Manufacturing methods for producing Anti-TNF antibody compositions

Abstract

To provide an anti-TNF antibody or a fragment thereof for use as a therapeutic agent for a disease mediated by TNFα.SOLUTION: An anti-TNF antibody expressed in Chinese Hamster Ovary cells (CHO cells), including a heavy chain (HC) including a specific amino acid sequence and a light chain (LC) including a specific amino acid sequence, wherein preferably, the oligosaccharide profile of the anti-TNF antibody includes more than 99.0% of total neutral oligosaccharide species and less than 1.0% of total charged oligosaccharide species.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Reference to electronically submitted sequence listing) This application is filed on February 5, 2019 under the title "JBI6055USPSP1SEQL The sequence list will be sent via EFS-Web in ASCII format with the file name "IST.TXT". The EFS-W contains a sequence listing that is electronically submitted with a size of 21,508 bytes. The sequence listing submitted via eb is incorporated herein by reference in its entirety. is incorporated into the book.

[0002] FIELD OF THEINVENTION The present invention relates to anti-TNF antibodies, e.g., anti-TNF antibodies having the amino acid sequence of SEQ ID NO:36. A recombinant vector having a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:37 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:38. A method for producing an anti-TNF antibody and a composition comprising the recombinant anti-TNF antibody Pertaining to things. [Background technology]

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

[0004] 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 Transgenic T and B cell lines also produce TNFα.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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 These are useful targets for certain biological treatments in diseases such as cancer. Beneficial effects of successful retreatment after relapse in rheumatoid arthritis and Crohn's disease are associated with TNFα This has been reported in an open-label study using a monoclonal antibody against Beneficial results in rheumatoid arthritis have also been reported in randomized, double-blind, placebo-controlled trials.

[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 induce immune complexes of antibodies or fragments circulating in the blood. This can result in clearance mediated by IL-1, making repeated administration unsuitable for therapy, This reduces the therapeutic effectiveness of the antibody or fragment and limits its administration to the patient. For example, repeated administration of antibodies or fragments containing non-human portions may result in serum sickness and / or anaphylaxis. To avoid these and other problems, techniques well known in the art may be used. To reduce the immunogenicity of such antibodies and portions thereof, including chimerization and humanization, However, these and other approaches are still However, some of them may have low immunogenicity, low affinity, low avidity, or may be difficult to culture in cell culture, scale-up, or This can result in antibodies or fragments with problems in production and / or low yields. However, such antibodies or fragments may not be ideally suited for production or use as therapeutic proteins. It is possible that they have not. Summary of the Invention [Problem to be solved by the invention]

[0013] Thus, the present invention provides a method for the treatment of diseases mediated by TNFα. It is desired to provide an anti-TNF antibody or fragment thereof. [Means for solving the problem]

[0014] For brevity, the independent and dependent claims appended hereto are incorporated herein by reference. The appended claims define respective embodiments of the invention. The embodiments, features and advantages of the present invention are described in the following detailed description taken in conjunction with the accompanying drawings. It becomes clear from the shape.

[0015] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells).

[0016] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); The oligosaccharide profile of the anti-TNF antibody was characterized by >99.0% total neutral oligosaccharide species and >99.0% total charged oligosaccharide species. It is composed of <1.0% sugar species.

[0017] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); The oligosaccharide profile of anti-TNF antibodies showed G0F>60 for individual neutral oligosaccharide species. 0%, G1F<20.0%, and G2F<5.0%.

[0018] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); When measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA), The anti-TNF antibodies do not contain disialylated glycan species.

[0019] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); Anti-TNF antibodies have a longer half-life compared to anti-TNF antibodies expressed in Sp2 / 0 cells or has improved antibody-dependent cell-mediated cytotoxicity (ADCC).

[0020] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); The anti-TNF antibody is a biosimilar.

