gene. "TNVs" indicates that the sequence shown is that of TNV14, TNV15, TNV148, and TNV196. The first three nucleotides of the TNV sequence define the translation initiation Met codon. The dotted line in the TNV mAb gene sequence indicates that the nucleotide is the same as in the germline sequence. The first 19 nucleotides of the TNV sequence (underlined) correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single letter code) starting with the mature mAb is shown only for the germline gene. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. 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. [Diagram 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. The dotted lines in the TNV mAb gene sequence indicate that the nucleotide is the same as in the germline sequence. The first 16 nucleotides (underlined) of the TNV sequence correspond to the oligonucleotides used to PCR amplify the variable region. The amino acid translation (single letter code) of the mature mAb is shown for the germline gene only. The three CDR domains in the germline amino acid translation are shown in bold and underlined. The line labeled TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to sequences that are unknown or not present in the germline gene. The TNV mAb light chain uses the J3 binding region. [Figure 4]Figure 1 shows the predicted amino acid sequence of the TNV mAb heavy chain variable region. The amino acid sequence shown (single letter code) was deduced from DNA sequence determined from both uncloned and cloned PCR products. The amino acid sequence is shown divided into secretory signal sequence (signal), framework (FW), and complementarity determining region (CDR) domains. The amino acid sequence of the DP-46 germline gene is shown in the line above each domain. Dotted lines indicate that the amino acid in the TNV mAb is identical to the germline gene. TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. "TNV" indicates that the sequence shown is for all TNV mAbs unless a different sequence is shown. Dashed lines in the germline sequence (CDR3) indicate that the sequence is not known or is not present in the germline gene. [Diagram 5] The deduced amino acid sequence of the TNV mAb light chain variable region is shown. The amino acid sequence shown (single letter code) was deduced from DNA sequence determined from both uncloned and cloned PCR products. The amino acid sequence is shown divided into secretory signal sequence (signal), framework (FW), and complementarity determining region (CDR) domains. The amino acid sequence of the Vg / 38K type light chain germline gene is shown in the line above each domain. The dotted lines indicate that the amino acid in the TNV mAb is identical to the germline gene. TNV148(B) indicates that the sequence shown is for both TNV148 and TNV148B. "All" indicates that the sequence shown is for TNV14, TNV15, TNV148, TNV148B, and TNV186. [Figure 6]Schematic diagrams of the heavy and light chain expression plasmids used to generate rTNV148B expressing C466 cells are shown. p1783 is the heavy chain plasmid and p1776 is the light chain plasmid. The rTNV148B variable and constant region coding domains are shown in black boxes. The immunoglobulin enhancer in the JC intron is shown in grey boxes. Relevant restriction sites are indicated. The plasmids are shown oriented such that transcription of the Ab genes 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-generated cell lines is shown. Cultures were initiated on day 0 by seeding cells in I5Q+MHX medium in T75 flasks to have a viable cell density of 1.0×105 cells / ml in a volume of 30 ml. The cell cultures used in these studies were continuous cultures as transfections and subcloning were performed. The cells in the T flasks were then thoroughly resuspended and 0.3 ml aliquots of culture were removed. The growth curve studies were terminated when the cell count dropped below 1.5×105 cells / ml. The number of viable cells in the aliquots was determined by typan blue exclusion and the remaining aliquots were kept for later mAb concentration determination. ELISA for human IgG was performed on all sample aliquots at the same time. [Figure 8]Figure 1 shows a graphical representation of a comparison of cell growth rates in the presence of various MHX selection concentrations. Cell subclones C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for an additional 2 days. Both cell cultures were then split into three cultures that contained either no MHX, 0.2X MHX, or 1X MHX. After one day, new T75 flasks were seeded with the cultures at a starting density of 1x105 cells / ml and cells were counted at 24 hour intervals for one week. The doubling times for the first 5 days were calculated using the formula in SOP PD32.025 and are shown above the bars. [Figure 9] Graphical representation of the stability of mAb production over time from two rTNV148B producing cell lines. After transfection and subcloning, subclones of cells that were in continuous culture were used to initiate long-term continuous culture in 24-well culture dishes. Cells were cultured in I5Q medium with or without MHX selection. Cells were serially passaged by splitting cultures every 4-6 days to maintain new viable cultures while simultaneously exhausting previous cultures. Aliquots of exhausted cell supernatants were collected immediately after the cultures were exhausted and stored until mAb concentrations were determined. ELISA for human IgG was performed on all sample aliquots at the same time. [Figure 10] Figure 1 shows the weight change of mice Tg197 in the arthritis mouse model in response to anti-TNF antibodies of the invention compared to the control of Example 4. Approximately 4-week-old Tg197 study mice were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus of Dulbecco's PBS (D-PBS) or an anti-TNF antibody of the invention (TNV14, TNV148, or TNV196) at either 1 mg / kg or 10 mg / kg. When weight was analyzed as the change from pre-treatment, animals treated with 10 mg / kg cA2 consistently showed higher weight gain than D-PBS-treated animals throughout the study. This weight gain was significant from weeks 3 to 7. Animals treated with 10 mg / kg TNV148 also achieved significant weight gain at week 7 of the study. [Figure 11A]1 shows the progression of disease severity based on the arthritis index as shown in Example 4. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to show a significant decrease in AI from week 3 onwards when compared to the D-PBS treated group. There was no significant difference between the 10 mg / kg treatment groups when each was compared to the others at similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148 and 196). When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI at weeks 3, 4 and 7 than 1 mg / kg cA2. 1 mg / kg TNV148 was also significantly lower at weeks 3 and 4 than the 1 mg / kg TNV14 treatment group. While TNV196 showed a significant reduction in AI by week 6 of the study (when compared to the D-PBS treated group), TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 11B] 1 shows the progression of disease severity based on the arthritis index as shown in Example 4. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to show a significant decrease in AI from week 3 onwards when compared to the D-PBS treated group. There was no significant difference between the 10 mg / kg treatment groups when each was compared to the others at similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148 and 196). When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI at weeks 3, 4 and 7 than 1 mg / kg cA2. 1 mg / kg TNV148 was also significantly lower at weeks 3 and 4 than the 1 mg / kg TNV14 treatment group. While TNV196 showed a significant reduction in AI by week 6 of the study (when compared to the D-PBS treated group), TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 11C]1 shows the progression of disease severity based on the arthritis index as shown in Example 4. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7). Animals treated with 1 mg / kg TNV14 and 1 mg / kg cA2 failed to show a significant decrease in AI from week 3 onwards when compared to the D-PBS treated group. There was no significant difference between the 10 mg / kg treatment groups when each was compared to the others at similar doses (10 mg / kg cA2 compared to 10 mg / kg TNV14, 148 and 196). When comparing the 1 mg / kg treatment groups, 1 mg / kg TNV148 showed significantly lower AI at weeks 3, 4 and 7 than 1 mg / kg cA2. 1 mg / kg TNV148 was also significantly lower at weeks 3 and 4 than the 1 mg / kg TNV14 treatment group. While TNV196 showed a significant reduction in AI by week 6 of the study (when compared to the D-PBS treated group), 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 mice Tg197 in the arthritis mouse model in response to anti-TNF antibodies of the present invention compared to the control of Example 5. Tg197 study mice, approximately 4 weeks of age, were assigned to one of eight treatment groups based on body weight and treated with an intraperitoneal bolus of control article (D-PBS) or 3 mg / kg of antibody (TNV14, TNV148) (week 0). Injections were repeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for efficacy of the test article. Serum samples obtained from animals in groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3, and 4. [Figure 13A]1 is a graph depicting the progression of disease severity in Example 5 based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS treatment group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and was also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13B]1 is a graph depicting the progression of disease severity in Example 5 based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS treatment group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and was also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13C]1 is a graph depicting the progression of disease severity in Example 5 based on arthritis index. The arthritis index of the group treated with 10 mg / kg cA2 was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5). Animals treated with 1 mg / kg or 3 mg / kg cA2 and animals treated with 3 mg / kg TNV14 failed to achieve any significant reduction in AI at any time point throughout the study when compared to the d-PBS control group. Animals treated with 3 mg / kg TNV148 showed a significant reduction starting at week 3 and continuing through week 5 when compared to the d-PBS treatment group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study, and was also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 14] Figure 1 shows the weight change of arthritic mouse model mice Tg197 in response to anti-TNF antibodies of the present invention compared to the control of Example 6. Tg197 study mice, approximately 4 weeks of age, were assigned to one of six treatment groups based on sex and weight and treated with a single intraperitoneal bolus of either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148). The study utilized D-PBS and 10 mg / kg cA2 control groups. [Figure 15]Figure 1 depicts the progression of disease severity based on the arthritis index as shown in Example 6. All treatment groups showed some protection at early time points, with 5 mg / kg cA2 and 5 mg / kg TNV148 showing significant reductions in AI at weeks 1-3, and all treatment groups showing significant reductions at week 2. Later in the 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 arthritis mouse model mice Tg197 in response to anti-TNF antibodies of the present invention compared to the control of Example 7. To compare the efficacy of a single intraperitoneal administration of TNV148 (derived from hybridoma cells) and rTNV148B (derived from transfected cells). Approximately 4-week-old Tg197 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]1 depicts the progression of disease severity based on arthritis index as shown in Example 7. The arthritis index of the group treated with 10 mg / kg cA2 was lower than the D-PBS control group starting at week 4 and continuing throughout the remainder of the study (week 8). Both the TNV148-treated group and the 1 mg / kg cA2-treated group showed a significant reduction in AI at week 4. A previous study (P-099-017) showed that TNV148 was slightly more effective in reducing the arthritis index after a single 1 mg / kg intraperitoneal bolus, but this study showed slightly higher AI from groups treated with both versions of the TNV antibody. The 1 mg / kg cA2 treated group (except at week 6) did not increase significantly when compared with the 10 mg / kg cA2 group, and the TNV148 treated group was significantly higher at weeks 7 and 8, but there were no significant differences in AI between 1 mg / kg cA2, 1 mg / kg TNV148, and 1 mg / kg TNV148B at any time point in the study. [Figure 18] FIG. 1 shows a diagram of the study design for a trial of Simponi (golimumab) administered intravenously in subjects with active ankylosing spondylitis (AS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] 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 and devices, including the production and use of such nucleic acids and antibodies, as well as anti-idiotypic antibodies. The methods further include, but are not limited to:

[0046] 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" At least one of the heavy or light chain complements that can be incorporated into the antibody of the present invention. a heavy or light chain variable region, a heavy or light chain variable region, or light chain constant region, framework region, or any part thereof, or a TNF receptor Immunoglobulins, such as, but not limited to, at least one portion of a receptor or binding protein. Any protein or peptide-containing molecule, including molecules that contain at least a portion of a globulin molecule. Such antibodies can optionally be further targeted to specific ligands, including but not limited to However, such antibodies may be capable of detecting at least one of the following in vitro, in situ, and / or in vivo antibodies: Modulate, decrease, or inhibit one or more of TNF activity or binding or TNF receptor activity or binding. to increase, to antagonize, to activate, to reduce, to mitigate, to block, to inhibit, As non-limiting examples, preferred anti-TNF antibodies of the present invention, in particular The specified portion or variant comprises at least one TNF or a specified portion, variant or variant thereof. A preferred anti-TNF antibody, a specified portion or variant thereof, can bind to the TNF domain. The body may also optionally regulate RNA, DNA, or protein synthesis, TNF release, TNF These include receptor signaling, membrane TNF cleavage, TNF activity, TNF production, and / or synthesis. affect at least one of the TNF activities or functions, including but not limited to The term "antibody" also includes antibodies, digests, specified portions and variants thereof. This includes antibody mimetics, or may be based on the structure and / or function of an antibody. Single chain antibodies and their derivatives, including portions of antibodies that mimic their functions or specific fragments or parts thereof Functional fragments include antigen-binding fragments that bind to mammalian TNF. For example, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion) (e.g., by pepsin digestion), fac b (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion) Fd (e.g., by pepsin digestion, partial reduction and reassembly), Fv or sc Fv (e.g., by molecular biology techniques) fragments, Antibody fragments capable of binding to NF or portions thereof are encompassed by the invention (e.g., see above). (See Colligan, Immunology, supra.)

[0047] Such fragments may be used 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 were introduced upstream of the natural stop site, various truncations were For example, a combination of genes encoding the F(ab')2 heavy chain portion may be used. 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 coupled by conventional techniques, or It can be prepared as a contiguous protein using genetic engineering techniques.

[0048] As used herein, the term "human antibody" refers to an antibody in which substantially all portions of the protein (e.g., For example, CDR, framework, C L , C H Domain (e.g., C H 1. C H 2. C H 3 ), Hinge (V L , V H )) is substantially non-human in nature with only minor sequence changes or mutations. It refers to antibodies that are immunogenic. Similarly, primates (monkeys, baboons, chimpanzees, etc.), rodents, etc. mice, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals Antibodies designated as such refer to antibodies specific for such species, subgenus, genus, subfamily, and family. Chimeric antibodies include any combination of the above. Such changes or mutations are optionally and preferably retains immunogenicity in humans or other species compared to unmodified antibodies. Thus, a human antibody is distinct from a chimeric or humanized antibody. The organism contains functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. that can be produced by a non-human animal, or a prokaryotic or eukaryotic cell capable of expressing Moreover, when a human antibody is a single-chain antibody, it is pointed out that there is a difference between the structure of the single-chain antibody and that of the naturally occurring human antibody. For example, Fv can include a linker peptide connecting the variable region of the heavy chain and the variable region of the light chain. The linker peptide may include 2 to about 8 glycine or other amino acid residues following the Such linker peptides are considered to be of human origin.

[0049] Also preferred are monoclonal antibodies having binding specificities for at least two different antigens. or human or humanized antibodies, bispecific, heterospecific, heterobinding, or similar antibodies. In this case, one of the binding specificities is directed to 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 production of IgG antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains are have different specificities (Milstein and Cuello, Nature, 30 5:537(1983)) due to the random assortment of immunoglobulin heavy and light chains. These hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules. Of these, only one has the correct bispecific structure. Purification of the correct molecule ( The purification (usually performed by an affinity chromatography step) is quite laborious and The yield is low. Similar procedures are described, for example, in WO 93 / 08829, U.S. Pat. , No. 210,668, No. 6,193,967, No. 6,132,992, No. 6, No. 106,833, No. 6,060,285, No. 6,037,453, No. 6,0 No. 10,902, No. 5,989,530, No. 5,959,084, No. 5,95 No. 9,083, No. 5,932,448, No. 5,833,985, No. 5,821 , No. 333, No. 5,807,706, No. 5,643,759, No. 5,601, No. 819, No. 5,582,996, No. 5,496,549, No. 4,676,9 80, International Publication No. 91 / 00360, International Publication No. 92 / 00373, European Patent No. 0 No. 3089, Traunecker et al., EMBO J.10:3655(1 991), Suresh et al., Methods in Enzymology 121:210 (1986), each of which is incorporated herein by reference in its entirety. INCORPORATED INTO THE SPECIFICATION.

[0050] Anti-TNF Antibodies (also referred to as TNF Antibodies) Useful in the Methods and Compositions of the Invention The antibody is selected to have high affinity binding to TNF, and optionally and preferably low toxicity. Specifically, individual regions such as variable regions, constant regions, and framework regions can be characterized as desired. The components, individually and / or collectively, optionally and preferably have low immunogenicity. The antibodies of the invention, specified fragments, or variants thereof, which have the above-mentioned functions, are useful in the present invention. The antibodies that can be used in the invention optionally provide measurable relief of symptoms as well as reduced risk of infection. These therapeutic options are characterized by the ability to treat patients for extended periods of time with acceptable toxicity and / or acceptable toxicity. Low or acceptable immunogenicity and / or high affinity, as well as other favorable properties, "Low immunogenicity" as used herein can contribute to the therapeutic outcome obtained. Less than about 75% of patients, or preferably less than about 50% of patients, have significant HAHA or HAC. A or HAMA responses and / or low titers (double An increase of less than about 300, preferably less than about 100, as measured by antigen enzyme immunoassay (Elliott et al., Lancet 344:1 125-1127 (1994), which is incorporated herein by reference in its entirety.

[0051] Utility: The isolated nucleic acids of the present invention can be used in cells, tissues, organs or animals (including mammals and humans). The present invention is directed to measuring or acting on immune disorders or diseases, cardiovascular disorders or diseases, infectious diseases, and , malignant and / or neurological disorders or diseases, The present invention relates to a method for diagnosing, monitoring, modulating, treating, ameliorating, preventing, or treating at least one TNF condition, including, but not limited to, a method for treating at least one TNF condition. At least one compound that can be used to help prevent or reduce the symptoms of It may be used to generate anti-TNF antibodies or specified variants thereof.

[0052] Such methods include those for modulating, treating, ameliorating, preventing, or reducing symptoms, effects, or mechanisms. The present invention relates to a method for administering at least one anti-TNF antibody to a cell, tissue, organ, animal or patient in need thereof. The effective amount may include administering an effective amount of a composition or pharmaceutical composition comprising the compound of formula (I) or (II) as described herein. The method may be performed and determined using known methods, such as those described in the literature or known in the relevant art, and may be used to determine the For example, about 0.001 to 500 mg / kg per bolus, multiple doses, or continuous administration. or serum concentration of 0.01 to 5000 μg / mL per single, multiple, or continuous administration The present disclosure may include amounts that achieve a desired degree of efficacy, or any range or value therein. All publications or patents cited herein are incorporated by reference in their entirety. It represents the state of the art at the time of the invention and / or provides an explanation and enabling of the invention. A publication is 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 formulas. , 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).

[0053] Antibodies of the 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 sequence numbers 4, 5, and 6. The TNF antibody can be optionally administered to a cell line, mixed cell line, or other cell line, as is well known in the art. The antibody may be produced by a line, an immortalized cell, or a clonal population of immortalized cells. el,et al.,ed.,Current Protocols in Molec. ular Biology,John Wiley&Sons,Inc.,NY,NY( 1987-2001), Sambrook, et al., Molecular Clo ning:A Laboratory Manual,2nd Edition,Col d Spring Harbor, NY (1989), Harlow and Lane ,antibodies,a Laboratory Manual,Cold Spr. ing Harbor, NY (1989), Colligan, et al., eds. ,Current Protocols in Immunology,John Wi ley & Sons, Inc., NY (1994-2001), Colligan et al. al.,Current Protocols in Protein Science ,John Wiley&Sons,NY,NY,(1997-2001) No. 6,399,413, each of which is incorporated herein by reference in its entirety.

[0054] 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.

[0055] In one approach, hybridomas are generated using a suitable immortalized cell line (e.g., Sp2 / 0 , Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3 X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 S A5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT , WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 1 Myeloma cells, such as, but not limited to, 44, NAMAIWA, and NEURO 2A strains, or heteromylomas, their fusion products, or any derived therefrom. or any other suitable cell line known in the art. For example, www.atcc.org, www.li See fetech.com. Isolated or cloned spleen, peripheral Such as, but not limited to, blood, lymph, tonsillar, or other immune or B cell-containing cells. Untreated antibody-producing cells, or recombinant or endogenous, viral, bacterial, algal, prokaryotic animals, amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep, goats, sheep, Primates, eukaryotes, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single stranded, double stranded endogenous or non-endogenous, such as single stranded or triple stranded, hybridized, etc., or combinations thereof. heavy or light chain constant or variable or frame, either as a heterologous or heterologous nucleic acid or any other cell expressing the CDR sequences. See Subel and Colligan, Immunology chapter 2. No. 6,399,433, which is incorporated herein by reference in its entirety.

[0056] Antibody producing cells can also be isolated from the peripheral blood of humans or other suitable animals immunized with an antigen of interest, or The cells may be preferably obtained from the spleen or lymph nodes. The cells also include heterologous or endogenous antibodies encoding the antibodies, specified fragments, or variants thereof of the invention. The fusion cells (hybridomas) or recombinant cells can be used to express the nucleic acid of The cells are isolated using selective culture conditions or other suitable known methods and grown by limiting dilution or cell culture. The desired specificity can be obtained by cloning the antibody by cell sorting or other known methods. The cells giving rise to the body can be selected by a suitable assay (e.g., ELISA). do.