[0021] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); Anti-TNF antibodies have a longer half-life compared to anti-TNF antibodies expressed in Sp2 / 0 cells or has improved antibody-dependent cell-mediated cytotoxicity (ADCC).

[0022] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); The oligosaccharide profile of the anti-TNF antibody was characterized by >99.0% total neutral oligosaccharide species and >99.0% total charged oligosaccharide species. Anti-TNF antibodies were detected in the anti-TNF expressed in Sp2 / 0 cells. Longer half-life or improved antibody-dependent cell-mediated cytotoxicity (ADC) compared to antibodies C).

[0023] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); The oligosaccharide profile of anti-TNF antibodies showed G0F>60 for individual neutral oligosaccharide species. 0%, G1F<20.0%, and G2F<5.0%, and the anti-TNF antibody is Sp2 Longer half-life or improved antibody-dependent activity compared to anti-TNF antibodies expressed in 1 / 0 cells. It has anti-cancer cell-mediated cytotoxicity (ADCC).

[0024] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); When measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA), Anti-TNF antibodies do not contain disialylated glycan species, and anti-TNF antibodies are expressed in Sp2 / 0 cells. Longer half-life or improved antibody-dependent cell-mediated inhibition compared to previously demonstrated anti-TNF antibodies It has cytotoxicity (ADCC).

[0025] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); Anti-TNF antibodies have a longer half-life compared to anti-TNF antibodies expressed in Sp2 / 0 cells or has improved antibody-dependent cell-mediated cytotoxicity (ADCC).

[0026] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); Anti-TNF antibodies have a longer half-life compared to anti-TNF antibodies expressed in Sp2 / 0 cells or improved antibody-dependent cell-mediated cytotoxicity (ADCC), and anti-TNF antibodies are biocompatible. It is a successor product.

[0027] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); Anti-TNF antibodies have a longer half-life compared to anti-TNF antibodies expressed in Sp2 / 0 cells or improved antibody-dependent cell-mediated cytotoxicity (ADCC), and anti-TNF antibodies are biocompatible. It is a successor product.

[0028] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); The oligosaccharide profile of the anti-TNF antibody was characterized by >99.0% total neutral oligosaccharide species and >99.0% total charged oligosaccharide species. Anti-TNF antibodies were detected in the anti-TNF expressed in Sp2 / 0 cells. Longer half-life or improved antibody-dependent cell-mediated cytotoxicity (ADC) compared to antibodies C), and the anti-TNF antibody is a biosimilar.

[0029] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); The oligosaccharide profile of anti-TNF antibodies showed G0F>60 for individual neutral oligosaccharide species. 0%, G1F<20.0%, and G2F<5.0%, and the anti-TNF antibody is Sp2 Longer half-life or improved antibody-dependent activity compared to anti-TNF antibodies expressed in 1 / 0 cells. It has active cell-mediated cytotoxicity (ADCC), and the anti-TNF antibody is a biosimilar.

[0030] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); When measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA), Anti-TNF antibodies do not contain disialylated glycan species, and anti-TNF antibodies are expressed in Sp2 / 0 cells. Longer half-life or improved antibody-dependent cell-mediated inhibition compared to previously demonstrated anti-TNF antibodies It has cytotoxicity (ADCC), and the anti-TNF antibody is a biosimilar.

[0031] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37, The TNF antibody was expressed in Chinese hamster ovary cells (CHO cells); Anti-TNF antibodies have a longer half-life compared to anti-TNF antibodies expressed in Sp2 / 0 cells or improved antibody-dependent cell-mediated cytotoxicity (ADCC), and anti-TNF antibodies are biocompatible. It is a successor product.

[0032] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The composition is produced by a manufacturing method comprising the steps of:

[0033] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The oligosaccharide profile of the anti-TNF antibody is Consists of >99.0% total charged oligosaccharide species and <1.0% total charged oligosaccharide species, In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The oligosaccharide profile of the anti-TNF antibody is determined by a method comprising the steps of: For oligosaccharide species, G0F>60.0%, G1F<20.0%, and G2F<5.0% Further includes.