[0057] Selecting recombinant antibodies from a peptide or protein library (e.g., bacteriophage Display live phage, 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 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 / 1444 3, International Publication No. 90 / 14424, International Publication No. 90 / 14430, International Application PCT / US94 / 1234, WO 92 / 18619, WO 96 / 07754 No. 614 989 (MorphoSys), International Patent Publication No. WO 95 / 16027 (BioInvent), Publication No. WO 88 / 06630, ...88 / 06630, Publication No. WO 8 Publication No. 90 / 3809 (Dyax), U.S. Pat. No. 4,704,692 (Enzon) , International Application PCT / US91 / 02989 (Affymax), International Publication No. 89 / 06 283, European Patent No. 371 998, European Patent No. 550 400, (Xoma), European Patent No. 229046, International Application No. PCT / US91 / 07149 (Ixsys), or stochastically generated peptides or proteins - U.S. Pat. No. 5,723,323; Same No. 5763192, Same No. 5814476, Same No. 5817483, Same No. 58245 14, International Publication No. 5976862, International Publication No. 86 / 05803, European Patent No. 59068 Issue 9 (Ixsys, now Applied Molecular Evolution ( AME), each of which is incorporated herein by reference in its entirety), or known in the art. To generate a repertoire of human antibodies, the methods are known in the art and / or described herein. The method relies on immunization of transgenic animals capable of expressing the gene (e.g., SCID mice, Nguyen et al.,Microbiol.Immunol.41:901-9 07(1997), Sandhu et al.,Crit.Rev.Biotechn ol.16:95-118(1996), Eren et al., Immunol.9 3:154-161 (1998), each of which is incorporated by reference in its entirety, as well as The present invention relates to a method for producing or administering antibodies of the required specificity, including, but not limited to, methods ... Other suitable methods for isolating the ribosomal protein may be used. Prey (Hanes et al., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997), Hanes et al., Proc. .Natl.Acad.Sci.USA,95:14130-14135(Nov.19 98)), single cell antibody generation technology (e.g., Selected Lymphocyte Antibody Method ("SLAM") (U.S. Patent No. 5,627,052, Wen et al., J. Immunol. 17:8 87-892(1987), Babcook et al., Proc. Natl. Ac. Ad. Sci. USA 93:7843-7848(1996)), gel microdrop gel microdroplet, and flow cytometry (Powell et al. .,Biotechnol.8:333-337(1990), One Cell Sy. stems, Cambridge, MA, Gray et al., J. Imm. Met h.182:155-163(1995), Kenny et al., Bio / Tec hnol. 13:787-790 (1995)), B cell selection (Steenbakke rs et al.,Molec.Biol.Reports 19:125-134( 1994), Jonak et al., Progress Biotech, Vol. 5,In Vitro Immunization in Hybridoma Tec hnology,Borrebaeck,ed.,Elsevier Science Publishers BV,Amsterdam,Netherlands(19 88)) but are not limited to these.

[0058] Methods for engineering or humanizing non-human or human antibodies can also be used, Generally, humanized or engineered antibodies are known in the art. Such animals include, but are not limited to, mice, rats, rabbits, non-human primates, or other mammals. These human amino acids have one or more amino acid residues from a non-human source. The acid residues are often referred to as "import" residues and are typically the "import" residues of known human sequences. The antibody can be taken from the variable region, constant region or other domain of a known human Ig sequence. The present invention is disclosed, for example, at www.ncbi.nlm.nih.gov / entrez / query.fcgi, www.atcc.org / phage / hdb.html, w www.sciquest.com / , www.abcam.com / , www.anti bodyresource.com / onlinecomp.html, www.pub lic.iastate.edu / ~pedro / research_tools.ht ml, www.mgen.uni-heidelberg.de / SD / IT / IT.h tml, www.whfreeman.com / immunology / CH05 / ku by05.htm, www.library.thinkquest.org / 1242 9 / Immune / Antibody.html, www.hhmi.org / gran ts / lectures / 1996 / vlab / , www.path.cam.ac.u k / ~mrc7 / mikeimages.html, www.antibodyreso urce.com / , mcb.harvard.edu / BioLinks / Immun ology.html.www.immunologylink.com / , pathb ox.wustl.edu / ~hcenter / index.html, www.bio tech.ufl.edu / ~hcl / , www.pebio.com / pa / 3409 13 / 340913.html、www.nal.usda.gov / awic / pub s / antibody / 、www.m.ehime-u.ac.jp / ~yasuhit o / Elisa.html、www.biodesign.com / table.asp 、www.icnet.uk / axp / facs / davies / links.html 、www.biotech.ufl.edu / ~fccl / protocol.html 、www.isac-net.org / sites_geo.html、aximt1. imt.uni-marburg.de / ~rek / AEPStart.html、ba serv.uci.kun.nl / ~jraats / links1.html、www. recab.uni-hd.de / immuno.bme.nwu.edu / 、www. mrc-cpe.cam.ac.uk / imt-doc / public / INTRO.h tml、www.ibt.unam.mx / vir / V_mice.html、imgt .cnusc.fr:8104 / 、www.biochem.ucl.ac.uk / ~m artin / abs / index.html、antibody.bath.ac.uk / 、abgen.cvm.tamu.edu / lab / wwwabgen.html、w ww.unizh.ch / ~honegger / AHOseminar / Slide01 .html、www.cryst.bbk.ac.uk / ~ubcg07s / 、www. nimr.mrc.ac.uk / CC / ccaewg / ccaewg.htm、www. path.cam.ac.uk / ~mrc7 / humanisation / TAHHP. html, www.ibt.unam.mx / vir / structure / stat_ aim.html, www.biosci.missouri.edu / smithgp / index.html, www.cryst.bioc.cam.ac.uk / ~fm olina / Web-pages / Pept / spottech.html, www.j erini.de / fr_products.htm, www.patents.ibm .com / ibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest,USD Etc. Health (1983), each of which is incorporated herein by reference in its entirety. (It can be included.)

[0059] Such imported sequences may be used to reduce immunogenicity or to improve the efficacy and safety of recombinant proteins. As known in the art, the terms binding, affinity, on-rate, off-rate, avidity, specificity, affinity, etc. may be used. The compounds may be used to reduce, enhance or modify the shelf life, or any other suitable property. Generally, some or all of the non-human or human CDR sequences can be used in place of the non-human and constant region CDRs. The human sequences are maintained while replacing them with human or other amino acids. If desired, the antibody can be produced by the method of the present invention while retaining high affinity for the antigen and other favorable biological properties. To this end, if desired, humanized antibodies can be produced by combining the parental and human sequences. The process of analysis of 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 that can easily illustrate and display three-dimensional conformations. Inspection of these representations allows for the identification of the role of the residues in the function of the candidate immunoglobulin sequence and Analysis of the likely candidates, i.e., the ability of the candidate immunoglobulin to bind to its antigen. In this way, it is possible to analyze the residues that affect the affinity for the target antigen(s). Consensus sequences and import sequences are optimized to achieve desired antibody properties, such as increased affinity. From the sequence, FR residues can be selected and combined. Generally, CDR residues are responsible for antigen binding. The humanization of the antibody of the present invention or The engineering process is described by Winter (Jones et al., Nature 321:52 2 (1986), Riechmann et al., Nature 332:323( 1988), Verhoeyen et al., Science 239:1534( 1988)), Sims et al., J. Immunol. 151:2296(19 93), Chothia and Lesk, J. Mol. Biol. 196:901( 1987), Carter et al., Proc. Natl. Acad. Sci. U .SA89:4285(1992), Presta et al., J. Immun ol.151:2623(1993), U.S. Patent No. 5723323, U.S. Patent No. 597686 No. 2, No. 5824514, No. 5817483, No. 5814476, No. 57 No. 63192, No. 5723323, No. 5,766886, No. 5714352 , No. 6204023, No. 6180370, No. 5693762, No. 5530 No. 101, No. 5585089, No. 5225539, No. 4816567, International Applications PCT / :US98 / 16280, US96 / 18978, US91 / 0963 No. 0, No. US91 / 05939, No. US94 / 01234, No. GB89 / 01334, G B91 / 01134, GB92 / 01755, International Publication No. 90 / 14443, International Publication No. 90 / 14424, International Publication No. 90 / 14430, European Patent No. 229246 (each of which is incorporated by reference in its entirety, including the references cited therein) This may be done using any known method, such as, but not limited to, can.

[0060] Anti-TNF antibodies may also be optionally used as described herein and / or in the art. Transgenic animals capable of producing a repertoire of human antibodies, which are known to (e.g., mice, rats, hamsters, non-human primates, etc.) Cells that produce human anti-TNF antibodies can be isolated from such animals and used as described herein. The cells may be immortalized using any suitable method, such as those described above.

[0061] Transgenic mice capable of generating a repertoire of human antibodies that bind to human antigens The cuminoids can be produced by known methods, including, but not limited to, However, U.S. Patents 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 25, 5,633,425, 5,661,016, and 5,789, No. 650, WO 98 / 50433 by Jakobovits et al. ts et al., International Publication No. 98 / 24893; Lonberg et al., International Publication No. 98 / 2488 No. 4, International Publication No. 97 / 13852 by 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. Pat. No. 5,313,233 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. 0438 474(B1) to 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 Acids Research 20(23):6287-6295(1992), Tu aillon et al., Proc Natl Acad Sci USA 90( 8) 3720-3724 (1993), Lonberg et al., Int Rev. Immunol 13(1):65-93(1995), and Fishwald et al. al., Nat Biotechnol 14(7):845-851(1996), (Each of which is incorporated herein by reference in its entirety.) In general, these mouse The sequence comprises at least one functionally rearranged or functionally rearrangeable gene. at least one transgene comprising DNA from a human immunoglobulin locus The endogenous immunoglobulin loci of such mice can be disrupted or deleted to produce endogenous genes. This can eliminate the ability of the animal to produce the antibody encoded by the offspring.

[0062] Screening of antibodies for specific binding to similar proteins or fragments can be performed using peptide This can be successfully achieved using a display library. Screening a large sample of peptides for individual members with novel functions or structures Antibody screening of peptide display libraries involves the use of a method for screening antibodies. The length of the displayed peptide sequence is 3,000 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 generating 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 a nucleotide sequence that encodes a particular displayed peptide sequence. Such methods are described in WO 91 / 17271, WO 91 / 18980 ... The peptide lysates are described in US Pat. Nos. 91 / 19818 and 93 / 08278. Other systems for generating libraries include those based on both in vitro chemical synthesis and recombinant methods. The surface of the fluororesin is preferably a fluororesin. See U.S. Pat. No. 5,658,754 and U.S. Pat. No. 5,645,756. See also Peptide Display Libraries, Vectors, and Screens. The screening kits were purchased from Invitrogen (Carlsbad, CA) and Cambria 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. 5,837,500, No. 5,427,908 assigned to Affymax, and No. No. 5580717, Cambridge antibody Technologies No. 5885793 assigned to Genentech; No. 575037 assigned to Genentech 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.

[0063] The antibodies of the present invention may also be used in transgenic animals, such as goats, cows, horses, sheep, etc., which produce such antibodies in their milk. To provide a transgenic animal or mammal, a nucleic acid encoding at least one Such animals can be prepared using one or more anti-TNF antibodies. For example, but not limited to, those disclosed in U.S. Pat. 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. 5,313,633 and 5,313,725, each of which is incorporated herein by reference in its entirety.

[0064] The antibodies of the present invention can be used to detect the specific Transgenic plants and cultured plant cells (e.g., In order to provide at least Further, the present invention can be prepared using one or more anti-TNF antibody-encoding nucleic acids. For example, inducible promoters can be used to express recombinant proteins in transfectants. Feline tobacco leaves have been used successfully to provide large amounts of recombinant proteins. For example, Cramer et al.,Curr.Top.Microbol.Immunol. 240:95-118 (1999) and references cited therein. Transgenic maize may be produced in other recombinant systems or from natural sources. A commercially produced mammalian protein with biological activity equivalent to that of proteins purified from For example, Hood et al., Adv. Ex. p. Med. Biol. 464:127-147 (1999) and references therein Antibodies have also been isolated from single chain antibodies (scFv) including from tobacco seeds and potato tubers. ) have also been produced in large quantities from transgenic plant seeds containing antibody fragments such as Conrad et al., Plant Mol. Biol. 38:101-10 9 (1998) and references cited therein. Thus, the antibodies of the present invention They can also be produced using transgenic plants, by known methods. For example, Fischer et al.,Biotechnol.Appl.Bioche m.30:99-108(Oct.,1999),Ma et al.,Trends Biotechnol.13:522-7(1995), Ma et al., Plan t Physiol.109:341-6(1995), Whitelam et al. ., Biochem. Soc. Trans. 22:940-944 (1994), and See also the references cited in, but not limited to, the following articles on plant expression of antibodies in general: Each of the above documents is incorporated herein by reference in its entirety.

[0065] 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 is optionally human TN For example, human mAbs can bind human TNF with high affinity at approximately 10 - 7 M or less, e.g., 0.1 to 9.9 (or any range or value therein) x 10 -7 , 1 0 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 Or any of them Such as, but not limited to, the range or value of K D can be combined with

[0066] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (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 literature.) 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 The measurement of , standardized solutions of antibodies and antigens, and standardized buffers, such as those described herein. This is carried out using

[0067] A nucleic acid molecule comprising at least one contiguous nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. a nucleotide sequence encoding at least 70-100% of the amino acids of a sequence identified; 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. The 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.

[0068] The nucleic acid molecules of the present invention may be in the form of mRNA, hnRNA, tRNA or any other suitable 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 it may be called the antisense strand. It may be a non-coding strand.

[0069] The isolated nucleic acid molecule of the present invention may be an open-loop nucleic acid molecule, optionally having one or more introns. An ORF, including, but not limited to, at least one overlapping CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR20, CDR21, CDR22, CDR23, CDR24, CDR25, CDR26, CDR27, C Identification of at least one CDR, such as CDR2 and / or CDR3. A nucleic acid molecule comprising a portion of the coding sequence of an anti-TNF antibody or a variable region (e.g., a sequence Nucleic acid molecules that contain the above-mentioned nucleic acid molecules but are substantially different from the above-mentioned nucleic acid molecules, By virtue of the degeneracy of the above, at least one of the following is provided: The nucleic acid molecule may comprise a nucleic acid molecule comprising a nucleotide sequence encoding yet another anti-TNF antibody. Of course, the genetic code is well known in the art. It is within the scope of the present invention to provide a method for the production of such degenerate nucleic acid variants that encode the specific anti-TNF antibodies of the present invention. It would be routine to do so. See, e.g., Ausubel et al., supra, for such nucleic acids. Mutants are encompassed by the present invention. Non-limiting examples of isolated nucleic acid molecules of the present invention include, but are not limited to, , 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 sequence numbers include: 10, 11, 12, 13, 14, and 15.

[0070] As provided herein, the nucleic acid molecules of the invention include nucleic acids encoding anti-TNF antibodies. The antibody fragment itself encodes the amino acid sequence of an antibody fragment, a whole antibody or a part thereof. Coding sequence, the coding sequence for the antibody, fragment or portion, as well as additional sequences, e.g., at least Non-coding sequences, with or without the additional coding sequences mentioned above, such as one or more introns 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 NA. together with additional non-coding sequences, including but not limited to transcribed non-translated sequences that serve to At least one signal leader or fusion peptide coding sequence, additional amino acids For example, additional coding sequences encoding amino acids that provide additional functionality can be included. Thus, the antibody encoding sequence can be, but is not limited to, an antibody fragment. A sequence encoding a peptide that facilitates purification of the fused antibody containing the fragment or portion thereof. The gene can be fused to a marker sequence.

[0071] Polynucleotides that selectively hybridize to the polynucleotides described herein The present invention relates to 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 that contain such polynucleotides. For example, the polynucleotides of the present invention can be used to quantify By identifying, isolating, or amplifying partial or full-length clones in the accumulated library, In some embodiments, the polynucleotides are isolated or Alternatively, a genomic sequence complementary to a cDNA from a human or mammalian nucleic acid library may be used. The nucleic acid sequence may be a sequence or a cDNA sequence.

[0072] Preferably, the cDNA library contains at least 80% of the full-length sequence, preferably the entire 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 favor sequences with higher identity. Low stringency conditions are those with a sequence identity of about 70%. and allows selective hybridization of sequences having orthologous or paralogous sequences. Can be used to identify the column.

[0073] Optionally, the polynucleotide of the present invention is a polynucleotide as described herein. The polypeptide of the present invention encodes at least a portion of an antibody encoded by the polypeptide of the present invention. The oligonucleotides are selective hybrids to the polynucleotides encoding the antibodies of the present invention. For example, the Ausube See Colligan, supra, each of which is incorporated herein by reference in its entirety. Be absorbed.

[0074] Construction of Nucleic Acids. The isolated nucleic acids of the invention can be produced by any method known in the art, including (a) recombinant methods; (b) synthetic techniques; (c) purification techniques; or a combination thereof. Cut.

[0075] The nucleic acid may conveniently comprise a sequence 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 is also possible to insert a translatable sequence into the vector to aid in the isolation of the polynucleotide. 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 invention. The nucleic acid of the present invention (excluding the coding sequence) may, if desired, be A vector, an adapter or a linker for cloning and / or expression.

[0076] 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. It may be possible to improve the introduction of polynucleotides into cells. The use of vectors, adapters, and linkers is well known in the art. (See, for example, Ausubel, supra, or Sambrook, supra.)

[0077] Recombinant methods for constructing nucleic acids. RNA, cDNA, genomic DNA, or any of these. The isolated nucleic acid compositions of the present invention, such as any combination of clones, may be 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 extraction procedures. 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 the art. (See, e.g., Ausubel, supra, or Sambrook, supra. and.)

[0078] Nucleic Acid Screening and Isolation Methods. A probe based on the nucleotide sequence is used to screen a cDNA or genomic library. Probes can be used to identify homologous genes in the same or different organisms. To isolate a polypeptide, one can hybridize 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 washing medium may be stringent. It is clear that as the conditions for hybridization become more stringent, For duplex formation to occur, there must be a high degree of complementarity between the probe and the target. The degree of stringency can be determined by a variety of factors, such as temperature, ionic strength, pH, and formamide. or the presence of a partially denaturing solvent. The stringency of the reduction can be, for example, between 0% and 50% formaldehyde. This can be successfully altered by changing the polarity of the reaction solution through manipulation of the amide concentration. The degree of complementarity (sequence identity) necessary for successful binding depends on the hybridization medium and and / or the stringency of the washing medium. The degree of complementation is optimally 10 0%, or 70-100%, or any range or value therein. However, Small sequence variations in the probes and primers may affect hybridization and / or washing. It should be understood that this can be compensated for by reducing the stringency of the purification medium. do.

[0079] 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.

[0080] Known methods for amplifying DNA or RNA include the polymerase chain reaction (PCR). PCR) and related amplification processes (see, e.g., U.S. Pat. No. 5,333,393, 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. 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 to Biswas, No. 4,766,067 to 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. No. 5,130,238, which is also known by the trade name NASBA. (the entire contents of which are incorporated herein by reference). See, for example, Ausubel, supra, or Sambrook, supra.)

[0081] For example, genomic DNA or cDNA fragments can be generated using polymerase chain reaction (PCR) technology. 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 amplify, for example, the protein to be expressed. cloning a nucleic acid sequence encoding a gene; detecting the presence of a desired mRNA in a sample; for use as probes for detecting nucleotides, for nucleic acid sequencing, or for other purposes. These methods may be useful for guiding the skilled artisan through in vitro amplification methods. Examples of sufficient technology are Berger, supra; Sambrook, supra; and Ausub, supra. el and Mullis et al., U.S. Pat. No. 4,683,202 (1987), and Inni s, et al., PCR Protocols A Guide to Method s and Applications,Eds.,Academic Press I nc, San Diego, CA (1990). Such kits are known in the art. For example, Advantage-GC Geno See the MicroPCR Kit (Clontech). Long PCR was performed using gene 32 protein (Boehringer Mannheim). The product yield can be improved.

[0082] Synthetic Methods for Constructing Nucleic Acids. The isolated nucleic acids of the present invention can be prepared by direct chemical synthesis using known methods. (See, for example, Ausubel et al., supra). Generally, the amplification is performed by hybridization with a complementary sequence or by using a single strand as a template. It is a single-stranded oligonucleotide that can be converted into double-stranded DNA by polymerization with the DNA polymerase used in the Those skilled in the art will appreciate that chemical synthesis of DNA produces oligonucleotides 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.

[0083] Recombinant Expression Cassettes. The present invention further provides recombinant expression cassettes comprising a nucleic acid of the invention. Using a nucleic acid sequence of the invention, e.g., a cDNA or genomic sequence encoding an antibody of the invention, and constructing a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically is capable of expressing a polynucleotide in an 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 can be used to express the nucleic acid of the present invention. This can lead to the development of acid.

[0084] In some embodiments, it functions as a promoter, enhancer, or other element. The isolated nucleic acid can be used in a method of the invention to up- or down-regulate expression of a polynucleotide of the invention. Introducing a non-heterologous version of the polynucleotide into an appropriate location (upstream, downstream, or within an intron) For example, the gene can be modified in vivo or in vitro by mutation, deletion and / or substitution. This allows the endogenous promoter to be altered.

[0085] Vectors and host cells. The present invention relates to vectors, recombinant vectors, and host cells comprising the isolated nucleic acid molecules of the present invention. The host cells can be genetically engineered with a recombinant vector, and the recombinant vectors can be produced by 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, the method of Sambroo et al., supra. See, K. et al., Ausubel et al., supra, each of which is incorporated herein by reference in its entirety. Be absorbed.

[0086] The polynucleotide may optionally be expressed as 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 vesicle or into a complex with a charged lipid. If so, package it in vitro using an appropriate packaging cell line; It can then be transduced into a host cell.

[0087] The DNA insert should be operably linked to a suitable promoter. Lactococci contain transcription initiation sites, transcription termination sites, and, within the transcribed region, ribosomal enzymes for translation. The coding portion of the mature transcript expressed by the construct preferably contains a Preferably, the mRNA comprises a translation initiation site at the beginning and a stop codon (e.g., In mammalian or eukaryotic cells, the ribosomal RNA (UAA, UGA, or UAG) will be appropriately positioned. For cellular expression, UAA and UAG are preferred.

[0088] The expression vector preferably contains at least one selectable marker, although this is optional. Such markers include, for example, methotrexate (MTX) for eukaryotic cell culture, dimers, and the like. Hydrofolate reductase (DHFR, U.S. Pat. Nos. 4,399,216 and 4,634 , No. 665, No. 4,656,134, No. 4,956,288, No. 5,149, No. 636, No. 5,179,017, ampicillin, neomycin (G418), mycobacterium tuberculosis Cophenolic acid or glutamine synthetase (GS, US Patent No. No. 5,122,464, No. 5,770,359, No. 5,827,739) Resistance Genes and tetracycline for cultivation in E. coli and other bacteria or prokaryotes These include, but are not limited to, phosphate or ampicillin resistance genes (see, for example, US Pat. No. 6,333,623). (The entire contents of which are incorporated herein by reference.) Appropriate culture media and The conditions are well known in the art. Suitable vectors are readily apparent to those skilled in the art. Introduction of the vector construct into the host cell was performed using calcium phosphate transfection. Transfection, DEAE-dextran mediated transfection, cationic lipid The method may be by plasmid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods can be affected by the method described in Sambrook, supra, 1-4. Chapters 1, 9, 13, 15, 16, etc., of Ausubel, supra. It has been described in the art.

[0089] At least one antibody of the present invention may be expressed in a modified form, such as a fusion protein. In addition to the secretion signal, the polypeptide may also contain additional heterologous functional regions. For example, a region of additional amino acids. In order to prevent the addition of charged amino acids to the N-terminus of the antibody, it is necessary to add a region, particularly a charged amino acid, to the N-terminus of the antibody to prevent the antibody from being depleted during purification or subsequent processing and storage. It is possible to improve the stability and persistence in the host cell. In addition to the antibody, the antibody or at least one fragment thereof may be used to facilitate purification. Such regions can be removed prior to final preparation of the strip. Such a method is described in Sambu et al., supra. rook, Chapters 17.29-17.42 and 18.1-18.74, Ausu bel, Chapters 16, 17 and 18, and many other standard laboratory manuals. do.

[0090] Those of skill 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.

[0091] Alternatively, a nucleic acid of the invention can include endogenous DNA encoding an antibody of the invention. To be expressed in a host cell by being switched on (by engineering) in the host cell Such methods are described in U.S. Pat. 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 entireties. do.

[0092] One example of a cell culture useful for the production of antibodies, specified portions or variants thereof is a 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. Several suitable host cell lines have been developed in the art, including COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-165 1), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., A TCC CRL1610) and BSC-1 (e.g. ATCC CRL-26) cell lines, os-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0 -Ag14, 293 cells, HeLa cells, etc., which are, for example, a Type Culture Collection,Manassas,Va(w Suitable host cells include myeloma and lysosomes. Particularly preferred host cells include cells of lymphoid origin, such as lymphoma cells. g8.653 cells (ATCC registration number CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851). In a particularly preferred embodiment, the recombinant cell are P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0093] Expression vectors for these cells include an origin of replication, a promoter (e.g., late or early SV 40 promoter, CMV promoter (U.S. Pat. No. 5,168,062, U.S. Pat. No. 5,3 85,839), HSV tk promoter, pgk (phosphoglycerate kinase) Promoter, EF-1α promoter (U.S. Patent No. 5,266,491), at least Another human immunoglobulin promoter, enhancer, and / or ribosome binding site positions, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag polyadenylation sites, addition sites), as well as processing information sites such as transcription termination sequences, but are not limited to these. The expression control sequences may include one or more of the expression control sequences described in, for example, Ausubel et al., supra. See Sambrook et al., supra. Useful for producing the nucleic acids or proteins of the invention Other such cells are known and / or may be used, for example, in the American Type The Culture Collection's catalog of cell lines and hybridomas (www atcc.org) or other known or commercial sources.

[0094] 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 can be included as well. 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, gene sequences for controlling replication within a host cell can be used as a vector. It can be incorporated into the controller.

[0095] 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 Chromatography, hydrophobic interaction chromatography, affinity chromatography, Hydroxyl apatite chromatography and lectin chromatography. and recovering and purifying the protein from recombinant cell culture by well-known methods, including but not limited to. High performance liquid chromatography ("HPLC") can also be used for purification. For example, Colligan, Current Protocols in Immunology ology or Current Protocols in Protein Scie nce, John Wiley & Sons, NY, NY (1997-2001), See, for example, Chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety. INCORPORATED INTO THE SPECIFICATION.

[0096] Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, as well as products obtained by, e.g., Produced by recombinant techniques from eukaryotic hosts, including yeast, higher plants, insect and mammalian cells. Depending on the host employed in a recombinant production procedure, the antibody of the present invention may be glycosylated or unglycosylated. It may be sylated or unglycosylated, but is preferably glycosylated. The method is 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. 6,399,413, all of which are incorporated herein by reference in their entireties.

[0097] 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 antibody of the present invention comprising all six light chain variable CDR regions can be synthesized 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 antibody against human TN F, thereby partially or substantially inhibiting at least one biological activity of the protein. At least one biologically active agent of at least one TNF protein or fragment is neutralized. Antibodies or specified portions or variants thereof that partially or preferably substantially neutralize the activity. The variant binds to a protein or fragment, thereby blocking TNF through binding to the TNF receptor. and inhibiting activities mediated through other TNF-dependent or -mediated mechanisms. As used herein, the term "neutralizing antibody" refers to a neutralizing antibody that is capable of binding to at least about 20% of the antibody, depending on the assay. ~120%, preferably at least about 10, 20, 30, 40, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, It refers to an antibody that is capable of inhibiting TNF-dependent activity by 99, 100% or more. The ability of the anti-TNF antibody to inhibit TNF-dependent activity is preferably determined by the method described herein and and / or at least one suitable TNF protein or receptor known in the art. The human antibodies of the present invention can be evaluated by immunoassays of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype, and may contain κ or λ light chains In one embodiment, the human antibody may comprise an IgG heavy chain or a defined fragment, such as Contains at least one of the isotypes IgG1, IgG2, IgG3, or IgG4. Antibodies of this type include those 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 the transgenes γ1, γ2, γ3, γ4, γ5, γ6, γ7, γ8, γ9, γ10, γ11, γ12, γ13, γ14, γ15, γ16, γ17, γ18, γ19, γ20, γ21, γ22, γ23, γ24, γ2 In another embodiment, the present invention can be prepared by using a non-human mammal. The anti-human TNF human antibody comprises an IgG1 heavy chain and an IgG1 light chain.

[0098] At least one antibody of the present invention binds to at least one TNF protein, subunit At least one specific epitope specific for the nucleotide sequence, fragment, portion, or any combination thereof. The at least one epitope binds to at least a portion of the protein. and the epitope can preferably comprise at least one antibody binding region comprising , at least one extracellular portion of the protein, a soluble portion, a hydrophilic portion, an external portion, At least one identified epitope is At least 1 to 3 amino acids for the entirety of the specified portion of adjacent amino acids in sequence number 9 It may comprise any combination of at least one amino acid sequence.

[0099] Generally, the human antibodies or antigen-binding fragments of the invention contain at least one human complementarity determining region. (CDR1, CDR2 and CDR3) or at least one variant of the heavy chain variable region, and At least one human complementarity determining region (CDR1, CDR2, and CDR3) or at least A non-limiting example of an antibody that includes an antigen-binding region that includes a variant of a light chain variable region is an antibody that includes an antibody having a light chain variable region that is a mutated light chain variable region. or the antigen-binding portion or variant comprises 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 and at least a portion of the heavy chain CDRs (i.e., CDR1, CDR2, and / or CDR3) of In another particular embodiment, the antibody or antigen-binding domain may comprise The portion or variant may be a CDR1, 2, and / or 3 amino acid sequence, e.g., At least one light chain CDR (i.e., CDR1) having SEQ ID NO: 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 The light chain CDRs of mAbs TNV148, TNV14, TNV1 5, at least one of TNV196, TNV118, TNV32, and TNV86 Such antibodies can be produced by conventional techniques of recombinant DNA technology. by preparing and expressing a nucleic acid molecule (i.e., one or more) encoding an antibody using By using the conventional techniques, or by using any other suitable method, By chemically linking various parts of the body (e.g., CDRs, frameworks) together It can be more easily prepared.

[0100] Anti-TNF antibodies are characterized by the presence of 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 can include at least one of: Optionally, a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and / or optionally At least one of the light chain variable regions having the amino acid sequence of SEQ ID NO:8. Antibodies that bind human TNF and contain defined heavy or light chain variable regions can be produced by any suitable method, including the use of For example, phage vectors 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, They can be prepared using any suitable method, for example functionally rearranged human immunoglobulins. A human immunoglobulin heavy chain transgene and a human immunoglobulin light chain capable of undergoing functional rearrangement are and a transgenic mouse comprising a transgene comprising DNA from a human Immunization with TNF or a fragment thereof can be used to induce the production of antibodies. Somatic cell proliferation and differentiation may be achieved by isolating somatic cells, as described herein and / or by methods known in the art. As is known in the art, hybridomas or other immortalized antibody-producing cells can be prepared. 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.

[0101] The present invention also provides amino acids within sequences which 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 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. The amino acids are selected from the group consisting of a first amino acid and a second amino acid, and a second amino acid. Conservative refers to the substitution of a second amino acid with a more favorable amino acid sequence (e.g., conformation, polarity, hydrophobicity / hydrophilicity). Substitutions include replacing one amino acid with another amino acid within the following group: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamine (G). phosphate (E); asparagine (N), glutamine (Q), serine (S), threonine (T ), tyrosine (Y), K, R, H, D, and E; alanine (A), valine (V), leucine (L), L, isoleucine (I), proline (P), phenylalanine (F), tryptophan (T). vanadium (W), methionine (M), cysteine ​​(C), and glycine (G); Y; C, S, and T.

[0102] Amino Acid Codes The amino acids that make up the anti-TNF antibodies of the invention are often abbreviated. The amino acid may be represented by its one-letter code, its three-letter code, its name, or by three nucleotides. Codons can be used to denote amino acids, and are well known in the art. is well understood in the field (Alberts, B., et al., Molecular Biology of The Cell,Third Ed.,Garland P (see Publishing, Inc., New York, 1994).

[0103] [Table 2]

[0104] The anti-TNF antibodies of the invention may be of naturally occurring or human origin, as specified herein. It may involve the substitution, deletion or addition of one or more amino acids by any of the manipulations.

[0105] Of course, the number of amino acid substitutions that one of skill in the art can make is limited, including many of those listed above. Generally speaking, for any given anti-TNF antibody, fragment or variant, The number of amino acid substitutions, insertions, or deletions in the sequence may be 40, 30, 20, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 2 0, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, Not exceeding 5, 4, 3, 2, 1, for example, 1 to 30, or any range or value therein .

[0106] Amino acids within the anti-TNF antibodies of the invention that are functionally essential 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, Chapter 8, 15; Cunningham, am and Wells,Science 244:1081-1085(1989) The latter procedure introduces one alanine substitution mutation at every residue in the molecule. The resulting mutant molecule may have, for example, at least one TNF neutralizing activity. The proteins are tested for biological activity, including but not limited to: Critical 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)).

[0107] 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, combinations.

[0108] The anti-TNF antibody may further comprise at least one adjacent amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8. It may contain at least one polypeptide that is 70 to 100% amino acids.

[0109] In one embodiment, an immunoglobulin chain or a portion thereof (e.g., a variable region, a CDR) The amino acid sequence of at least one of the corresponding chains of SEQ ID NOs: 7 and 8 is 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 the above For example, the amino acid sequence of the light chain variable region may be selected from the group consisting 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 identity is 70 to 100% (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range therein The value of the sigma-based ... The decision is made based on the above.

[0110] 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, the subsequence 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. The integer number can be any integer number that is supported.

[0111] As will be apparent to one of skill in the art, the present invention includes a method for the preparation of a medicament for the treatment of a cancer, comprising administering to the patient a therapeutically effective amount of at least one biologically active antibody of the present invention. Biologically active antibodies include those that are naturally occurring (non-synthetic), endogenous or related and known. At least 20%, 30% or 40%, and preferably at least 50% of that of the antibody , 60% or 70%, and most preferably at least 80%, 90% or 95% to 100% The assay and quantification methods for enzyme activity and substrate specificity are described in detail in this paper. It is well known to businesses.

[0112] In another aspect, the present invention provides a method for the preparation of a compound according to any one of the preceding claims, wherein the compound is modified by the covalent attachment of an organic moiety. Such modifications may result in improved pharmacokinetic properties (e.g., In some embodiments, the antibody or antigen-binding fragment has an 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 5000. Approximately 120,000 daltons, polyalkane glycol (e.g., polyethylene glycol) Polypropylene glycol (PEG), polypropylene glycol (PPG), carbohydrate polymers, amines The copolymer may be a polyvinylpyrrolidone or a polyvinyl acid polymer, and may also be a fatty acid or fatty acid ester group. can contain from about 8 to about 40 carbon atoms.

[0113] The modified antibodies and antigen-binding fragments of the invention are directly or indirectly covalently attached to the antibody. 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 mono- and di-carboxylic 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, the antibodies modified by the covalent bond of polylysine are Suitable hydrophilic polymers for modifying the antibodies of the invention may be linear or branched. For example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycols (mPEG, PPG, etc.), carbohydrates (e.g., dextran, cellulose , oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, etc.), polyalkane oxide (e.g., polyethylene oxide, polyaspartic acid, etc.) Polyvinylpyrrolidone, polypropylene oxide, etc., and polyvinylpyrrolidone. The hydrophilic polymer that modifies the antibody of the present invention has an individual molecular entity of about 800 to about 150,000. 00 Daltons. For example, PEG 5000 and PEG 20,000 Use where the subscript is the average molecular weight of the polymer in Daltons. The mer group can be substituted with 1 to about 6 alkyl groups, fatty acid groups, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared using suitable methods. For example, a polymer containing amine groups can be prepared by reacting it with a fatty acid or can be linked to the carboxylate of a fatty acid ester, Activated carboxylates (e.g., activated with N,N-carbonyldiimidazole) ) can be linked to a hydroxyl group on the polymer.

[0114] The fatty acids and fatty acid esters suitable for modifying the 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-Octadecanoic acid salt (C 18 oleate), all cis-Δ5,8,11,14- Eicosatetraenoic acid salt (C 20 , arachidonate), octanedioic acid, tetradecane Suitable fatty acid esters include ethyl ester, octadecanedioic acid, docosanedioic acid, and the like. The esters include monoesters of dicarboxylic acids containing straight or branched chain lower alkyl groups. The lower alkyl group can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0115] 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 the methods described above. Suitable organic groups containing activating groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) are intended to be An "activating group" is a group that, under appropriate conditions, reacts with a second chemical group, thereby forming a modifier. A chemical moiety or functional group that is capable of forming a covalent bond with a second chemical group. For example, amine-reactive activating groups include tosylate, mesylate, halo (chloro, bromo, fluoro). Electrophilic groups such as fluorine, iodo, and N-hydroxysuccinimidyl ester (NHS) Examples of activating groups capable of reacting with thiols include maleimide, iodoacetate, etc. Acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol The aldehyde functional group can be an amine or hydrazine. The azide group can be linked to an azide-containing molecule, and the azide group can react with a trivalent phosphorus group to form a phosphoryl group. Amidate or phosphorimide bonds can be formed. Introducing activating groups into the molecule Suitable methods for this purpose are known in the art (e.g., Hermanson, ,GT,Bioconjugate Techniques,Academic P Ress: San Diego, CA (1996). The activating group is an organic Directly to a group (e.g., a hydrophilic polymer, a fatty acid, a fatty acid ester) or to a linker moiety For example, divalent C1 to C 12 A group in which one or more carbon atoms are oxygen, nitrogen, or sulfur The linker moiety can be a heteroatom such as The moiety is, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6- NH-, -(CH2)2-NH- and -CH2-O-CH2-CH2-O-CH2-CH 2-O-CH-NH-. Modifiers that include a linker moiety include, for example, 1-ethyl-3-( In the presence of 3-dimethylaminopropyl carbodiimide (EDC), mono-Boc- To alkyldiamines (e.g., mono-Boc-ethylenediamine, mono-Boc-diamine By reacting hexane with fatty acids, the reaction between free amines and fatty acid carboxylates is The Boc protecting group can be converted to trifluoroacetate by forming an amide bond. The product can be removed by treatment with acid (TFA) to give the alternative carboxylate salts as described. This exposes a primary amine that can be linked or reacted with maleic anhydride. and cyclizing the resulting product to produce an activated maleimide derivative of the fatty acid. (See, e.g., Thom, 2002, the teachings of which are incorporated herein by reference in their entirety. (See, for example, WO 92 / 16221 to P.S. et al.).

[0116] 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 the The disulfide bonds (e.g., intrachain disulfide bonds) of the antibody or antigen-binding fragment can be reduced. Modified human antibodies or antigen-binding fragments can also be prepared by cloning the antibodies or antigen-binding fragments. The reduced antibody or antigen-binding fragment is then reacted with a thiol-reactive modifying agent to form the compound 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., 2002). 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.

[0117] Anti-idiotypic antibodies to anti-TNF antibody compositions. Monoclonal or chimeric anti-TN In addition to F antibodies, the present invention also provides anti-idiotypes (anti-Id) specific for such antibodies of the present invention. Anti-Id antibodies generally refer to antibodies that bind to 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 human strain (such as a human immunized ... The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody. The anti-Id antibodies can also be used to induce an immune response in yet another animal, known as "immunoglobulins." It can be used as an "antigen" to generate so-called anti-anti-Id antibodies.

[0118] Anti-Tnf antibody compositions. The present invention also relates to compositions comprising any of the compositions 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. 1, at least 2, at least 3, at least 4, at least 5, at least At least one anti-TNF antibody composition comprising six or more of said anti-TNF antibodies is also provided. Such compositions include those comprising the contiguous amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8. Selected from the group consisting of 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 non-naturally occurring compositions, including N-terminal deletion variants, domains, fragments, or variants thereof, as specified. A preferred anti-TNF antibody composition comprises a compound selected from the group consisting of 70 to 1 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. 00% of an anti-TNF antibody, or a specified fragment, domain or variant thereof, At least one or two full-length, fragment, domain, or fragments as CDR or LBR-containing portions. Further preferred compositions include the 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 0-100%, or at least one of the specified fragments, domains, or variants thereof The percentages of such compositions are known in the art. As used herein, weight, volume, concentration, molarity, or Molar mass as a liquid or dry solution, mixture, suspension, emulsion, or colloid It depends on the concentration.

[0119] The anti-TNF antibody compositions of the present invention further comprise at least one anti-TNF antibody directed against a cell, tissue, organ, animal, or patient, Optionally, at least one TNF antagonist (e.g., a TNF antibody or fragment, a soluble TNF NF receptors or fragments, fusion proteins thereof, or small molecule TNF antagonists. but are not limited to), antirheumatic drugs (e.g., methotrexate, auranofil, aurothioglucose, azathioprine, etanercept, sodium gold thiomalate Medications, hydroxychloroquine sulfate, leflunomide, sulfasalidine), muscle relaxants, narcotics, Nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular Blockers, antibacterial agents (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, Penems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonavir antibacterials, antipsoriatics, corticosteroids, anabolites, steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related Hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietics (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, cyclomodulators, alkylating agents, Metabolic agents, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanics, antipsychotics, anxiolytics, Hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta agonists, inhaled steroids loids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or similar Select from drugs, dornase alfa (Pulmozyme), cytokines or cytokine antagonists. Any suitable and effective amount of the composition or pharmaceutical composition further comprising at least one of the following: Non-limiting examples of such cytokines include at least one of I The appropriate dose is, but is not limited to, any of IL-1 to IL-23. , which are well known in the art. See, for example, Wells et al., eds., P Harmacotherapeutic Handbook, 2 nd Edition,Appl eton and Lange, Stamford, CT (2000), PDR Pha rmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing , Loma Linda, CA (2000), each of which is incorporated by reference. The entire contents of which are incorporated herein.

[0120] Such anti-cancer or anti-infective agents also may be associated, bound, or inhibited by at least one antibody of the invention. Coformulation or combination of toxin molecules may also be included. Toxins selectively kill diseased cells or tissues. The diseased cells may be cancer cells or other cells. Such toxins include, but are not limited to, ricin, diphtheria, At least one of the toxins is selected from at least one of the following: bacterial toxins, snake toxins, or bacterial toxins. It can be a purified or recombinant toxin or toxin fragment that contains one functional cytotoxic domain. The term toxin is also used to describe a substance that can cause toxic shock, which can be fatal, in humans and other mammals. Any naturally occurring, mutated or recombinant bacteria or organism that can cause any disease condition, including These include both endotoxins and exotoxins produced by viruses. Such toxins include enterotoxins, Enterotoxin Escherichia coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST) , Shigella cytotoxins, Aeromonas enterotoxins, toxic shock syndrome toxin-1 ( TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (S EC), streptococcal enterotoxins, and the like. Such bacteria include enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., serotype 0 157 strains: H7), Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus pyogenes, ognes), Shigella species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella spp.), Salmonella spp. (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella Nera enteritidis), Clostridium species (e.g., Clostridium parvum , Clostridium difficile, Clostridium botulinum), Pyrobacter species (e.g., Campylobacter jujuni, Campylobacter feta) spp.), Helicobacter spp. (e.g., Helicobacter pylori), Aeromonas spp. (e.g., , Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae) , Plesiomonas shigelloides, Yersinia enterocolitica, Vibrio species (e.g., Vibrio cholerae, Vibrio bryo-parahaemolyticus), Klebsiella spp., Pseudomonas aeruginosa, and Streptococcus spp. strains. For example, see Stein, ed., INTERNAL MEDI CINE, 3rd ed., pp 1-13, Little, Brown and Co. .,Boston,(1990), Evans et al.,eds.,Bacter. ial Infections of Humans:Epidemiology an d Control,2d.Ed.,pp 239-254,Plenum Medicine al Book Co., New York (1991), Mandell et al. ,Principles and Practice of Infectious D iseases,3d.Ed.,Churchill Livingstone,New York (1990), Berkow et al, eds., The Merck Manual,16th edition,Merck and Co.,Rahway ,NJ,1992,Wood et al,FEMS Microbiology Immunology,76:121-134(1991), Marrack et a. See, for example, J. Med., Science, 248:705-711 (1990) (these references (the contents of which are incorporated herein by reference in their entirety).