[0034] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. and the resulting product is analyzed by high performance liquid chromatography (HPLC) or reduced mass spectrometry. The anti-TNF antibodies are free of disialylated glycan species as determined by analysis (RMA).

[0035] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The anti-TNF antibody is produced by a production method comprising the steps of: F antibody, longer half-life or improved antibody-dependent cell-mediated cytotoxicity (AD CC).

[0036] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. and the anti-TNF antibody is a biosimilar product.

[0037] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The oligosaccharide profile of the anti-TNF antibody is The anti-TNF antibody was composed of S Longer half-life or improved antibody activity compared to anti-TNF antibodies expressed in p2 / 0 cells It has dependent cell-mediated cytotoxicity (ADCC).

[0038] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The oligosaccharide profile of the anti-TNF antibody is determined by a method comprising the steps of: For oligosaccharide species, G0F>60.0%, G1F<20.0%, and G2F<5.0% Further, the anti-TNF antibody is more potent than the anti-TNF antibody expressed in Sp2 / 0 cells. have a long half-life or improved antibody-dependent cell-mediated cytotoxicity (ADCC).

[0039] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. and the product is produced by a manufacturing method including the steps of: Anti-TNF antibodies do not contain disialylated glycan species as measured by RMA analysis. The TNF antibodies showed a longer half-life or longer survival time compared to the anti-TNF antibodies expressed in Sp2 / 0 cells. has improved antibody-dependent cell-mediated cytotoxicity (ADCC).

[0040] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The anti-TNF antibody is produced by a production method comprising the steps of: F antibody, longer half-life or improved antibody-dependent cell-mediated cytotoxicity (AD CC).

[0041] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a method for producing an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; The present invention provides a method for producing an anti-TNF antibody, comprising: a. a. culturing Chinese hamster ovary cells (CHO cells) using the c. expressing an anti-TNF antibody in a cell; and c. purifying the anti-TNF antibody. The anti-TNF antibody is produced by a production method comprising the steps of: F antibody, longer half-life or improved antibody-dependent cell-mediated cytotoxicity (AD CC), and the anti-TNF antibody is a biosimilar.

[0042] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). It is something.

[0043] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The oligosaccharide profile of the anti-TNF antibody was >99.0% total neutral oligosaccharide species and and total charged oligosaccharide species <1.0%.

[0044] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The oligosaccharide profile of anti-TNF antibodies was characterized by G Further includes G0F>60.0%, G1F<20.0%, and G2F<5.0%.

[0045] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). When measured by high performance liquid chromatography (HPLC), anti-TNF antibodies Does not contain disialylated glycan species.

[0046] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The anti-TNF antibody was more potent than the anti-TNF antibody expressed in Sp2 / 0 cells. These antibodies have a longer half-life or improved antibody-dependent cell-mediated cytotoxicity (ADCC).

[0047] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The anti-TNF antibody is a biosimilar.

[0048] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The oligosaccharide profile of the anti-TNF antibody was >99.0% total neutral oligosaccharide species and The anti-TNF antibody was expressed in Sp2 / 0 cells and consisted of <1.0% total charged oligosaccharide species. Longer half-life or improved antibody-dependent cell-mediated cell proliferation compared to anti-TNF antibodies It has ADCC.

[0049] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The oligosaccharide profile of anti-TNF antibodies was characterized by G 0F>60.0%, G1F<20.0%, and G2F<5.0%, and further comprising an anti-TNF antibody. The antibody showed a longer half-life or improved potency compared to the anti-TNF antibody expressed in Sp2 / 0 cells. It has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC).

[0050] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). When measured by high performance liquid chromatography (HPLC), anti-TNF antibodies Anti-TNF antibodies were not found to be related to anti-TN Fc-expressed in Sp2 / 0 cells, as they did not contain disialylated glycan species. F antibody, longer half-life or improved antibody-dependent cell-mediated cytotoxicity (AD CC).