[0121] The anti-TNF antibody compounds, compositions or mixtures of the present invention may further comprise a diluent, binder, stabilizer, buffer, etc. 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. For example, see Gennaro, Ed., Remington's Pharmaceut. Physical Sciences, 18 th Edition, Mack Publication Examples of commercially available products include, but are not limited to, 1990. Anti-TNF antibodies, fragments, or antibodies as known in the art or as described herein. A pharma- ceutically acceptable carrier suitable for the mode of administration, solubility, and / or stability of the variant composition. can be selected on a daily basis.

[0122] Pharmaceutical excipients and additives useful in the present compositions include, but are not limited to, tannins, glycerols, sorbitol, sorbitol derivatives, sorbitol ester ... Proteins, peptides, amino acids, lipids and carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and sugars including oligosaccharides, derivatized sugars such as alditols, aldonic acids, and esterified sugars; and polysaccharides or sugar polymers), which may be present alone or in combination. , alone or in combination, in amounts of 1 to 99.99% by weight or volume. Excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (RAA), and erythrocyte sedimentation agents. Representative examples of buffering capacity include rHA, gelatin, and casein. Amino acids / antibody building blocks include alanine, glycine, arginine, betaine, and histidine. , glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, Preferred amino acids include amine, methionine, phenylalanine, and aspartame. One of the acids is glycine.

[0123] Carbohydrate excipients suitable for use in the present invention include, for example, fructose, maltose, and the like. Monosaccharides such as saccharides like saccharide, galactose, glucose, D-mannose, sorbose, lactose, Disaccharides such as saccharides like sugar, trehalose, cellobiose, raffinose, melezitose, maltose, Polysaccharides such as dextrin, dextran, starch, mannitol, xylitol, Maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol Preferred carbohydrate excipients for use in the present invention include alditols such as tallow. are mannitol, trehalose, and raffinose.

[0124] 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. Representative buffers include citric acid, ascorbic acid, Organic acid salts such as salts of carboxylic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Preferred for use in the composition include phosphate buffer, tromethamine hydrochloride, or phosphate buffer. A preferred buffering agent is an organic acid salt such as citrate.

[0125] In addition, the anti-TNF antibody composition of the present invention is a polymer that is -sugars), dextrates (e.g., 2-hydroxypropyl-β-cyclodextrin, etc. Cyclodextrin), polyethylene glycol, flavoring, antibacterial, sweetener, antioxidant agents, antistatic agents, surfactants (e.g., "TWEEN 20" and "TWEEN 80" polysorbates), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol and polymeric excipients / additives such as chelating agents (e.g., EDTA). obtain.

[0126] These and further compounds suitable for use in the 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 Pha'' rmacy", 19 th ed., Williams & Williams, (1995) and Physician's Desk Reference, 52. nd ed, Listed in Medical Economics, Montvale, NJ (1998) No. 5,393,633, the disclosures of which are incorporated herein by reference in their entireties. Excipient materials include carbohydrates (e.g., monosaccharides and alditols) and buffers (e.g., citrate) or polymeric reagent.

[0127] As mentioned above, the present invention preferably provides a formulation comprising saline or a selected salt. Stable formulations that are phosphate buffers, as well as preservative-containing storage solutions and formulations, and pharmaceutical preparations A pharmaceutical or veterinary preparation comprising at least one anti-TNF antibody in a therapeutically acceptable formulation. The present invention provides a multi-use preserved formulation suitable for use in a wide range of applications. The preserved formulation comprises at least one At least one of known phenols, m-cresol, p-cresol, o-cresol, sol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanoic acid ethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), Alkylparaben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, Benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof The composition may contain a preservative selected from the group consisting of: Sea urchin, 0.001 to 5%, or 0.001, 0.003, 0.005, 0.009, 0. 01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, Such as, but not limited to, 4.8, 4.9, or any range or value therein. Any suitable concentration or mixture of any range or value therein may be used. Typical examples include no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0. 3, 0.4, 0.5, 0.9, 1.0%), about 0.1 to 3% benzyl alcohol (e.g. For example, 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001 to 0. 5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.000 5-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02 ,0.05,0.075,0.09,0.1,0.2,0.3,0.5,0.75,0. 9, 1.0%).

[0128] As mentioned above, the present invention provides a pharmaceutical composition comprising a packaging material and, optionally, a buffer formulated in an aqueous diluent. At least one vial containing a solution of at least one anti-TNF antibody with / or 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 relates to a packaging material and a label that indicates that the product can be preserved for a long period of time. a first vial containing at least one anti-TNF antibody and a formulated buffer or preservative; and a second vial containing an aqueous diluent of the preservative, the packaging further comprising at least At least one anti-TNF antibody is reconstituted in an aqueous diluent for at least 24 hours. The composition includes a label instructing the patient to form a solution that can be administered intravenously.

[0129] At least one anti-TNF antibody used in accordance with the present invention may be any of those described herein. or produced from mammalian cells or transgenic preparations known in the art. The polypeptide may be produced by recombinant means, including by purification from other biological sources.

[0130] The range of at least one anti-TNF antibody included in the product of the present invention is a wet / dry system. When reconstituted, the amount is such that a concentration in the range of about 1.0 μg / mL to about 1000 mg / mL is obtained. However, lower and higher concentrations are possible and these concentrations are not intended. It depends on the delivery vehicle used, e.g., for solution formulations, transdermal patches, pulmonary, transmucosal, or is distinct from osmotic or micropump methods.

[0131] Preferably, the aqueous diluent optionally further comprises a pharma- ceutically acceptable preservative. New preservatives include phenol, m-cresol, p-cresol, o-cresol, and chloroform. Croxysol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl ethyl, etc.), 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.

[0132] Other excipients, such as isotonicity agents, buffers, antioxidants, preservative enhancers, are optionally included. An isotonic agent such as glycerin may 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 in the range of 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).

[0133] 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 pharma- ceutically acceptable solubilizer. Sorbate 20 or 80 or Poloxamer 184 or 188, Pluronic Nonionic surfactants such as (registered trademark) Polyols, other block copolymers, and and chelating agents such as EDTA and EGTA may be optionally added to the formulation or composition. These additives can be used in conjunction with a pump or pres- Page 13 13 13 13 14 15 16 17 18 19 20 21 22 23 24 25 26 37 27 28 38 39 40 This is particularly useful when a plastic container is used. The presence of the protein reduces its tendency to aggregate.

[0134] The formulations of the present invention comprise at least one anti-TNF antibody and phenol, m-cresol, p -Cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl para Benzyl chloride (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride from the group consisting of thimerosal, sodium dehydroacetate, and thimerosal or mixtures thereof 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 applications. Dissolution and mixing procedures are used to prepare suitable formulations. For example, buffer solutions may be used. A quantity of at least one anti-TNF antibody in a liquid is added to the desired concentration of protein and a preservative. The process is combined with the desired preservative in a sufficient amount of buffer solution to provide. The form will be recognized by one of ordinary skill in the art. For example, the order of addition of the components, additional addition The use of any preservatives, the temperature and pH during preparation of the formulation, all relate to the concentration and method of administration used. It is a factor that can be optimized for

[0135] The claimed formulations may be administered 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 reconstituting in 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 Any solution vials or combination vials 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.

[0136] The claimed products are useful for administration from immediately to over a period of 24 hours or more. Thus, the products claimed in this invention provide great benefits to patients. The formulation may be safely stored at any temperature between about 2°C and about 40°C to maintain the biological activity of the protein over time. The packaging label therefore indicates that the solution can be stored for 6, 12, 18, 24, 36, Demonstrate that it can be retained and / or used for periods of 48, 72, or 96 hours or more. When using stored diluents, such labels may contain up to 1-1 This may include up to 2 months, 6 months, 1.5 years, and / or 2 years of use.

[0137] The solutions of at least one anti-TNF antibody of the present invention comprise at least one antibody in an aqueous diluent. The mixture can be prepared by a process including mixing in a conventional dissolving and To prepare a suitable diluent, for example, water or a buffer solution may be used. a desired concentration of protein, and optionally a preservative. or in an amount sufficient to provide a buffer. Variations of this process are known to those of skill in the art. For example, the order of addition of the components, whether additional additives are used, etc. The temperature and pH of the formulation are all optimized for the dosage concentration and means of administration used. This is a factor that can be used.

[0138] 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 vial that is reconstituted in a lyophilized solution. It can be provided to patients as a single solution vial or as a combination solution requiring reconstitution. Each vial can be reused multiple times to complete single or multiple patient treatment cycles. This provides a more convenient treatment regimen that can meet the patient's needs and is therefore more convenient than those currently available.

[0139] The claimed product is a clear solution or a second vial containing an aqueous diluent. a combination vial containing at least one lyophilized vial of an anti-TNF antibody, , pharmacies, clinics, or other such institutions and facilities to provide Indirect delivery of the clear solution may be achieved in volumes of up to 1 liter or even more. and dispense a smaller amount of at least one antibody solution from the larger container at once. or multiple withdrawals into smaller vials and distributed to customers and / or clinics by the pharmacy or clinic. Or it can be provided to the patient.

[0140] Approved devices that contain these single-vial systems include BD Pens, BD A Utojector (registered trademark), Humaject (registered trademark), NovoPen (registered trademark) BD® Pen, AutoPen®, and OptiPe n(registered trademark), GenotropinPen(registered trademark), Genotronorm Pen(registered trademark), Humatro Pen(registered trademark), Reco-Pen(registered trademark) (trademark), Roferon Pen (registered trademark), Biojector (registered trademark), ije ct(registered trademark), J-tip Needle-Free Injector(registered trademark) ), Intraject®, Medi-Ject®, and other solution delivery Examples of such pen-type injector devices include those available from Becton Dickinson. 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 Mediject, Inc. (Police, MN, www.mediject.com) Approved devices that contain combination vials include HumatroPen (registered trademark) ) to reconstitute a lyophilized drug in a cartridge for delivering the reconstituted solution. Examples of such systems include pen-type injector systems.

[0141] The products claimed herein include packaging. The packaging may be required by a regulatory agency. In addition to providing information about the product, the packaging material of the present invention provides the conditions under which the product can be used. The anti-TNF antibody is reconstituted with an aqueous diluent to form a solution, and then incubated for a period of 2 to 24 hours or more. Provide patients with instructions to use this solution for the wet / dry two vial product. For single vial solution products, the label shall state that the solution is durable for 2 to 24 hours or longer. The products claimed herein are useful for human pharmaceutical product applications. It is useful.

[0142] The formulations of the present invention comprise at least one anti-TNF antibody and a selected buffer, preferably a biocompatible buffer. by a process which includes mixing saline or a phosphate buffer containing a 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, for example: A quantity of at least one antibody in water or buffer is added to the desired concentration of protein and buffer. and the desired buffer in a sufficient amount of water to provide. 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 administration concentration and means used It is a factor that can be optimized.

[0143] The claimed stable or preserved formulations are either present as clear solutions or in aqueous diluents containing preservatives or or a second vial containing a buffer and excipients. Both drugs can be provided to patients as a combination vial, containing one vial of anti-TNF antibody each. Any single solution vials or combination vials requiring reconstitution may be reused multiple times. can satisfy single or multiple patient treatment cycles, and therefore, To provide a more convenient treatment regimen than is available.

[0144] At least one of the antibodies of any of the stable or preserved formulations or solutions described herein. Anti-TNF antibodies can be administered by SC or IM injection, intradermally, or via the pulmonary route, as is well known in the art. , transmucosal, implant, osmotic pump, cartridge, micropump or any other suitable device known in the art. via a variety of delivery methods, including other means well known in the art and understood by those of skill in the art; The present invention can be administered to a patient.

[0145] Therapeutic Applications The present invention also relates to therapeutic applications of compounds known in the art or described herein. The present invention provides a method for the detection and / or identification of a cell, tissue, organ, or tissue fragment using at least one dual integrin antibody of the present invention. Methods for modulating or treating at least one TNF-related disorder in a host, animal or patient It also provides the law.

[0146] 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. 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 TNF-related disorder in a mammal.

[0147] The present invention also relates to a method for treating rheumatoid arthritis, juvenile, systemic-onset juvenile rheumatoid arthritis, ankylosing spondylitis, and the like. , 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, A Allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplantation, organ transplant rejection, Graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, gram-positive sepsis, gram-negative sepsis Fungal sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, urinary sepsis, meningococcal blood injury, trauma / bleeding, burns, exposure to ionizing radiation, acute pancreatitis, adult respiratory distress syndrome, alcohol-related Hepatitis, chronic inflammatory diseases, sarcoidosis, Crohn's disease, sickle cell anemia, diabetes, Nephrosis, atopic diseases, hypersensitivity reactions, allergic rhinitis, hay fever, perennial rhinitis, Uteritis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolysis Thrombocytopenia, any organ or tissue transplant rejection, kidney transplant rejection, cardiac transplant rejection Transplant rejection, Liver transplant rejection, Pancreas transplant rejection, Lung transplant rejection, Bone marrow transplant (BMT) ) Rejection, Skin allograft rejection, Cartilage graft rejection, Bone graft rejection, Small intestine transplant rejection Rejection of fetal thymus transplants; rejection of parathyroid transplants; xenotransplantation of any organ or tissue Rejection, allograft rejection, antireceptor hyperactivity, Graves' disease, Raynaud's disease, type B insulin insulin-resistant diabetes, asthma, myasthenia gravis, antibody-mediated cytotoxicity, type III hypersensitivity reactions, Systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organomegaly, endocrine disorder, Monoclonal gammopathy, and skin syndrome), polyneuropathy, organomegaly, Endocrine disorders, monoclonal gammopathy, skin syndrome, antiphospholipid syndrome, Smallpox, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, Acute biliary cirrhosis, vitiligo, vasculitis, post-MI open-heart syndrome, type IV hypersensitivity, contact skin Inflammation, hypersensitivity pneumonitis, allograft rejection, granulomas caused by intracellular organisms, drug hypersensitivity, metabolic / idiopathic Wilson's disease, hemacromatosis, alpha-1-antitrypsin deficiency, diabetic retina thyroiditis, Hashimoto's thyroiditis, osteoporosis, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cysts Neonatal chronic pulmonary disease, chronic obstructive pulmonary disease (COPD), familial hematopoietic lymphoma Histiocytosis, dermatological conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerulonephritis , acute renal failure, hemodialysis, uremia, toxicity, preeclampsia, OKT3 therapy, anti-CD3 therapy, rhinoplasty Tocain therapy, chemotherapy, and radiation therapy (e.g., asthenia, anemia, cachexia, etc.) at least one of the following: chronic salicylate intoxication, At least one immune response in a cell, tissue, organ, animal or patient, including but not limited to, Methods for regulating or treating associated disorders are also provided. ,12th~17th Editions,Merck & Company,Rahw ay,NJ(1972,1977,1982,1987,1992,1999),Pha rmacotherapy handbook, Wells et al., eds. Second Edition, Appleton and Lange, Stamfo rd, Conn. (1998, 2000), each of which is incorporated herein by reference. The entire system is incorporated.

[0148] The present invention relates to a method for treating cardiac stun syndrome, myocardial infarction, congestive heart failure, and the like. , stroke, ischemic attack, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic artery Sclerotic diseases, hypertension, arterial hypertension, renal vascular 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-reflux syndrome, inflammatory response to cardiopulmonary bypass, disorganized or multifocal atrial tachycardia , regular narrow QRS tachycardia, specific arrhythmia hythmias, ventricular fibrillation, His bundle arrhythmias, atrioventricular block, bundle branch block Lock, myocardial ischemic disease, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive myocardium Cardiac disease, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumors, aortic aneurysms and peripheral arterial Aneurysm, aortic dissection, inflammation of the aorta, occlusion of the abdominal aorta and its branches, peripheral vascular disease, occlusive Arterial disease, peripheral atherosclerosis, thromboangiitis obliterans, functional peripheral arterial disease, Inno phenomenon and diseases, acrocyanosis, erythromelalgia, venous diseases, venous thrombosis, varicose veins, arteriovenous Venous fistula, lymphedema, lipedema, unstable angina, reperfusion injury, post pump syndrome p syndrome, ischemia-reperfusion injury, etc. Methods for modulating or treating cardiovascular disease in a cell, tissue, organ, animal or patient Such methods include administering to the patient an effective amount of a composition or medicament comprising at least one anti-TNF antibody. The pharmaceutical composition is administered to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. The method may optionally include administering the compound to a subject.

[0149] 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, haematitis Streptococcus aureus, toxic shock syndrome, streptococcal myositis, gas gangrene, tuberculosis, mycobacterium Mu-avium intracellulare, Pneumocystis carinii pneumonia, Pelvic inflammatory disease , orchitis / epididymitis, Legionnaires' disease, Lyme disease, influenza A, Epstein-Barr virus -Viruses, virus-associated hemophagocytic syndrome, viral encephalitis / aseptic meningitis, etc. at least one of, but not limited to, in a cell, tissue, organ, animal or patient Also provided are methods for modulating or treating at least one infectious disease using the method.

[0150] The present invention also relates to leukemia, acute leukemia, acute lymphoblastic leukemia, and ukemia, ALL), B cell, T cell or FAB ALL, acute myeloid leukemia (acute myeloid leukemia) oid leukemia (AML), chronic myelocytic leukemia (CML) , chronic lymphocytic leukemia (CLL), hairy cell leukemia, Myelodyplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant Lymphoma, Non-Hodgkin's lymphoma, Burkitt's lymphoma, Multiple myeloma, Kaposi's sarcoma, Rectal Colon cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, malignant paraneoplastic syndrome / hypercalcemia solid tumors, adenocarcinomas, sarcomas, malignant melanomas, hemangiomas, metastatic disease, cancer-associated bone resorption, cancer-associated bone and / or the like. Also provided are methods for modulating or treating at least one malignant disease in a patient.

[0151] The present invention also relates to a method for treating neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia syndrome, demyelinating diseases, and the like. multiple sclerosis and acute transverse myelitis, extrapyramidal and cerebellar disorders, e.g. corticospinal Lesions of the spinal cord, 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., those that block 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, Friedrichshafen Ataxia, cerebellar cortical degeneration, multiple system degeneration (Mencel, Dejerine-Tho mas, Shi-Drager and Machado-Joseph), systemic diseases (Lef Susceptibility to schizophrenia, abetalipoproteinemia, ataxia, telangiectasia, and mitochondrial polymorphisms 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 pugilism. 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 neurological disorder in a subject. and administering to the patient an effective amount of a composition comprising at least one TNF antibody or specified portion or variant. The method comprises administering to a subject a pharmaceutical composition or a cell, tissue, organ or tissue in need of such modulation, treatment or therapy. For example, Merck Manu al,16 th Edition,Merck&Company,Rahway,NJ( See 1992).

[0152] Any of the methods of the present invention may be used to treat a cell, tissue, or organ in need of such modulation, treatment, or therapy. The organ, animal or patient is administered an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody. Such methods may optionally include administering a composition to treat such immune disorders. The method may further comprise the co-administration or combination therapy of at least one anti-TNF antibody, The administration of the portion or variant thereof may be in combination with at least one TNF antagonist (e.g., a TNF antibody or or fragments thereof, soluble TNF receptors or fragments thereof, fusion proteins thereof, or low molecular weight TNF antagonists, etc.), antirheumatic drugs (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldthioprine sodium malate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), Muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, topical anesthetics, neuromuscular blocking agents, antibacterials (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, Antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins antibacterials, antipsoriatics, corticosteroids, steroids, anabolic steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, 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), Immunotherapy 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, antimanics, antipsychotics Diagnosing drugs, anti-anxiety drugs, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma drugs, Beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, ethoxyquin, Pinephrine or analogues, dornase alfa (Pulmozyme), cytokines or or before, simultaneously with, and / or after administration of at least one selected from the group consisting of cytokine antagonists. Suitable dosages are well known in the art. For example, Wells et al., eds., Pharmacotherapy Han dbook,2 nd Edition, Appleton and Lange, Sta. mford, CT (2000), PDR Pharmacopoeia, Tarasco. n Pocket Pharmacopoeia 2000,Deluxe Editi on, Tarascon Publishing, Loma Linda, CA (200 0), each of which is incorporated herein by reference in its entirety.

[0153] The compositions, combination therapies, co-administrations, devices and / or methods of the invention (at least one of the invention) Suitable TNF antagonists include, but are not limited to, any of the antibodies, specified portions thereof, and variants thereof: Anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules that specifically bind to TNF, TNF synthases compounds that block and / or inhibit the formation, release, or action of TNF on target cells; For example, thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxycycline, adenosine receptor agonists and rolipram, enhancers, compounds that block and / or inhibit TNF receptor signaling, e.g. MAP kinase inhibitors, which block and / or inhibit membrane TNF cleavage Compounds that inhibit TNF activity, such as metalloproteinase inhibitors, Compounds that inhibit the action of angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril, ), as well as compounds that block and / or inhibit TNF production and / or synthesis, e.g., MA P kinase inhibitors, but are not limited to these.

[0154] As used herein, "tumor necrosis factor antibody," "TNF antibody," "TNFα antibody" " or "fragment" and the like can be used to describe TN For example, the preferred TNF human antibodies of the present invention reduce, block, inhibit, abrogate or interfere with Fα activity. The body is capable of binding to TNFα and is capable of detecting anti-TNF antibodies, antigen-binding fragments thereof, and TNFα Preferred TNF antibodies or fragments thereof include those that specifically bind to The fragments were used to measure TNF RNA, DNA, or protein synthesis, TNF release, and TNF receptor signaling. Reduce, block, or inhibit signal transmission, membrane TNF cleavage, TNF activity, TNF production and / or synthesis. It may also be possible to block, interfere, prevent and / or inhibit the

[0155] 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 and increases the circulating serum half-life of cognate antibodies. This reduces the immunogenicity of the antibody. The binding activity 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 variable region is the A2 hybridoma cell line.

[0156] Chimeric A2 (cA2) suppresses the cytotoxic effects of both natural and recombinant human TNFα. Binding assay of chimeric antibody cA2 with recombinant human TNFα showed that chimeric antibody cA2 neutralized TNFα in a dose-dependent manner. The affinity constant of the mela antibody cA2 is 1.04 x 10 10 M -1 It was calculated that The preferred method for determining the specificity and affinity of a monoclonal antibody by immunohistochemistry is rlow,et al.,antibodies:A Laboratory Manu al,Cold Spring Harbor Laboratory Press,C old Spring Harbor, New York, 1988, Colligan et al., eds.,Current Protocols in Immuno logy,Greene Publishing Assoc.and Wiley I terscience, New York, (1992-2000), Kozbor et al., Immunol.Today, 4:72-79(1983), Ausub el et al., eds.Current Protocols in Molec ular Biology,Wiley Interscience,New York (1987-2000), and Muller, Meth. Enzymol., 92:58 9-601 (1983), which references are incorporated herein by reference in their entireties. will be incorporated into the subsection.

[0157] In a specific embodiment, the mouse monoclonal antibody A2 is a polypeptide designated c134A. The chimeric antibody cA2 is produced by a cell line called c168A. It is done.

[0158] Further examples of monoclonal anti-TNF antibodies that can be used in the present invention include It has been described in the art (e.g., U.S. Pat. No. 5,231,024, Moll er, A. et al., Cytokine 2(3):162-169(1990), U.S. Application No. 07 / 943,852 (filed September 11, 1992), Rathjen e t 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 1988) 26th), Liang, et al.,Biochem.Biophys.Res.Co mm.137:847-854(1986), Meager, et al., Hybri doma 6:305-311(1987), Fendly et al., Hybri doma 6:359-369(1987), Bringman,et al.,Hyb Ridoma 6:489-507(1987) and Hirai, et al., J.I. See, e.g., Munol. Meth. 96:57-62 (1987). (the entire reference is incorporated herein by reference).

[0159] TNF Receptor Molecules. Preferred TNF receptor molecules useful in the present invention have a high affinity for TNFα. (See, e.g., Feldmann et al., International Publication WO 92 / 07076 (1992) (published April 30), Schall et al., Cell 61:361-370(1 990), and Loetscher et al., Cell 61:351-359 ( 1990), which references are incorporated herein by reference in their entireties. ), and preferably has low immunogenicity. The TNF cell surface receptors of 75 kDa (p75 TNF-R) and 75 kDa (p75 TNF-R) are used in the present invention. These receptors, including the extracellular domain (ECD) of the receptor or a functional portion thereof, are useful. Truncated forms of the receptor (e.g., Corcoran et al., Eur. J. Biochem 223:831-840 (1994) are also useful in the present invention. Truncated forms of the TNF receptor containing CD41 were detected in urine and serum as 30 kDa and 40 kDa TNF receptors. It has been detected as an Fα inhibitor binding protein (Engelmann, H. et al. , J. Biol. Chem. 265:1531-1536 (1990)). TNF receptors Multimeric molecules and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, Further examples of TNF receptor molecules that are useful in the methods and compositions of the present invention. The TNF receptor molecule that can be used for this purpose provides good to excellent relief of symptoms and low toxicity to patients. It is characterized by low immunogenicity and / or high affinity as well as the ability to treat Undefined properties may contribute to the therapeutic outcome obtained.

[0160] The TNF receptor multimeric molecules useful in the present invention may be poly(ethylene 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. The compound may further include a signal peptide of a secretory protein to effect expression of the monomeric molecule. These multimeric molecules and methods for their production are described in U.S. application Ser. No. 08 / 437,533. (filed May 9, 1995), the contents of which are incorporated herein by reference in their entirety. is incorporated into.

[0161] TNF immunoreceptor fusion molecules useful in the methods and compositions of the invention include one or more 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 in a monovalent or multivalent manner. 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.USA 88:10535-10539(1991), Pepp el et al., J. Exp. Med. 174:1483-1489 (1991), Kolls et al.,Proc.Natl.Acad.Sci.USA 91:2 15-219(1994), Butler et al., Cytokine 6(6) :616-623(1994), Baker et al., Eur. J. Immuno 24:2040-2048 (1994), Beutler et al., U.S. Pat. No. 5,447 ,851 and U.S. Application No. 08 / 442,133 (filed May 16, 1995), which (Each of the references is incorporated herein by reference in its entirety). Methods for producing offspring are described in Capon et al., U.S. Pat. No. 5,116,964, Capon et al., U.S. Pat. Patent No. 5,225,538 and Capon et al., Nature 337:5 25-531 (1989), which are incorporated herein by reference in their entireties. INCORPORATED INTO THE SPECIFICATION.

[0162] 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 that can be 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 include those functional to the 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, a functional equivalent of the TNF receptor molecule is "SILE NT" codon, or one or more amino acid substitutions, deletions, or additions (e.g., one 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 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).

[0163] Cytokines include any known cytokine. See Cytokines.com. Cytokine antagonists include any antibody. , fragments or mimetics, any soluble receptor, fragments or mimetics, any small molecule antagonist , or any combination thereof.

[0164] Therapeutic Treatment Any of the methods of the invention can be used to treat a cell, tissue, or tissue in need of such modulation, treatment, or therapy. and administering to a tissue, organ, animal, or patient a safe and effective amount of at least one anti-TNF antibody. A method for treating a TNF-mediated disorder comprising administering a composition or pharmaceutical composition Such methods may optionally include co-administration or combination therapy for the treatment of such immune disorders. The method may further comprise administering to a patient a therapeutically effective amount of at least one anti-TNF antibody, specified portion or variant thereof. The administration of at least one TNF antagonist (e.g., a TNF antibody or fragment, a soluble T NF receptors or fragments, fusion proteins thereof, or small molecule TNF antagonists, including but not limited to, antirheumatic drugs (e.g., methotrexate, auranofin, Aurothioglucose, azathioprine, etanercept, sodium gold thiomalate, Hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxants, narcotics, non-stimulants Nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockade Antibiotics (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides , tetracyclines, other antibacterials), antipsoriatics, corticosteroids, anabolic Steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related drugs rumon, antidiarrheals, antitussives, antiemetics, antiulcer drugs, laxatives, anticoagulants, erythropoietin (e.g. e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), Sargramostim (GM-CSF, Leukine), immunizing agent, immunoglobulin, immune inhibitors (e.g., basiliximab, cyclosporine, daclizumab), growth hormone, Lumbar replacement drugs, estrogen receptor modulators, mydriatics, cycloplegics, alkylating agents, and anti-aging drugs. Antidepressants, antimanic drugs, antipsychotics, anxiolytics, hypnotics, Sleeping pills, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta-agonists, inhaled steroids ides, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or similar analogues, dornase alfa (Pulmozyme), cytokines or cytokine antagonists and administering at least one of the following to the patient before, simultaneously with, and / or after the patient: Further includes.

[0165] As used herein, the term "safe" refers to a compound that is compatible with the anti-TNF antibodies of the invention (e.g., TNF antibody golimumab) for compositions, dosages, administration regimens, treatments, or methods , compared with standard of care or another comparator, such as another anti-TNF agent, for a favorable risk Adverse events (AE) and serious adverse events (SA) E) refers to the benefit ratio with acceptable frequency and / or acceptable severity of adverse events. An undesirable medical occurrence in a patient who has been administered a medicinal product. Safety of compositions, dosages, administration regimens, treatments or methods using the disclosed anti-TNF antibodies Adverse events include, for example, infusion reactions, abnormal liver and biliary tests, infections including TB, and malignancies. This refers to an acceptable frequency and / or an acceptable severity of

[0166] As used herein in the context of a composition, dose, dosing regimen, treatment or method The terms "effect" and "effective" refer to the use of an anti-TNF antibody of the invention (e.g., an anti-TNF antibody). Effectiveness refers to the effectiveness of a particular composition, dose, administration, treatment or method using golimumab. The efficacy can be measured based on changes in the course of the disease in response to an agent of the invention. For example, The anti-TNF antibodies of the invention have at least one indicator that reflects the severity of the disorder being treated. and administering to the patient in an amount and for a time sufficient to cause improvement, preferably a sustained improvement, in To determine whether the amount and duration of treatment is adequate, the disease, Various indicators that reflect the extent of a disease or condition can be assessed. Such indicators include, for example: Clinically recognized risk of disease severity, symptoms, or manifestations of the disorder of interest The degree of improvement is generally assessed by a physician or other appropriately trained individual. They will be assessed based on signs, symptoms, biopsy, or other test results indicating clinical improvement, or The disease activity may be determined based on any other measure. For example, the anti-TNF antibody of the invention may be It may be administered to achieve improvement in a patient's condition associated with ankylosing spondylitis (AS). Improvement in a patient's condition associated with, for example, ankylosing spondylitis disease activity score (ASS). pondylitis Disease Activity Score (ASDAS), Bath Ankylosing Spondylitis Functional Index Bath Ankylosing Spondylitis Measurement Index (BASFI), Bath Ankylosing Spondylitis Measurement Index (BASMI), 36 items Short Form Health Survey Physical Health (SF-36 PCS), 36-Item Short Form Health Survey Mental Health severity (SF-36 MCS) and / or Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire The ASDAS can be assessed using one or more criteria, including the results of a questionnaire. , a disease activity index for use in AS developed by the International Ankylosing Spondylitis Assessment Society. The disease activity score (DAS) is a score for all back pain. , duration of morning stiffness, peripheral pain / swelling, and patient global assessment. The BASFI is a self-assessment of the subject expressed as the average of 10 questions. of which eight relate to the functional anatomy of the subject and two relate to everyday life It concerns the subject's ability to cope. BASMI measures lateral lumbar flexion, tragus-to-wall distance, lumbar flexion by converting the assessment into a score of five assessments including vertebral discrepancy, intermalleolar distance, and cervical rotation angle. The SF-36 is a multi-item scale consisting of eight scored items. The SF-36 PSA and SF-36 MCS are questionnaires consisting of different from the SF-36, allowing comparison of the relative burden of disease and the relative benefits of different treatments The ASQoL is a summary score obtained from the questionnaire that assesses sleep, mood, motivation, coping ability, and daily living. Answer questions related to the impact of pain on activities, autonomy, relationships, and social life. It is a self-administered patient-reported outcome instrument consisting of 18 required items.

[0167] Typically, treatment of a condition will require, on average, about 100 mg of the compound in total, depending on the specific activity contained in the composition. In the range of at least about 0.01 to 500 milligrams per kilogram of patient body weight per dose at least one anti-TNF antibody, preferably at least 100 mg / kg of patient weight per single or multiple doses At least one antibody in the range of at least about 0.1 to 100 milligrams of antibody per kg. This is accomplished by administering a safe and effective amount or dose of the TNF antibody composition. The effective serum concentration is 0.1 to 5000 μg / mL of blood per single or multiple doses. Suitable dosages will be known to medical practitioners and, of course, may include concentrations in the specific The specific activity of the administered composition and the particular patient undergoing treatment will depend on the underlying disease state. In some cases, repeated administration, i.e., specific monitored doses, may be required to achieve the desired therapeutic dose. It may be necessary to provide repeated individual doses of the same amount or amount, in which case the individual doses Doses are repeated until the desired daily dose or effect is achieved.

[0168] Preferred doses are 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 .7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 4 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 8 1, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99 and / or 100-500 mg / kg / dose, or Including any range, value or fraction thereof, or 0. 1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2. 9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6. 0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9. 5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 15, 15.5, 15 .9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18. 9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 7 0, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600 , 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 35 Serum concentrations of 00, 4000, 4500, and / or 5000 μg / mL, or any The present invention may include obtaining a range, value or fraction of the above.

[0169] Alternatively, the dose administered may be determined based on the pharmacokinetic characteristics of the particular drug and its method and route of administration. , age, health and weight of the recipient, nature and severity of symptoms, type of concurrent treatment, 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 administered, and the desired effect. It may be about 0.1 to 100 milligrams per kilogram of body weight. Usually, it is about 1 kilogram per dose. 0.1 to 50, preferably 0.1 to 10 milligrams per gram or in sustained release form is preferred. This is effective for obtaining better results.

[0170] As a non-limiting example, human or animal treatment may be performed using a 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 additionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 5 In at least one of weeks 1 or 52, or in addition, , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or in at least one of the twentieth years, or any combination thereof, 0.1 to 100 mg / kg per day, for example, 0.5, 0.9, 1.0, 1.1, 1.5, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 , 45, 50, 60, 70, 80, 90, or 100 mg / kg of the composition of the present invention It may be provided as a single or periodic dose of an antibody.

[0171] Dosage forms (compositions) suitable for internal administration generally contain about 0.1 milligrams per unit or container. In these pharmaceutical compositions, the active ingredient is is typically present in an amount of about 0.5 to 99.999% by weight, based on the total weight of the composition.

[0172] For parenteral administration, the antibody may be administered in combination with or without a pharma- ceutically acceptable parenteral vehicle. The composition may be formulated as a solution, suspension, emulsion or lyophilized powder. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils are used. The vehicle or lyophilized powder may contain additives that maintain isotonicity and chemical stability. Additives (e.g., sodium chloride, mannitol for isotonicity; The formulation may contain sterilization agents (buffers and preservatives) by known or suitable techniques. It will be sterilized.

[0173] Suitable pharmaceutical carriers are described in Remington's, a standard reference text in this field. Described in the latest edition of Pharmaceutical Sciences, A. Osol It has been done.

[0174] Alternative Administration. A pharma- ceutical effective amount of at least one anti-TNF antibody according to the invention is administered. Many known and developed administration methods can be used in accordance with the present invention to deliver the Although pulmonary administration is used in the following description, other modes of administration may be used in accordance with the present invention. may be used to obtain suitable results.

[0175] The TNF antibody of the present invention may be present in the carrier as a solution, emulsion, colloid or suspension. or as a dry powder, by inhalation, or by administration as 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.

[0176] Parenteral formulations and administration. Parenteral formulations contain sterile water or saline as the common vehicle. Water, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogen Naphthalene fluoride and the like may be included. Aqueous or oily suspensions for injection may be prepared according to known methods. Injections can be prepared by using appropriate emulsifying or wetting agents and suspending agents. Non-toxic parenterally acceptable diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, and isotonic saline. As a typical solvent or suspension medium, a sterile, non-volatile oil is used. For these purposes, fatty oils or fatty acids, natural or synthetic or semi-synthetic, natural or synthetic or semi-synthetic mono- or di- or triglycerides Any type of fixed oil or fatty acid may be used. Parenteral administration is known in the art. Conventional injection means known in the art, such as those described in U.S. Pat. No. 5,851,198, are also known in the art. 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 those described herein. is incorporated herein by reference in its entirety.

[0177] Alternative Delivery. The present invention further relates to parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, , intracapsular, intracartilaginous, intrasinusoidal, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, Intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal 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 as creams and suppositories. For use in vaginal or rectal administration in semi-solid form, including but not limited to: Buccal or sublingual administration, such as, but not limited to, tablets or capsules For use in the treatment of skin diseases, or as a powder, nasal drops or aerosol, or as a certain drug, etc. Intranasally, or by modifying the skin structure or in a transdermal patch, in a form such as, but not limited to, or chemical facilitators such as dimethyl sulfoxide to either increase the drug concentration. Drug Permeation Enhancement”;Hsieh,DS,Eds.,pp.59-90(M Arcel Dekker, Inc. New York 1994, the entire book is incorporated by reference. (incorporated herein) or by applying to the skin formulations containing proteins and peptides. by using an oxidizing agent that enables the synthesis of 1,2-dichlorophenyl ether (WO 98 / 53847) or by electroporation. or to create a transient delivery pathway, such as iontophoresis, or to administer drugs to the skin, such as iontophoresis. Application of an electric field to increase the mobility of a charged drug through a membrane, or ultrasound, such as sonophoresis Using the application of the These include, but are not limited to, pharmaceuticals, ointments, lotions, suspensions or patch delivery systems. (The above publications and patents are incorporated herein by reference in their entireties.) (Incorporated into the Detailed Description).

[0178] 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 disclosed in a method according to the art for administering a therapeutic agent 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 that can be used include metered dose inhalers, nebulizers, dry powder generators, and sprayers. Other devices suitable for pulmonary or intranasal administration purposes are known in the art. All such devices are suitable for administration to dispense antibodies in an aerosol. Such aerosols may be used in the form of solutions (both aqueous and non-aqueous) or Ventolin® Metered Inhaler can be composed of either solid particles. 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. Turbuhaler™ (Astra), Rotahaler® (Gl axo), Diskus® (Glaxo), Spiros® inhaler (D ura), devices marketed by Inhale Therapeutics, and Dry powder inhalers, such as the Spinhaler® powder inhaler (Fisons) The device uses breath actuation of a mixed powder (U.S. Pat. No. 4,668,218 (Astra) and European State Patent No. 237507 (Astra), International Publication No. WO 97 / 25086 (Glaxo) , WO 94 / 08552 (Dura), U.S. Pat. No. 5,458,135 (Inha le), WO 94 / 06498 (Fisons), incorporated herein by reference in its entirety. AERx™ (Aradigm), Ultravent® ) nebulizer (Mallinckrodt), and Acorn II® nebulizer Nebulizers such as the Riser (Marquest Medical Products) (U.S. Patent No. 5,404,871 (Aradigm), International Publication No. 97 / 22376) The above references (all of which are incorporated herein by reference in their entireties) generate aerosols from solutions. In contrast to metered dose inhalers and dry powder inhalers, which generate small particle aerosols. These specific examples of inhalation devices are representative of the particular devices suitable for practicing the present invention. It is intended as an illustration of the invention and is not intended as a limitation on the scope of the invention. Preferably, a composition comprising at least one anti-TNF antibody is administered by a dry powder inhaler or a spray. An inhalation device for administering at least one antibody of the present invention, the antibody being delivered by a nebulizer. There are several desirable features of inhalation devices. For example, delivery by inhalation devices is advantageously reliable. The inhalation device is highly reliable, reproducible and accurate. For example, small dry particles of less than about 10 μm, preferably about 1 to 5 μm, can be delivered. It is possible.

[0179] Administration of the TNF antibody composition in a 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 size and configuration of the nozzle, the applied pressure and the liquid supply The velocity can be selected to achieve a desired output and particle size. Electrospray can be produced by an electric field in conjunction with a nozzle or nozzle feed. Preferably, the particles of at least one anti-TNF antibody composition protein delivered by the nebulizer The diameter 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 μm range.

[0180] At least one anti-TNF antibody composition protein suitable for use with a nebulizer. The formulations are typically in aqueous solution, e.g., 0.1, 0.2., 0.3, 0.4, 0.5 , 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or is, but is not limited to, 100 mg / mL or mg / gm, 1 ml or From about 0.1 mg to about 100 mg per mg / gm of solution, or any range therein The antibody composition protein is included at a concentration of at least one of the anti-TNF antibody composition protein values. The formulation may contain any suitable additives, such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and preferably zinc. The formulation may contain any suitable agent, such as a buffer, a reducing agent, bulk protein or carbohydrate. The antibody composition may also include an excipient or agent for stabilizing the antibody composition protein. Bulk proteins useful in protein formulations include albumin and protamine. Exemplary carbohydrates useful in formulating antibody composition proteins include cholinesterases, Examples of the sugar include sugar, mannitol, lactose, trehalose, and glucose. Protein formulations also have the potential to reduce the antibody composition tumour caused by atomisation of the solution during aerosol formation. The polysaccharide may also include a surfactant, which can reduce or prevent surface-induced aggregation of proteins. Oxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol A variety of conventional surfactants can be used, such as fatty acid esters. The amounts are generally: The range of the surfactant is 0.001 to 14% by weight of the formulation. The antibacterial agents are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorb Proteins such as TNF antibodies or specified portions or variants The formulations may also include additional agents known in the art.

[0181] 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 supply is used to blow the gas through the nozzle. As the gas expands beyond the nozzle, a low pressure area is created. This draws a solution of the antibody composition protein through a capillary tube connected to a liquid reservoir. The liquid flowing 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 the 1990s, high frequency electrical energy was used to generate vibrations, typically using piezoelectric transducers. This energy is transferred directly or through a coupling fluid to the The antibody composition protein is then transferred to a preparation containing the antibody composition protein by any of the methods described above. Advantageously, the antibody composition protein delivered by the nebulizer is The particles of the substance 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 about 3 μm to about 5 μm.