[0051] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The anti-TNF antibody was more potent than the anti-TNF antibody expressed in Sp2 / 0 cells. Anti-TNF antibodies have long half-lives or improved antibody-dependent cellular cytotoxicity (ADCC), Longer half-life or improved antibody activity compared to anti-TNF antibodies expressed in p2 / 0 cells It has dependent cell-mediated cytotoxicity (ADCC).

[0052] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The oligosaccharide profile of the anti-TNF antibody was >99.0% total neutral oligosaccharide species and The anti-TNF antibody was expressed in Sp2 / 0 cells and consisted of <1.0% total charged oligosaccharide species. Longer half-life or improved antibody-dependent cell-mediated cell proliferation compared to anti-TNF antibodies It has ADCC inhibitors, and the anti-TNF antibody is a biosimilar.

[0053] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). The oligosaccharide profile of anti-TNF antibodies was characterized by G 0F>60.0%, G1F<20.0%, and G2F<5.0%, and further comprising an anti-TNF antibody. The antibody showed a longer half-life or improved potency compared to the anti-TNF antibody expressed in Sp2 / 0 cells. The anti-TNF antibody has a strong antibody-dependent cell-mediated cytotoxicity (ADCC) effect, and is a biosimilar. be.

[0054] In a particular embodiment, the invention relates to a polypeptide comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) providing a composition comprising an anti-TNF antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 37; Here, the anti-TNF antibody was expressed in Chinese hamster ovary cells (CHO cells). When measured by high performance liquid chromatography (HPLC), anti-TNF antibodies Anti-TNF antibodies were not found to be related to anti-TN Fc-expressed in Sp2 / 0 cells, as they did not contain disialylated glycan species. F antibody, longer half-life or improved antibody-dependent cell-mediated cytotoxicity (AD CC), and the anti-TNF antibody is a biosimilar. [Brief description of the drawings]