[0182] At least one anti-TN antibody suitable for use with either a jet or ultrasonic nebulizer Formulations of F antibody typically contain at least about 0.1 mg to about 100 mg per mL of solution. The formulation also includes a concentration of an anti-TNF antibody protein. The formulation also includes an excipient, a buffer, an isotonicity agent, Agents such as preservatives, surfactants, and preferably zinc may be included in the formulation. 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 stabilization of the protein. Bulk proteins useful in product protein formulations include albumin, protamine, etc. Exemplary carbohydrates useful in the formulation of 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 pulmonary function induced by atomization of the solution upon formation of the aerosol. A surfactant capable of reducing or preventing surface-induced aggregation of at least one anti-TNF antibody. Polyoxyethylene fatty acid esters and alcohols, as well as polyoxyethylene A variety of conventional surfactants such as ethylene sorbital fatty acid esters can be used. The amount is generally in the range of 0.001 to 4% by weight of the formulation. A preferred surfactant is 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.

[0183] Administration of the TNF antibody composition 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 may contain a liquefied compressed gas. The mixture is contained in a canister. By actuating the throttle valve, the mixture is preferably dispensed at about 10 μl. The size is preferably in the range of about 1 μm to about 5 μm, and most preferably in the range of about 2 μm to 3 μm. The mixture is released as an aerosol containing particles of the metal. Antibody composition proteins produced by a variety of methods known to those skilled in the art, including critical point condensation. By using a formulation of the desired quality, the desired aerosol particle size can be obtained. The inhaler is manufactured by 3M or Glaxo and uses hydrofluorocarbon propellants. Examples include those that use

[0184] Formulations of at least one anti-TNF antibody for use in a metered dose inhaler device generally comprise At least one anti-TNF antibody is suspended in a propellant, for example with the aid of a surfactant. The propellant is trichlorofluoromethyl ether. ethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1, 2-Tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227) and other chlorofluoro Carbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons The propellant may be any conventional material used for this purpose. The surfactant is a hydrofluorocarbon. The surfactant is a hydrofluorocarbon. selected for stabilization as a suspension of the active agent, protection of 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.

[0185] 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 accomplished by pulmonary administration of the anti-TNF antibody composition.

[0186] Oral Formulations and Administration. Formulations for oral administration may be prepared using an agent that artificially increases the permeability of the intestinal wall. Adjuvants (e.g., resorcinol, and polyoxyethylene oleyl ether and Simultaneous administration of a non-ionic surfactant such as n-hexadecyl polyethylene ether, and In addition, enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl The solid formulation for oral administration is 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 the following: glycerides, albumin, synthetic or semi-synthetic polymers, and glycerides; These dosage forms can also be mixed with other types of additives, e.g. For example, inert diluents, lubricants (magnesium stearate), paraben preservatives (sorbic acid ), ascorbic acid, α-tocopherol, antioxidants (cysteine), disintegrants, binding agents The composition may also contain flavoring, thickening, buffering, sweetening, flavoring and perfuming agents.

[0187] 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) for drug delivery It has been used to manufacture the fluororesin (U.S. Patent No. 4,925,673). The carrier compounds described in U.S. Pat. Nos. 79,681 and 5,5871,753 are It is known in the art that it can be used to orally deliver biologically active agents. do.

[0188] Mucosal Formulations and Administration. At least one anti-TNF antibody is administered 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 viscoelastic emulsion (see U.S. Pat. No. 5,514,670). Mucosal surfaces suitable for application of the emulsion of the present invention include the cornea, conjunctiva, oral, sublingual, These include nasal, vaginal, pulmonary, gastric, intestinal and rectal routes of administration. The preparations, e.g., suppositories, may contain, as excipients, e.g., polyalkylene glycols, petrolatum, cocoa, etc. The formulation for intranasal administration may be solid and may contain excipients such as butter. For example, it may contain lactose or may be an aqueous or oily solution nasal spray. For oral administration, the excipients are sugar, calcium stearate, malic acid stearate, and glycerin. calcium, alpha starch (pregelinatined starch), etc. (U.S. Patent No. 5, No. 849,695).

[0189] Transdermal Formulations and Administration. For transdermal administration, at least one anti-TNF antibody is administered in a liposomal formulation. or polymer nanoparticles, microparticles, microcapsules or microspheres (specially those not specified) Unless otherwise specified, the drug is encapsulated in a delivery device such as a polysaccharide (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, alginates and Many suitable devices, including microparticles made from other polysaccharides, as well as combinations thereof, are available. A chair is known (U.S. Pat. No. 5,814,599).

[0190] Prolonged Administration and Formulations. The compounds of the invention can be administered in a single dose over an extended period of time, e.g. It may sometimes be desirable to deliver the drug to a subject for periods ranging from one week to one year. For example, the dosage form may be an implantable formulation containing a compound that is poorly soluble in body fluids. Pharmaceutically acceptable non-toxic salts of the compounds, for example, (a) phosphoric, sulfuric, citric, tartaric, Tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfone (b) acid addition salts with polybasic acids such as zinc, calcium, bismuth , barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc. or, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine. (c) a salt having an organic cation formed from (a) and (b) ) combination, for example, zinc tannate. In addition, the compounds of the present invention, Preferably, the relatively insoluble salts, such as those mentioned above, are combined with an aqueous solution suitable for injection, such as sesame oil. It can also be formulated in a gel, such as, for example, an aluminum monostearate gel. Particularly preferred salts are zinc salts, zinc tannate, and zinc pamoate. Injectable sustained release depot formulations include, for example, polylactic acid formulations described in U.S. Pat. No. 3,773,919. / Ca in slowly degrading, non-toxic, non-antigenic polymers such as polyglycolic acid polymers The compound or salt is preferably dispersed in the capsule. Relatively insoluble salts such as those listed above are useful for the preparation of cholesterol matrices, particularly for use in animals. It may also be formulated in a silastic pellet of 100 mg / kg. Inclusion formulations, such as gas or liquid liposomes, are described in the literature (U.S. Pat. No. 5,770,222). , and "Sustained and Controlled Release Dr g Delivery Systems”, JR Robinson ed., Mar. cel Dekker, Inc., NY, 1978).

[0191] Having generally described the invention, the like 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. Wax.

[0192] 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. A promoter element, the antibody coding sequence, and required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and RNA splices. It contains intervening sequences flanking the donor and acceptor sites for isolating. The transcription rate is controlled by early and late promoters from SV40, retroviruses, e.g., RS V, HTLVI, and HIVI long terminal repeats (LTRS), This can be achieved by using the early promoter of cytomegalovirus (CMV). However, cellular elements can also be used (e.g., human actin promoter). Suitable expression vectors for use in the practice of the present invention include, for example, , pIRES1neo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, CA), pcDN A3.1(+ / -), pcDNA / Zeo(+ / -) or pcDNA3.1 / Hygro (+ / -) (Invitrogen), PSVL and PMSG (Pharmacia, USA) ppsala, Sweden), pRSVcat (ATCC 37152), pSV2d hfr (ATCC 37146) and pBC12MI (ATCC 67109) The mammalian host cells that can be used include human Hela29 3. H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Co s7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells (Chinese hamster ovary, CHO) cells.

[0193] Alternatively, the gene can be expressed in a stable cell line that contains the gene integrated into a chromosome. dhfr, gpt, neomycin or hygromycin. Co-transfection with a selectable marker allows identification and identification of transfected cells. Allows for separation.

[0194] 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. It is further useful to develop cell lines that have several thousand copies. The enzyme glutamine synthase (GS) is the chem.J.227:277-279(1991), Bebbington,et a l., Bio / Technology 10:169-175(1992)). The marker is used to grow mammalian cells in selective medium and select the cells with the highest resistance. These cell lines contain the amplified gene(s) integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are used to produce antibodies. This is often the case.

[0195] 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., Cell 41:521-530(1985)). For example, the restriction enzyme cleavage site B A multiple cloning site with amHI, XbaI and Asp7l8 is provided for the insertion of the gene of interest. The vector further contains the 3' end of the rat preproinsulin gene. It contains introns, polyadenylation and termination signals.

[0196] Cloning and expression in CHO cells. The vector pC4 was used to express the TNF antibody. Plasmid pC4 is amplified from plasmid pSV2-dhfr (ATCC Accession No. 37 146) is a derivative of the mouse SV40 early promoter. The plasmids transfected with these vectors contain the DHFR gene. Chinese hamster ovary cells or other cells lacking folic acid activity have been shown to inhibit the chemotherapeutic agent methotrexate. Selective medium supplemented with Lexate (e.g., α-MEM, Life Technologies, The cells were selected by growing them in a 5% CO2 medium (Laboratory of Pharmacia, Gaithersburg, MD). Amplification of the DHFR gene in methotrexate (MTX)-resistant cells has been well documented (e.g., FWAlt, et al., J. Biol. Chem.253:1357-1370(1978), J.L.Hamlin and CMa,Biochem.et Biophys.Acta 1097:107-143 (1990) and MJ Page and MA Sydenham, Biotech (See Biochemistry 9:64-68 (1991)). Cells grown in this environment produce excessive amounts of the target enzyme, DHFR, as a result of amplification of the DHFR gene. Overproduction leads to the development of resistance to drugs. A second gene is linked to the DHFR gene. When genes are expressed in amplified sequences, they are usually co-amplified and overexpressed. Ability to develop cell lines with more than 1,000 copies of the gene(s) are known in the art. A cell line is obtained that contains the amplified gene integrated into one or more chromosomes of the cell. do.

[0197] The plasmid pC4 contains the Rous sarcoma virus long terminal repeat fragment (LTF) for expression of a 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 (C MV) immediate early gene enhancer (Boshart, et al., Cell 41 The promoter downstream is , BamHI, XbaI and Asp718 restriction sites allowing gene integration Following these cloning sites, the plasmid contains the rat preproinsulin Contains the 3' intron and polyadenylation site of the gene. Other highly efficient promoters , for example, the human β-actin promoter, the SV40 early or late promoter, or Long terminal repeats from other retroviruses, such as HIV and HTLV-I, can also be used for expression. Clontech's Tet-Off and Tet-On gene expression systems, and similar systems are used to express TNF in a regulated manner in mammalian cells. (M. Gossen and H. Bujard, Proc. Natl. A cad. Sci. USA 89:5547-5551(1992)). For denylation, other signals, e.g., from the human growth hormone or globin genes, can also be used. Stable cell lines carrying a gene of interest integrated into a chromosome can be produced by: Co-transfection with a selection marker such as gpt, G418 or hygromycin Alternatively, the selection can be performed by first adding more than one selectable marker, e.g., G418. In addition, methotrexate is advantageously used.

[0198] The plasmid pC4 is digested with restriction enzymes and then purified according to procedures known in the art. The vector was then dephosphorylated using calf intestinal phosphatase. It is isolated from a sugar gel.

[0199] The isolated variable and constant region-encoding DNA and the dephosphorylated vector were then transformed with T4 DNA. A ligase was used to ligate the DNA. Then, E. coli HB101 or XL-1 Blue cells were transformed with For example, restriction enzyme analysis can be used to identify cells containing the fragment inserted into plasmid pC4. Identify the bacteria.

[0200] For transfection, Chinese hamsters lacking an active DHFR gene were used. CHO cells were used. 5 μg of the expression plasmid pC4 was transfected with lipofectin. The cells are co-transfected with 0.5 μg of plasmid pSV2-neo using the The plasmid pSV2-neo expresses 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 malaria. The cells were seeded in alpha minus MEM supplemented with 1 μg / mL G418. After 2 days, the cells were cultured. The cells were then treated with liposin and transferred to hybridoma cloning plates (Greiner, German y) 10, 25 or 50 ng / mL methotrexate + 1 μg / mL G418 After about 10-14 days, single clones were isolated by trypsinization. After treatment, different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 40 0 nM, 800 nM) are used to seed 6-well Petri dishes or 10 mL flasks. Next, clones grown in the highest concentration of methotrexate were cultured in even higher concentrations of methotrexate. Prepare a new 6-well plate containing Rexate (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 desired gene product can be confirmed by, for example, SDS-PAGE and Western blot analysis. The products are analyzed by HPLC or by reverse phase HPLC analysis.

[0201] Example 2: Using transgenic mice, a high affinity human TNF-reactive TNF receptor was generated Generation of IgG monoclonal antibodies. SUMMARY: Transgenic mice containing human heavy and light chain immunoglobulin genes have been developed. and / or a combination of these, comprising a therapeutic agent for inhibiting TNF action for the treatment of one or more TNF-mediated diseases. The heavy chain and the human monoclonal antibody are then combined to produce high affinity, fully human monoclonal antibodies that can be used therapeutically. The antibody contains human variable and constant region antibody transgenes for both the β and light chains (CBA / JxC57 / B L6 / J)F2 hybrid mice were immunized with human recombinant TNF (Taylor et al. t al., Intl. Immunol. 6:579-591 (1993), Lonbe rg, et al., Nature 368:856-859(1994), Neube rger, M., Nature Biotech. 14:826 (1996), Fish wild,et al.,Nature Biotechnology 14:845- 851 (1996). Several fusions were produced using fully human TNF-reactive IgG monoclonal antibodies. One or more panels of antibodies have been generated. Fully human anti-TNF antibodies are to be further characterized. All is IgG1κ. Such antibodies are approximately 1×10 9 ~9×10 12 With an affinity constant of The unexpected high affinity of these fully human monoclonal antibodies has led to They represent good candidates for therapeutic use in TNF-related diseases, conditions or disorders.

[0202] 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-interadermal, Ig -Immunoglobulin, TNF-tissue necrosis factor alpha, IP-intraperitoneal, IV-intravenous, Mab-monoclonal 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 amount.

[0203] Materials and Methods. Animals. Transgenic mice capable of expressing human antibodies are known in the art. (e.g., GenPharm International, San Jose These are commercially available from companies such as Abgenix, Freemont, CA, and others. For example, such a strain expresses mouse immunoglobulins but does not express mouse IgM or Igκ. Transgenic mice are characterized by V(D)J joining, heavy chain class switching and somatic mutations. containing human sequence transgenes, which are then subjected to (Lonberg, et al., Nature 368:856-859(199 4) The light chain transgene may, for example, contain, in part, approximately half of the germline human Vκ region. In addition, the heavy chain transgene can be derived from a yeast artificial chromosome clone. Both (Fishwild, et al., Nature Biotechnology 14:845-851 (1996)) and / or a γ3 constant region. Mice from the appropriate genotopic strain are used in the immunization and fusion process. It is possible to generate fully human monoclonal antibodies against TNF.

[0204] Immunization. One or more immunization schedules may be used to generate anti-TNF human hybridomas. The following exemplary immunization protocol can be used to administer the first few The fusion of the numbers can be performed, but other similar known protocols can also be used. 14-20 week old female and / or surgically castrated male transgenic mice were Then add an equal volume of TITERMAX or Complete Buffer to a final volume of 100-400 μl (e.g., 200). 1-1000 μg of recombinant human TNF emulsified in whole Freund's adjuvant was administered IP or I Each mouse is also inoculated with 100 μL of physiological saline in each of 2 SQ sites, if desired. Mice can also receive 1–10 μg in saline. , IP (1-400 μg) and SQ after 10-18, 17-25, and / or 21-34 days. (1-400μg x 2) with an equal amount of TITERMAX or complete Freund's adjuvant After 12-25 and 25-40 days without anticoagulants, Mice can be bled by retro-orbital puncture. Blood is then allowed to clot at room temperature for 1 hour. The serum is collected and titrated using a TNF EIA assay according to known methods. If the injection does not result in an increase in titer, a fusion is performed. At that time, the mouse is injected with 100 μL of saline. Give a final IV booster injection of 1 to 400 μg of TNF diluted in standard saline. After 3 days, the mice were euthanized by cervical dislocation, and the spleens were aseptically removed and U / mL penicillin, 100 μg / mL streptomycin and 0.25 μg / mL 10 mL of cold phosphate buffered saline (PBMS) containing 10 mL of amphotericin B (PSA) The spleen can be immersed in PSA-PBS to obtain splenic cells. Cells were washed once in cold PSA-PBS and isolated using trypan blue dye exclusion. The cells are then counted and resuspended in RPMI 1640 medium containing 25 mM Hepes.

[0205] Cell fusion. Cell fusion can be carried out according to known methods, for example, 1:1 to 1: Fusions can be performed at a ratio of 10 mouse myeloma cells to viable spleen cells. The spleen cells and myeloma cells can then be pelleted together. The pellet was slowly transferred to 1 mL of 50% (w / v) PEG / PBS solution (PEG molecule) at 37 °C. The solution can then be resuspended in 1,450 mL of 100 mM NaCl (Sigma) for 1 minute. Slowly add 10.5 mL of RPMI 1640 medium (37°C) containing pestle. Fusion can be stopped by rotating the fused cells at 500-1500 rpm for 5 min. The cells were then soaked in HAT medium (25 mM Hepes, 10% fetal clone I serum, 10% fetal bovine ... Hyclone, 1 mM sodium pyruvate, 4 mM L-glutamine, 10 μ g / mL gentamicin, 2.5% Origen culture supplement (Fisher); 10% 653-adjusted RPMI 1640 / Hepes medium, 50 μM 2-mercaptoethanol 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine After resuspending in RPMI 1640 medium, place in 15 wells of a 96-well flat-bottom tissue culture plate. Plate 200 μL / well on a 10-well plate. Then incubate in 5% CO2 and 95% air for 7-10 days. Place the plate into a humidified 37 °C incubator containing

[0206] Detection of human IgG anti-TNF antibodies in mouse serum. Mouse sera can be screened for human IgG antibodies specific for F. Plates can be simply 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 1% (w / v) BSA in PBS, 200 μL / well for 1 h at room temperature. Plates can be used immediately or frozen at -20°C for future use. Mouse serum dilutions were plated at 50 μL / well onto TNF-coated plates in a 30-well plate culture. Incubate at room temperature for 1 hour. Wash the plate and then resuspend the plate in 1:3 PBS containing 1% BSA. 50 μL / well Fc-specific HRP-conjugated goat anti-human IgG diluted at 0,000 Probe for 1 hour at room temperature. Allow plate to wash again, 100 μL / well citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0 0.01% H2O2 and 1 mg / mL OPD) is added over 15 minutes at room temperature. Add 25 μL / well of reaction stop solution (4N sulfuric acid) and measure the reaction using an automated plate spectrophotometer. Read OD at 490 nm.

[0207] Detection of fully human immunoglobulins in hybridoma supernatants using a suitable EIA Thus, growth positive hybridomas secreting fully human immunoglobulins can be detected. Briefly, 96-well pop-out plates (VWR, 610744) were incubated at 4°C overnight. 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 Incubate for 1 hour at 7° C. Wash the plate and resuspend in 1:10 PBS with 1% BSA. Probe with HRP-labeled goat anti-human kappa diluted at 1:10,000 for 1 hour at 37°C. The plates are then incubated with substrate solution as described above.

[0208] Determination of fully human anti-TNF reactivity. The above hybridomas 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. After incubation at room temperature for 1 hour, the cells were probed with radiolabeled TNF at the appropriate count per well. The wells are washed twice with PBS and the bound radiolabeled antibody is counted using a suitable counter. Quantify TNF.

[0209] 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.

[0210] Isotype. Determination of the antibody isotype was performed by screening mouse immune sera for specific titers. This can be accomplished using an EIA similar in format to that used to clean the 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.

[0211] Binding kinetics of human anti-human TNF antibodies with human TNF. The binding characteristics of the antibodies can be determined, for example, by The purified NF capture EIA and BIAcore technology can be used to suitably evaluate the The concentrations of human TNF antibodies administered were graded at 2 μg / mL in the assay as described above. Binding to TNF-coated EIA plates can be assessed. The OD can then be expressed as a semi-log plot showing the relative binding efficiency.

[0212] Quantitative binding constants can be determined, for example, as follows, or by any other known suitable method: The BIAcore CM-5 (carboxymethyl) chip was used for the BIA Place in core 2000 unit. HBS buffer (0.01M HEPES, 0. 15M NaCl, 3mM EDTA, 0.005% v / v P20 surfactant, pH 7.4) on the flow cell of the chip at 5 μl / min until a stable baseline is obtained. 15 mg of EDC (N-ethyl-N'-(3-dimethyl-amino)phenyl) in 200 μL of water was Dissolve 100 μL of a solution of 2. Add 3 mg of NHS (N-hydroxysuccinimide) to a solution of 100 μL. Inject 40 μL of the solution obtained as above onto the chip. 15 μg / mL in 1 mM sodium acetate, pH 4.8) was injected onto the chip, and approximately 500 R The buffer was changed to TBS / Ca / Mg / BSA running buffer (20 mM Tris , 0.15M sodium chloride, 2mM calcium chloride, 2mM magnesium acetate, 0.5 % Triton X-100, 25 μg / mL BSA, pH 7.4) and place on the chip overnight Equilibrate it by running it through 1000 ml of ... .

[0213] 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 derivatized (sample) and the second underivatized (blank). 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 injecting guanidine dihydrochloride twice sequentially (to prevent tissue necrosis) Dissociation of the α-antibody complex.

[0214] Analysis of the data was performed using BIA evaluation 3.0 or CLAMP2, which are known in the art. For each antibody concentration, a blank sensogram is compared with the sample sensogram. Dissociation (k d, sec -1 ) and meeting (k a ,mol -1 sec -1 )of A global fit was performed for both, and 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 Further dilutions of the body are performed.

[0215] Results and Discussion Generation of anti-human TNF monoclonal antibodies. Several fusions were made to identify the specific target of human TNF. Each fusion yielding several dozen antibodies was seeded onto 15 plates (1440 wells / fusion). Some of these are known to consist of combinations of human and mouse Ig chains. The remaining hybridomas secrete anti-TNF antibodies consisting of only human heavy and light chains (s All human hybridomas are predicted to be IgG1κ.

[0216] Binding kinetics of human anti-human TNF antibodies. ELISA analysis showed that most of these hybridomas Confirm 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 efficiency of these antibodies. Measures the binding activity to the antigen (epitope). TNF is directly bound to the EIA plate. This can lead to denaturation of the protein, and the apparent binding affinity can be reduced relative to that of the native protein. It should be noted that the 50 percent binding cannot reflect the binding to Found across a wide range of concentrations.

[0217] 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 a very high affinity Reveals that it is sexual.

[0218] Conclusion. Some fusions incorporate human variable and constant region antibody transgenes that are immunized with human TNF. This is carried out 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 are further characterized. Some of the antibodies generated were at 1×10 9 ~9×10 12The unexpected properties of these fully human monoclonal antibodies are The high affinity makes them suitable for therapeutic use in TNF-dependent diseases, pathologies or related conditions. It becomes something.

[0219] Example 3: Generation of human IgG monoclonal antibodies reactive to human TNFα. Summary. A human antibody containing both heavy and light chain human variable and constant region transgenes (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 produce fully human IgG1κ monoclonal antibodies. The strain was submitted to Molecular Biology for further characterization. Since the sequences of these Mabs are completely human, they are the same as those of human cA2 (Remicad It is predicted to be less immunogenic than e).

[0220] 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-monoclonal 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.

[0221] 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 immunogenicity-related side effects. and can be used therapeutically to inhibit inflammatory processes involved in TNFα-mediated diseases. It is expected that these specific antibodies can be used since they are used in

[0222] Materials and Methods. Animal transfection that expresses human immunoglobulins but does not express mouse IgM or Igκ The genetic mice were developed by GenPharm International. These mice have undergone V(D)J joining, heavy chain class switching and somatic mutations. The aim of this study was to develop a functional human antibody vector that generates a repertoire of antigen-specific human immunoglobulins (1). The light chain transgene contains, in part, approximately half of the germline human Vκ locus. It is derived from a yeast artificial chromosome clone containing several VH genes as well as heavy chain (H C) The transgene encodes both human μ and human γ1(2), and / or γ3 constant regions. Immunization and fusion protocols for generating the monoclonal antibodies described herein In the process, mice from the HCo12 / KCo5 genotype strain were used.

[0223] 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 media from C237A cells by affinity chromatography. The supernatant was purified by diluting the cell supernatant in 1 / 9 of its volume of 10X Dulbecco's PBS. The mixture was mixed with (D-PBS) and passed through the column at 4°C at 4 mL / min. The column was washed and TNFα was eluted with 0.1 M sodium citrate, pH 3.5, and then with 2 M Tris The purified TNFα was neutralized with HCl, pH 8.5. The solution was buffer exchanged to sodium chloride, pH 7.5, and filtered through a 0.2 um syringe filter. It was passed.

[0224] Immunization. Female GenPharm mice, approximately 16 weeks of age, were immunized with equal doses of 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 1000 μg ... Blood was allowed to clot at room temperature for 1 hour, and serum was collected and analyzed using a TNFα solid-phase EIA assay. After injection on the 28th day, mice were allowed to rest for 7 weeks before titrating GenTNV. The fusion, named IL-1, was then performed with a specific human IgG titer of 1:160 against TNFα. Vaccinate mice and inject 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 phosphate buffer containing 0.25 μg / mL amphotericin B (PSA). The spleen was then immersed in saline-buffered saline (PBS). Spleen cells were harvested 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.

[0225] Cell lines. The Cell Biology Services (CBS) group On May 14, Centocor's Product Development Group The cell lines received the non-secreting mouse 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% FBS and 5% D After cryopreservation in MSO (Sigma), the tissue was stored in a vapor phase liquid nitrogen freezer at CBS. The cell bank was sterile (Quality Control Centocor, Mal vern) and mycoplasma (Bionique Laboratories) were not present. Cells were maintained in logarithmic growth culture until confluence. Before confluence, they were washed in PBS. Cells were washed, counted, and viability determined by trypan blue dye exclusion (>95%).

[0226] Human TNFα was produced by a recombinant cell line, designated C237A, from Centococcus cerevisiae. The cell lines were generated at Molecular Biology, OR. BS (Cell Culture Labs), 2 mM L-glutamine (all from JRH (from Biosciences) and supplemented with 0.5 μg / mL mycophenolic acid. Expanded in MDM medium (JRH Biosciences), 95% FBS and 5% DM After freezing and preserving in SO (Sigma), the specimens were stored in a vapor-phase liquid nitrogen freezer in CBS (13). The cell bank was sterile (Quality Control Centocor, Malvern), and mycoplasma (Bionique Laboratories). There wasn't.

[0227] Cell fusion. Cell fusion was performed using 653 mouse myeloma cells and mouse viable spleen cells in 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 pellet was resuspended in 10.5 mL of R Fusion was induced by slowly adding PMI medium (without additives) (JRH) (37 °C). The fused cells were centrifuged at 750 rpm for 5 min. 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 (Fis her), 50 μM 2-mercaptoethanol, 1% 653-adjusted RPMI medium, 10 RP containing 0 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine After resuspending in 100 mL of 1000 mM MgCl / HEPES medium, five 96-well flat-bottom tissue culture plates were The plates were then incubated at 37 °C for 7-10 days in 5% CO2 and 95% air. Plates were placed in a humidified 37° C. incubator.

[0228] Detection of human IgG anti-TNFα antibodies in mouse serum. Mouse sera were screened for human IgG antibodies specific for NFα. Plates were coated overnight with 1 μg / mL TNFα in 0.02% (v / v ) After washing with 0.15M saline containing Tween 20, the wells were washed with 1% The plates were blocked with 200 μL / well (w / v) BSA for 1 hour at room temperature. The cells were either used or frozen at -20°C for later use. Serum was incubated with human TNFα coating in serial two-fold dilutions at 50 μL / well for 1 h 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 h at room temperature. Wash and add 100 μL / well of citrate-phosphate substrate solution (0.1 M citric acid and 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 and the reaction was automatically stopped. The OD was read at 490 nm using a moving plate spectrophotometer.

[0229] Detection of fully human immunoglobulins in hybridoma supernatants. GenPharm Mouse can produce both mouse and human immunoglobulin chains, thus producing two distinct Growth positive hybrids for the presence of both human light chains and human heavy chains using an EIA assay. Plates were coated as above and undiluted hybridoma clones were tested. Lydoma supernatant was incubated on the plate for 1 hour at 37° C. The plate was washed and HRP-conjugated yRP was incubated at 37°C for 1 h ... Gy anti-human κ (Southern Biotech) antibody or in 1% BSA-HBSS either HRP-conjugated goat anti-human IgG Fc-specific antibody diluted 1:30,000 in The plates were then incubated with substrate solution as described above. Both the anti-human κ and anti-human IgG Fc EIA formats did not yield a positive signal. Hybridoma clones that were not isolated were discarded.

[0230] Isotyping. Determination of the antibody isotype was performed by screening mouse immune sera for specific titers. This was accomplished using an EIA format similar to that used for cleaning. EIA with goat anti-human IgG (H+L) at 10:g / mL in sodium carbonate buffer overnight at 4°C. Plates were coated and blocked as described above. Undiluted cultures from 24-well cultures The supernatant was incubated on the plate for 1 hour at room temperature. The plate was washed and diluted with 1% BS A-HRP-conjugated goat anti-human IgG1, IgG2, Ig ​​diluted 1:4000 in PBS The plate was probed with G3 or IgG4 (Binding Site) for 1 hour at room temperature. The plates were washed again and incubated with substrate solution as above.

[0231] Results and Discussion. Generation of fully human anti-human TNFα monoclonal antibodies. Recombinant human TNF A fusion virus named 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. (>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.

[0232] Parental cells harvested from wells of 24-well culture dishes for each of the eight cell lines were transfected with For transfection and further characterization, the product was submitted to Molecular It was handed over to the Biology group.

[0233] [Table 3]

[0234] 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 purchased from Molecular Biology for further characterization and development. One of these new human antibodies was transferred to the Retina Biology group. Compared with micade, it has the potential benefit of reducing immunogenicity and allergic-type complications. may be useful in anti-inflammatory purposes.

[0235] References Taylor,et al.,International Immunology 6:579-591(1993). Lonberg,et al.,Nature 368:856-859(1994) . Neuberger, M. Nature Biotechnology 14:826 (1996). Fishwild,et al.,Nature Biotechnology 14 :845-851(1996). Scallon, et al., Cytokine 7:759-770 (1995) .

[0236] Example 4: Cloning and preparation of cell lines expressing human anti-TNFα antibodies. Summary. A panel of eight human monoclonal antibodies (mAbs) expressing TNV showed The eight mAbs were found to bind 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 of mAbs were identified. The original panel of mAbs was represented by TNV14, TNV15, TNV148, and TNV196. The predicted amino acid sequence of each mAb was also shown to be the only one of 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.

[0237] A database search identified a proline residue at position 75 (framework 3) of the TNV148 heavy chain. Since it was not found in that position in any other human antibody of the same subgroup, it was identified as a known germline variant. 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 amplified DNA fragments encoding the heavy and light chain variable regions of TNV148B and TNV14 were NA was cloned to the recently cloned heavy and light chain genes of another human mAb (12B75). Based on the IL-12 Antibodies published in International Publication No. WO 02 / 12500, Dies, Compositions, Methods and Uses No. 60 / 236,827, filed October 7, 2000, which is incorporated by reference in its entirety. The resulting cDNA was cloned into a newly prepared expression vector (herein incorporated).

[0238] P3X63Ag8.653 (653) cells or Sp2 / 0-Ag14 (Sp2 / 0) mice Mouse myeloma cells were transfected with the respective heavy and light chain expression plasmids and expressed at high levels. Bell's recombinant TNV148B and TNV14 (rTNV148B and rTNV14) mAbs Cell lines producing β were screened by two rounds of subcloning. Growth curve and stability evaluation of mAb production in 653 transfectant clone C4 66D and C466C stably expressed rTNV1 at approximately 125:g / mL in spent cultures. 48B mAb was generated, whereas Sp2 / 0 transfectant 1.73-12-122 (C467A) was stable in spent cultures at approximately 25:g / ml of rTNV148B Similar analyses showed that Sp2 / 0 transfectant clones produced mAbs. showed that rTNV14 produced 18 μg / ml of rTNV14 in spent cultures. did.

[0239] Introduction. Human TNFα-immunized GenPharm / Medarex mice (HCo12 / K A panel of eight mAbs derived from the Co5 genotype bind to human TNFα and are fully human I We have previously shown 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 the exemplary mAbs may have TNFα neutralizing activity. Based on the results, DNA sequence results, and in vitro characterization of some of the mAbs, TNV148 was selected as the mAb to be characterized.

[0240] The DNA sequence encoding the TNV148 mAb was cloned and a suitable constant region was added. The well-characterized 653 and 654 genes were modified to fit into expression vectors encoding the and Sp2 / 0 mouse myeloma cells, and the resulting transfected The cell line was then subcloned to identify a subclone that produced 40-fold more mAb than the original hybridoma cell line. Screening was continued until

[0241] Materials and Methods. Reagents and cells. TRIZOL reagent was purchased from Gibco BRL. Proteinase K was obtained from Sigma Chemical Company. Reverse transcriptase was from Life Sciences, Inc. Taq DNA polymerase was obtained from Perkin E The restriction enzymes were obtained from either Imer 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 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 Ource International. The names, identification numbers, and sequences of the peptides are shown in Table 2.

[0242] Table 2. Antibodies used to clone, engineer, or sequence TNV mAb genes The oligonucleotides The amino acids encoded by oligonucleotides 5'14s and HuH-J6 are sequenced. The "M" amino acid residue represents the translation initiation codon. Oligonucleotide 5'14 The underlined sequences in s and HuH-J6 denote the BsiWI and BstBI restriction sites, respectively. The diagonal lines in HuH-J6 correspond to exon / intron boundaries. Note that the corresponding oligonucleotides are written in the 3'-5' orientation.

[0243] [Table 4]

[0244] One frozen vial of 653 mouse myeloma cells was obtained. The vial was thawed on the same day and incubated with T cells. The cells were expanded in IMDM, 5% FBS, and 2 mM glutamine (culture medium) in Lasco. These cells were transfected 2-3 weeks later with anti-TNF DNA as described herein. The cultures 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 incubated for 30 min at 4°C for 1 h. The cells were aliquoted into 100 ml, frozen, and stored for future use. One frozen vial of cells was obtained. The vial was thawed and a new freeze-dried sample was prepared as described above. The frozen vials were stored in freezer boxes AA and AB at the CBC. These cells were thawed and used for all Sp2 / 0 transfections described herein. Used.

[0245] Assay for inhibition of TNF binding to the receptor. Using dorsal cell supernatant, the mAb was used to detect the expression of recombinant TNF receptor fusion protein p55-sf2 to 125 The antibodies were assayed for their ability to block the binding of I-labeled TNFα (Scallon et al. (1995) Cytokine 7:759-770). 1 hour at 37°C During the incubation, 50 μl of p55-sf2 was added to Op Add to tiplates and coat wells. Dilute in PBS / 0.1% BSA Serial dilutions of eight TNV ​​cell supernatants were prepared as 100 μg / mL in 96-well round-bottom plates using 100 μg / mL of TNV cells as the 100 μg / mL antibody. Cell supernatant containing anti-IL-18 mAb was included as a negative control, and cA2 (Anti-TNF Chimeric Antibody, Remicade, U.S. Pat. No. 5,770,198, see US Pat. No. 5,770,198) The same anti-IL-18 supernatant spiked with IL-18 (which is incorporated herein in its entirety) served 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 μl of cell supernatant. The coated Optiplates were pre-incubated at room temperature for 1 hour. Remove unbound p55-sf2 by 50:1 125 I-TNFα / cell supernatant mixture After 2 hours at room temperature, the plates were washed three times with PBS-Tween and transferred to Optiplates. The plates were washed. 100:1 of Microscint-20 was added and TopC Bound cpm was determined using a Count gamma counter.

[0246] 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 tubes were shaken The samples were 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 shaken vigorously and incubated at room temperature for 10 minutes. The samples were then centrifuged for 10 min at 4°C. The pellet was then diluted with 1 ml of 70% ethanol. The RNA pellet was washed once with 500 µl of DEPC and briefly dried in a vacuum dryer. 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.

[0247] To prepare heavy and light chain cDNA, 3 μl of RNA and 1 μg of either oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light chain) A mixture containing either (see Table 1) was prepared. The mixture was heated at 70° C. for 1 hour in a water bath. After incubation for 0 min, the mixture was cooled on ice for 10 min. 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 then incubated at 42°C for 40 minutes. The cDNA synthesis reactions were stored in a -20°C freezer.

[0248] Unpurified heavy and light chain cDNAs were used as templates to clone the variable region coding sequences. Five oligonucleotides were assayed for their ability to prime amplification of heavy strand 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 qDNA polymerase. Each reaction contained 2 μl of cDNA A reaction: 10 pmol of each oligonucleotide, 0.2 mM dNTPs, 5 μl 10 The thermal cycler program contained 2 mM magnesium sulfate. The incubation was performed at 95°C for 5 min, followed by (94°C for 30 s, 62°C for 30 s, and 68°C for 1.5 The reaction was incubated at 68°C for 10 min for 30 cycles, followed by a final incubation at 68°C for 10 min. It was held.

[0249] To prepare PCR products for direct DNA sequencing, follow the manufacturer's protocol. 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 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 cooling program consisted of 25 cycles of 96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 20 seconds. The reaction products were then run on a polyacrylamide gel for 4 min at 4° C. were fractionated and detected using an ABI 377 DNA sequencer.

[0250] Site-directed mutagenesis to change the amino acid Pro in TNV148 mAb 75 To replace the nucleotide sequence with a serine residue, a single nucleotide of the TNV148 heavy chain variable region DNA sequence was The complementary oligonucleotides 399 and 400 (Table 1) were designed and prepared. QuikChange™ site-directed mutagenesis as described by the manufacturer This change was made using the method described above. The oligonucleotide was first fractionated and the major band was purified. 1 TNV148 heavy chain plasmid template (p1753), 5 μl of 10X reaction buffer reagent, 1 μl dNTP mix, 125 ng primer 399, 125 ng primer Mutagenesis reactions were prepared using 400 and 1 μl of Pfu DNA polymerase. Sterile water was added to bring the total volume to 50 μl. It was then heated at 95°C for 30 seconds, followed by Cycles of 95°C for 30 s, 55°C for 1 min, 64°C for 1 min, and 68°C for 7 min. The procedure was repeated 14 times, followed by incubation at 30°C for 2 min (1 cycle). The reaction mix was incubated in a programmed thermal cycler. The mutagenic oligonucleotide was then injected into an otherwise identical newly synthesized plasmid. To remove the original TNV148 plasmid, After adding 1 μl of DpnI endonuclease, which cuts only the chilled plasmid, the test The mixture was incubated at 37°C for 1 hour. 1 μl of the reaction was then used for standard thermal Shock method for Epicurian Coli XL1-Blue Super Competitive The transformants were transformed into Escherichia coli and plated on LB-ampicillin agar plates. The transformed bacteria were identified using the Wizard™ kit as described by the manufacturer. Plasmid minipreps were prepared by eluting the samples from the Wizard™ columns. Afterwards, the plasmid DNA was further purified by precipitating it with ethanol. After that, the DNA was sequenced to confirm the desired base changes. Identify plasmid clones that have nucleotide changes that were not inadvertently introduced within the TNV148 coding sequence. We confirmed that the same parameters were not introduced into the For the purpose, 1 μl of plasmid, 3 μl of BigDye mix, 1 μl of pUC19F forward primer, and 10 μl of sterile water were used to prepare cycle sequencing reactions. Ta.

[0251] Construction of an expression vector from the 12B75 gene. Several recombinant DNA steps were carried out to From previously cloned genomic copies of the 12B75-encoding heavy and light chain genes, A new human IgG1 expression vector and a new human κ expression vector were prepared, respectively. published in International Publication No. WO 02 / 12500, s, Compositions, Methods and Uses, 200 No. 60 / 236,827, filed Oct. 7, 2000, the entire contents of which are incorporated herein by reference. The final vector can be constructed using any appropriately designed P Designed to allow for simple, one-step replacement of existing variable region sequences with CR amplified variable regions. It was.

[0252] To modify the 12B75 heavy chain gene of plasmid p1560, a promoter and a possible The 6.85 kb BamHI / HindIII fragment containing the mutated region was cloned from p1560 to pU This plasmid was transferred to C19 to generate p1743, which is smaller in size than p1560. The plasmid was engineered to contain a unique BsiWI primer immediately upstream of the translation start site according to the manufacturer's protocol. Use of QuikChange™ mutagenesis to introduce cloning sites (Using oligonucleotides BsiWI-1 and BsiWI-2) The resulting plasmid was called p1747. A BstBI site was inserted 3' of the variable region. The 5' oligonucleotide primer was designed to introduce SalI and BstBI sites at the ends. This primer was used together with the pUC reverse primer to create p1747 A 2.75 kb fragment was amplified from the naturally occurring 12B75 variable region. The unique Bs The tB1 site was introduced. The resulting intermediate vector, designated p1750, It was able to accept a variable region fragment with siWI and BstBI ends. To prepare a version of the heavy chain vector derived from the 2B75 gene, The amHI-HindIII insert contains an EcoRI site downstream of the HindIII site. The resulting plasmid p1768 was then transferred to pBR322 to carry the was digested with HindIII and EcoRI to create a large BamHI fragment from p1560 to pBC. p174, a subclone obtained by cloning the -BamHI fragment The 5.7 kb HindIII EcoRI fragment from 4 was ligated to the The resulting plasmid, p1784, is a TNV A plasmid containing BsiWI and BstBI ends. b It was used as a vector for cDNA fragments. Further work was carried out to clone the 12B75 gene from It contains the IgG1 constant region and is classified according to the extent to which it contains the 12B75 heavy chain JC intron. This was carried out to prepare expression vectors p1788 and p1798, which differ from each other in terms of the sequence.

[0253] To modify the 12B75 light chain gene of plasmid p1558, the 12B75 promoter was The 5.7 kb SalI / AflII fragment containing the marker and variable regions was extracted from p1558 The new plasmid p174 was transferred to the XhoI / AflII sites of plasmid L28. 5 provided a smaller template for the mutagenesis step. Using the codes (C340salI and C340sal2), ) 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. The DNA fragment contained allI and AflII restriction sites. The variable region fragment will preferably be linked 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 can be used to The oligonucleotides BAHN-1 and BAHN-2 were annealed to each other to create the restriction site Bs Contains iW1, AflII, HindII, and NotI, and KpnI and SacI sites A double-stranded linker was formed containing ends that could be ligated to pBC This was cloned between the KpnI and SacI sites of the 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 cloned into the Af This new clone was cloned between the III and HindII sites to give p1762. The plasmid contains a BsiWI / AflII fragment containing the promoter and the variable region. It contains unique BsiWI and AflII sites that allow the two halves of the gene to be joined and transferred. there was.

[0254] cDNA cloning and assembly of expression plasmids. Additionally, all RT-PCR reactions (see above) were treated with Klenow enzyme. The PCR fragment was digested with the restriction enzymes BsiWI and BstBI and then transformed into plasmid L28( Since the 2B75-based intermediate vector p1750 had not yet been prepared, L28 was used. The cloned insert was cloned between the BsiWI and BstBI sites of 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.

[0255] The BsiWI / BstBI inserts in the heavy chains of TNV14, TNV148, and TNV148B The vector was transferred from the L28 vector to the newly prepared intermediate vector p1750. The identification numbers assigned to these intermediate plasmids are shown in Table 2. No further steps were performed on TNV15 and TNV196. The variable regions were then The vector was then cloned into two different human IgG1 expression vectors. The vector was then cloned into two different human IgG1 expression vectors using 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 The expression plasmids used were p1781 (TNV14), p1782 (TNV148), and p The variable region was also designated 12 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. They are listed in 3.

[0256] 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 cloned into an incomplete 12B75 vector to generate an intermediate plasmid. One additional transfer step allowed the DNA to be linearized and then introduced into cells. or used to purify mAb gene inserts prior to cell transfection The results were consistent with those reported in Table 1. ND = not performed.

[0257] [Table 5]

[0258] The light chain PCR product was digested with restriction enzymes SalI and SacII and then cloned into the plasmid pBC The two clones differed in one amino acid and 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. They were cloned between the SalI and AflII sites to give p1755 and p1 These 5' halves of the light chain genes 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 then ligated to produce the final expression plasmids p1775 and p17 76 were produced (Table 2).

[0259] Transfection, screening and subcloning of cells. A total of 15 mouse Myeloma cells were transfected with various TNV expression plasmids ( Results and (See Table 3 in the Discussion section.) These transfections are: (1) infecting the host cells; (2) the heavy chain constant region is a previous I of Centocor (3) the mAb was encoded with a gG1 vector or the 12B75 heavy chain constant region, or Was it 148B, TNV148, TNV14, or a 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 chances of screening.

[0260] 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 1 and 16, proper expression was confirmed by digestion with restriction enzymes prior to transfection. The current plasmid was linearized. For example, the SalI and NotI restriction enzymes were inserted into TN To linearize the V148B heavy chain plasmid p1783 and the light chain plasmid p1776 For the remaining transfections, the heavy chain plasmid was ligated with BamHI and Extract only the mAb gene by digesting the light chain plasmid with BsiWI and NotI. The DNA insert containing the nucleotide 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 selection marker. was co-transfected with the PstI-linearized pSV2gpt plasmid (p13) After electroporation, cells were cultured in IMDM, 15% FBS, 2 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, 2× MH X-selection (1X MHX = 0.5:g / mL mycophenolic acid, 2.5:g / mL Hypoxanthine, 50 μg / mL xanthine) were added and incubated for colony formation. The plates were incubated for 2 to 3 weeks.

[0261] Cell supernatants harvested from wells containing colonies were analyzed by ELISA as described. Briefly, polyclonal goat anti-human IgG was used. Various dilutions of cell supernatants were incubated in 96-well EIA plates coated with Fc fragments. After incubation, alkaline phosphatase-conjugated goat anti-human IgG (H+L) and appropriate Bound human IgG was detected using the appropriate color substrate. A standard curve using purified mAbs as standards allowed for the 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 then plated onto 24-well plates for further production determination in the spent cultures. The clones were then passaged into cultures to identify the highest producing parent clones.

[0262] Subcloning the parent clone with the highest yield was performed to identify the subclones with higher yields. To prepare a more homogenous cell line, 96-well tissue culture plates were cultured in IMDM, 5% F BS, 2mM glutamine, 1x MHX, 1 cell per well or 4 per well Seed one cell per culture and incubate in a 5% CO2 incubator for 12-20 days until colonies appear. The wells were incubated at 37°C for 1 h. The wells contained one colony per well. 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 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.

[0263] Characterization of cell subclones. The best subclones were selected from the second round and growth curves were performed. The mAb production levels and cell growth characteristics were evaluated. 1x1 in MDM, 5% FBS, 2mM glutamine, and 1X MHX (or serum-free medium) 0 5 Aliquots of 300 μl were removed at 24-h intervals to determine viable cells. The density was measured. The number of viable cells was 1 × 10 5 Analysis continued until the number of cells / ml was less than 1. Aliquots of the collected cell supernatants were assayed for the concentration of antibody present. ELISA assay using rTNV148B or rTNV14 JG92399 Polyclonal goat anti-human IgG Fc coated ELISA plates were used. The samples were incubated on a plate for 1 h and the bound mAb was then washed with 1:1000 dilution of alkaline phosphatase. Detection was with sulfatase-conjugated goat anti-human IgG (H+L).

[0264] 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 for the cell lines C466A and C466B in MHX-free medium. The cells were thawed in medium (IMDM, 5% FBS, 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 1000 mg / mL xanthine or 1000 mg / mL ethanol. After 1 day, new T7 5 flasks, 1 x 10 5 Cultures were seeded at a starting density of cells / ml and cells were cultured for 1 week, 24 Counts were taken at timed intervals. No aliquots were withdrawn for mAb production. SOP PD Doubling times for these samples were calculated using the formula provided in 32.025.

[0265] 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 cultures became confluent, fresh The cultures were split and the old cultures were then allowed to deplete. At this time, aliquots of the supernatant were taken and The cells were then removed and stored at 4°C. Aliquots were removed over a period of 55-78 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.

[0266] 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 β-lactamase to β-lactamase was inhibited by ELISA, a simple binding assay was performed. Standard ELISA analysis of human IgG revealed that TNV ​​mAbs in each cell supernatant were The concentration was first determined. Then, the recombinant p55 TNF receptor / IgG fusion protein p55 -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 TNV All but one of the mAbs (TNV122) inhibited the binding of TNFα to the p55 receptor. In fact, TNV mAb was spiked into negative control hybridoma supernatants and effectively blocked appeared to be more effective at inhibiting TNFα binding than the cA2 positive control mAb. These results demonstrate that TNV ​​mAbs inhibit the bioactivity of TNFα in cell-based assays and in vivo. indicates that the effect of the serotonin receptor agonist is likely to be disruptive and therefore requires further analysis. Interpreted.

[0267] Analysis of DNA sequences. 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 hybridomas that generate these mAbs, Total RNA was isolated from the dorma cell lines. Each RNA sample was then used to assay the complete expression of each mAb. A human antibody heavy chain comprising a signal sequence, a complete variable region sequence, and a portion of a constant region sequence, The light chain cDNA was then prepared. These cDNA products were then amplified in a PCR reaction to obtain the The PCR-amplified DNA was sequenced directly without cloning the fragment into The heavy chain cDNA is a clone of DP- 46 (Figure 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 were human. It was confirmed that the antibody heavy chain and human antibody light chain were encoded. The variable region contained a signal sequence code. The PCR amplification was performed 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 the sequences shown represent the actual sequences of the recombinant TNV ​​mAbs, although they may be different.

[0268] Specific neutralizing mAb. Analysis of the cDNA sequences of the entire variable regions of both the heavy and light chains of each mAb demonstrated that TNV32 is TNV15 is identical to TNV15, TNV118 is identical to TNV14, and TNV86 is identical to TNV1 48. The results of the receptor binding assay were consistent with the DNA sequence analysis. The results were consistent, i.e., both TNV86 and TNV148 were effective in blocking TNF binding. It was about four times better than both TNV118 and TNV14. are four unique TNV mAbs, TNV14, TNV15, TNV148, and We focused only on TNV196.

[0269] 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, the heavy chain CDR3 sequences were highly similar and of the same length. Because they all use the J6 exon, they are clearly 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 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 (Figure 3).

[0270] The deduced amino acid sequences of the four mAbs revealed the actual relatedness of the mAbs. Abs contain four distinct heavy chains (Figure 4) but only two distinct light chains (Figure 5) The differences between the TNV mAb and germline sequences 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 regions, the TN V14 is identical, TNV15 differs by one amino acid, and TNV148 differs by two amino acids. TNV196 differed by three amino acids, while TNV197 differed by only one amino acid.

[0271] 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 fitting 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 It was digested with SalI and AflII and cloned into the plasmid vector pBC. Individual clones are then sequenced to identify the locations of the potentially heterogeneous molecular population. The results were obtained from direct sequencing of PCR products revealing the most abundant nucleotide at the position. It was confirmed that the sequence was identical to the previous one.

[0272] Site-directed mutagenesis to modify TNV148. mAb TNV148 and TNV 196 was 4.4 times more effective than the next best mAb (TNV14) in neutralizing TNFα bioactivity. However, as mentioned above, TNV148 and The 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 The Pro residue at position 75 was shown to be a rare amino acid at that position.

[0273] A similar comparison of the TNV196 heavy chain reveals three distinct regions in framework 3 that differ from the germline sequence. These differences suggested that the amino acids may be rare in human mAbs. When administered to humans, 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 5'-amino acid residue was coded for in place of the Pro residue at position 75. A single nucleotide was changed in the p1753 plasmid. The plasmid was called p1760 (see Table 2). The mAb was designated TNV148B to distinguish it from the original TNV148 gene and mAb. called (see Figure 5).

[0274] Assembly of the final expression plasmid. 12B7 was previously cloned as a genomic fragment. 5. New antibody expression vectors based on heavy and light chain genes were prepared. Plasmids were prepared (see Table 2), in each case containing the 5' furan The king sequence, promoter, and intron enhancer are located in each 12B75 gene. The light chain expression plasmid contained the complete JC intron, the constant region coding region The sequence, and the 3' flanking sequence, were also derived from the light chain gene of 12B75. Regarding the heavy chain expression plasmids that gave rise to the The human IgG1 constant region coding sequence was used in the expression vectors used before Centocor. Importantly, the final product cell line reported here was derived from the original The hybridoma-derived TNV mAb (G1m(z)) was of a different allotype (Gm(f +)) TNV mAb, which is derived from the GenPharm mouse 12B The 75 heavy chain gene encodes an Arg residue at the C-terminal end of the CH1 domain, but The IgG1 expression vector p104 of cor encodes a Lys residue at that position. The JC intron, the complete constant region coding sequence, and the 3' flanking sequence are Other heavy chain expression plasmids derived from 75 heavy chain genes (e.g., p1786, p1788) were prepared, but cell lines transfected with these genes were not used as production cell lines. The vector was not selected. After PCR amplification, the final expression plasmid was obtained. It was carefully designed to allow one-step cloning of the V regions.

[0275] The PCR-amplified variable region cDNA was extracted from the L28 or pBC vector to the promoter region and and part of the JC intron were transferred to an intermediate 12B75-based vector (Pla (See Table 2 for midi identification numbers.) Next, Restriction fragments were extracted from these intermediate vectors to provide the 3' halves of each gene. Transfer the plasmid to the final expression vector and generate the final expression plasmid (see Table 2 for plasmid identification numbers). (see reference).

[0276] Transfection and subcloning of cells. Expression plasmids were purified by restriction digestion. Either the antibody gene insert in each plasmid was linearized by the plasmid backbone or the antibody gene insert in each plasmid was linearized by the plasmid backbone. Sp2 / 0 and 653 mouse myeloma cells were either purified from electroporated or electroporated. The heavy and light chain DNAs were transfected by troporation. Various transfections were performed, most of which were unique as defined by Ab. Whether the gene is on a linearized whole plasmid or a purified gene insert Regardless, specific characteristics of the Ab genes and host cell lines were (summarized in Table 4 Cell supernatants from clones resistant to mycophenolic acid were assayed for the presence of human IgG. Assayed by LISA, using purified rTNV148B as a reference standard curve Quantified.

[0277] 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) and subcloned To screen for higher producing cell lines, a more homogenous cell population was prepared. Two of the subclones of the parent strains 2.320, 2.320-17, and 2.320-20 produced approximately 50 μg / ml in spent 24-well cultures, which is comparable to their parent strains. The subcloned strains 2.320-17 and 2.320 This was the result of a second subcloning of -20.

[0278] The identification numbers of the heavy and light chain plasmids encoding each mAb are shown. Purified mAbs For transfections made with gene inserts, the source of the gpt selection marker The heavy chain constant region was from Remica The same human IgG1 expression vector ("old") used to encode 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 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 C4 67A were derived from transfection numbers 2 and 1, respectively. Cell line C476A was derived from transfection no. 3.

[0279] [Table 6]

[0280] ELISA assays with spent 24-well culture supernatants were performed to confirm these second rounds of subcloning. All of the clones produced 98–124 μg / mL, which is consistent with the initial subclones. These 653 cell lines were enriched in 100% IgG4-dependent IL-16 expression, resulting in at least a two-fold increase in IL-16 expression, as shown in Table 5. Assigned C chord notation.

[0281] Of the Sp2 / 0 parental strains, the highest yield from rTNV148B transfection 1 The parent strain 1.73 was subcloned twice 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).

[0282] Highest producing rTNV14 cell line Three of the best-generated Sp2 / 0 parental lines from rTNV14 transfection 3 The subclone 3.27-1 had a production of 19 μg / ml. and was found to be the highest producer in spent 24-well cultures. The strain was designated C476A (Table 5).

[0283] Table 5. Summary of selected generating cell lines and their C codes. The first digit of the original clone name indicates which transfection the cell line was derived from. All of the C-encoded cell lines reported herein contain heavy and light chains linearized with restriction enzymes. Derived from transfection with all plasmids.

[0284] [Table 7]

[0285] 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 numbers is 1.0×10 6 ~1.25×10 6 Peak cell density in cells / ml and 110 It was shown that a maximum mAb accumulation level of ~140 μg / mL was reached (Figure 7). The highest producing Sp2 / 0 subclone, C467A, produced 2.0 × 10 6 Peak cells / mL Cell density and maximum mAb accumulation level of 25 μg / mL were reached (Figure 7). Growth curve analysis , but not performed on the rTNV14-producing cell line C476A.

[0286] Further growth curve analysis was performed to compare growth rates at different concentrations of MHX selections. This comparison shows that C466 cells cultured in the absence of MHX are more sensitive to the growth factor than C466 cells cultured in the presence of normal amounts of MHX (1X). This was prompted by recent observations that cells in culture appear to grow faster than the same cells in culture. Cytotoxic concentrations of compounds such as mycophenolic acid tend to be measured over several orders of magnitude. Therefore, using lower concentrations of MHX may sacrifice stability in mAb production. It was believed that it would be possible to significantly speed up the cell doubling time without increasing the number of Strains C466A and C466B were cultured with no MHX, 0.2X MHX, or 1X MHX. Viable cell counts were performed at 24-hour intervals for 7 days. The MHX concentration-dependent cell growth rate was revealed (Figure 8). The cell line C466A was cultured at 1X M The doubling time was 25.0 hours in HX, but only 20.7 hours without MHX. Similarly, cell line C466B showed a doubling time of 32.4 hours in 1X MHX. The doubling time was 22.9 hours without MHX. In addition, the doubling times of both cell lines in 0.2X MHX were significantly longer than those in 1X MHX. This observation is more similar to that observed in the previous study (Fig. 8). Enhanced cell performance using less MHX in bioreactors However, the stability study results (see below) suggest that this could be achieved by The results showed that the cell line C466D was able to transfect rTNV148 for at least 60 days in the absence of MHX. Although this suggests that it is possible to stably produce B, stability studies have also shown that MHX Higher mAb production levels were also observed when cells were cultured in the presence of MHX compared to its absence. showed.

[0287] 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 the selection agent. After just 2 weeks of culture, clone C4 The production of 66A was about 45% lower than at the start of the study. The production from clone C466B was also However, clones C466C and C466D showed Fairly stable production was maintained, with C466D showing the highest absolute production level (FIG. 9).

[0288] conclusion From an initial panel of eight human mAbs against human TNFα, protein sequences and TNF Based on several criteria, including neutralizing potency, TNV148B as well as TNV14 are preferred. rTNV148B >100:g / mL and rTNV148B >19:g / mL A cell line producing TNV14 was prepared.

[0289] Example 5: Arthritic mice using anti-TNF antibodies and controls using a single bolus injection Research on Approximately 4-week-old Tg197 study 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 mg g / kg of any of the anti-TNF antibodies of the present invention (TNV14, TNV148, or TNV1 96) was administered as a single intraperitoneal bolus.

[0290] Results: When body weight was analyzed as a change from pre-treatment, treatment with 10 mg / kg cA2 Animals treated with D-PBS consistently showed higher weight gain 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 the seventh week of the study. (See Figure 10).

[0291] 11A-C show the progression of disease severity based on the arthritis index. The arthritis index for the A2 treated group began at week 3 and continued throughout the remainder of the study (week 7). The 1 mg / kg TNV14-treated animals and the 1 mg / kg TNV14-treated animals were significantly lower than the D-PBS control group. Animals treated with cA2 at 100 mg / kg had significantly greater pulmonary function after 3 weeks compared to the D-PBS treated group. No significant reduction in AI could be demonstrated. When compared (10mg / kg TNV14, 148 and 196 There was no significant difference between the 1 mg / kg and 10 mg / kg treatment groups. When compared, 1 mg / kg TNV148 was 3, 4 and 5 times more potent than 1 mg / kg cA2. The AI ​​was significantly lower at weeks 1 and 7. TNV148 at 1 mg / kg was also significantly lower than that at week 1. TNV196 was significantly lower than the V14-treated group at weeks 3 and 4. A Although TNV148 showed a significant decrease in I (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.

[0292] Example 6: Study of arthritic mice using anti-TNF antibodies and controls as multiple bolus doses research Tg197 study mice approximately 4 weeks of age 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 of 100 mg / kg / day (1 eye). Injections were administered at weeks 1, 2, 3, and 4 in all animals. Groups 1 to 6 were evaluated for efficacy of the test article. Serum samples obtained from the study were used to evaluate the induction of immune responses to TNV14 or TNV148 at weeks 2, 3, and 4. and pharmacokinetic clearance were evaluated.

[0293] Results: When body weight was analyzed as the change from pre-treatment, no significant differences were observed. Animals treated with cA2 at 100 mg / kg consistently outperformed D-PBS-treated animals throughout the study. They showed high weight gain (see FIG. 12).

[0294] 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 continuing throughout the remainder of the study (week 5). The cA2 levels at 1 mg / kg or 3 mg / kg were significantly lower than those in the D-PBS control group. Animals treated with d-PBS and 3 mg / kg TNV14 showed significantly improved pulmonary function compared to the d-PBS control group. was 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 difference compared to the d-PBS treated group. When administered at 10 mg / kg, a significant reduction was observed starting at week 3 and continuing through week 5. Animals treated with cA2 at 1 mg / kg / day had better survival at lower doses of both cA2 (1 mg / kg / day) at weeks 4 and 5 of the study. 2 mg / kg) showed a significant decrease in AI and The 3 mg / kg dose was significantly lower than that of animals treated with TNV14. There appeared to be no significant difference between the AI ​​for animals treated with 3 mg / kg TNV14. was significantly higher than 10 mg / kg at one time point, whereas animals treated with TNV148 had This was not significantly different from animals treated with 10 mg / kg cA2.

[0295] Example 7: Clinical Use of Anti-TNF Antibodies and Controls as a Single Intraperitoneal Bolus in Arthritic Mice Research on Tg197 study mice, approximately 4 weeks of age, were assigned to one of six treatment groups based on sex and weight. Assigned to receive either 3 mg / kg or 5 mg / kg of antibody (cA2 or TNV148) The study included treatment with D-PBS and 10 mg / kg c The A2 control group was utilized.

[0296] 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 were still significantly elevated 8 weeks after injection (see FIG. 14).

[0297] 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 protective effect. A significant reduction in AI was observed at ~3 weeks, with all treatment groups showing a significant reduction at 2 weeks Later in the study, animals treated with 5 mg / kg cA2 showed some protection,4,6 At the low dose (3 mg / kg), both cA2 and TNV148 significantly decreased All treatment groups showed significant reductions at week 6, and all treatment groups showed significant reductions at week 7. None of the treatment groups were able to maintain a significant reduction at the end (week 8). There were no significant differences between any of the treatment groups (except for the saline control group).

[0298] Example 8: Single intraperitoneal bolus administration of anti-TNF antibody and modified anti-TNF antibody A study of arthritic mice using anti-TNF antibodies and controls TNV148 (derived from hybridoma cells) and rTNV148B (transfectant To compare the efficacy of a single intraperitoneal injection of 1000 mg / kg of Tg mice (derived from transfected cells) with that of a single intraperitoneal injection of 1000 mg / kg of Tg mice (derived from transfected cells) at approximately 4 weeks of age. 197 Study mice were assigned to one of nine treatment groups based on sex and weight, and then Becco's PBS (D-PBS) or 1 mg / kg of antibody (TNV148, rTNV1 48B) was administered as a single intraperitoneal bolus.

[0299] 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 w...

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 pharma- ceutically acceptable carrier or diluent, for use in the safe and effective treatment of active ankylosing spondylitis, wherein the composition is administered by IV infusion, wherein the anti-TNF antibody is administered at a dose of 2 mg / kg administered over 30±10 minutes at weeks 0 and 4, then every 8 weeks (q8w) thereafter, and wherein the composition is used such that patients treated with the composition achieve ASDAS inactive disease (<1.3) within 4 weeks of treatment or within 2 weeks of treatment.

2. The composition described in claim 1, comprising an aqueous medium containing histidine, sorbitol, and polysorbate 80 at a pH of 5.

5.

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

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