[0055] [Figure 1] A graphical representation showing an assay of the ability of TNV mAbs in hybridoma cell supernatants to inhibit TNFα binding to recombinant TNF receptors is shown. 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 TNVmAb samples are tested in these experiments, but for simplicity, three mAbs shown by DNA sequence analysis to be identical to one of the other TNVmAbs 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 not known or not present in such germline gene at that time. 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 not known or not present in such germline gene at that time. 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 (single letter code) shown 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 in a clockwise direction. Plasmid p1783 is 19.53 kb in length and plasmid p1776 is 15.06 kb in length. The complete nucleotide sequences of both plasmids are known. The variable region coding sequence of p1783 can be easily replaced with another heavy chain variable region sequence by replacing the BsiWI / BstBI restriction fragment. The variable region coding sequence of p1776 can be replaced with another variable region sequence by replacing the SalI / AflII restriction fragment. [Figure 7] 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 trypan blue exclusion and the remaining aliquots were kept for later mAb concentration determination. ELISA for human IgG was performed on all sample aliquots at the same time. [Figure 8]Figure 1 shows a graphical representation of the comparison of cell growth rates in the presence of various MHX selection concentrations. Cell subclones C466A and C466B were thawed into MHX-free medium (IMDM, 5% FBS, 2 mM glutamine) and cultured for an additional 2 days. Both cell cultures were then split into three cultures that contained either no MHX, 0.2xMHX or 1xMHX. After one day, new T75 flasks were seeded with the cultures at a starting density of 1x105 cells / ml and cells were counted at 24 hour intervals for one week. The doubling times for the first 5 days were calculated using the formula in SOP PD32.025 and are shown above the bars. [Figure 9] Graphical representation of the stability of mAb production over time from two rTNV148B producing cell lines. After transfection and subcloning, subclones of cells that were in continuous culture were used to initiate long-term continuous culture in 24-well culture dishes. Cells were cultured in I5Q medium with or without MHX selection. Cells were serially passaged by splitting cultures every 4-6 days to maintain new viable cultures while simultaneously exhausting previous cultures. Aliquots of exhausted cell supernatants were collected immediately after the cultures were exhausted and stored until mAb concentrations were determined. ELISA for human IgG was performed on all sample aliquots at the same time. [Figure 10] Figure 1 shows the weight change of arthritic mouse model mice Tg197 in response to anti-TNF antibodies of the invention compared to the control of Example 4. Approximately 4-week-old Tg197 study mice were assigned to one of nine treatment groups based on sex and weight and treated with a single intraperitoneal bolus of Dulbecco's PBS (D-PBS) or an anti-TNF antibody of the invention (TNV14, TNV148, or TNV196) at either 1 mg / kg or 10 mg / kg. When weight was analyzed as the change from pre-treatment, animals treated with 10 mg / kg cA2 consistently showed higher weight gain than animals treated with D-PBS 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. TNV148 at 1 mg / kg was also significantly lower than the TNV14 at 1 mg / kg treated group at weeks 3 and 4. TNV196 showed a significant decrease in AI (when compared to the D-PBS treated group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [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. TNV148 at 1 mg / kg was also significantly lower than the TNV14 at 1 mg / kg treated group at weeks 3 and 4. TNV196 showed a significant decrease in AI (when compared to the D-PBS treated group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [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. TNV148 at 1 mg / kg was also significantly lower than the TNV14 at 1 mg / kg treated group at weeks 3 and 4. TNV196 showed a significant decrease in AI (when compared to the D-PBS treated group) through week 6 of the study, but TNV148 was the only 1 mg / kg treatment group that remained significant at the end of the study. [Figure 12] 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 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 are 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 treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study and were also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13B]1 is a graph depicting the progression of disease severity 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 treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study and were also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 13C]1 is a graph depicting the progression of disease severity 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 treated group. Animals treated with 10 mg / kg cA2 showed a significant reduction in AI when compared to both lower doses of cA2 (1 mg / kg and 3 mg / kg) at weeks 4 and 5 of the study and were also significantly lower than animals treated with TNV14 at weeks 3-5. Although there appeared to be no significant differences between any of the 3 mg / kg treatment groups, the AI ​​for animals treated with 3 mg / kg TNV14 was significantly higher than 10 mg / kg at some time points, whereas animals treated with TNV148 were not significantly different from animals treated with 10 mg / kg cA2. [Figure 14] Figure 1 shows the weight change of 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]Figure 1 depicts the progression of disease severity based on arthritis index as shown in Example 7. The arthritis index of the 10 mg / kg cA2 treatment group is 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] The nine steps in the golimumab manufacturing process are outlined. [Figure 19] FIG. 1 shows a flow diagram of the manufacturing process for Stage 1 of the pre-culture and growth steps, including in-process controls and process monitoring tests. [Figure 20] FIG. 1 shows a flow diagram of the manufacturing process for Stage 2, a step that includes in-process controls and process monitoring tests. [Figure 21] Representative HPLC chromatograms of golimumab expressed in Sp2 / 0 cells using an HPLC method with fluorescence detection are shown. Peaks associated with different species are labeled. * indicates system peaks not associated with golimumab. [Figure 22] 1 shows a representative deconvoluted mass spectrum of IRMA analysis of golimumab produced in Sp2 / 0 cells. [Diagram 23]A representative cIEF electropherogram profile of golimumab expressed in Sp2 / 0 cells is shown with four major peaks labeled C, 1, 2, and 3 and a minor peak labeled B. Internal standards of pI 7.6 and 9.5 are also labeled. A diagram depicting the general relationship between cIEF peaks and reduced negative charge / degree of sialylation is also shown. [Figure 24] An overview of some of the major N-linked oligosaccharide species of golimumab IgG is shown. The role of some enzymes in the glycosylation maturation process and the role of some divalent cations (e.g., Mn2+ as a cofactor and Cu2+ as an inhibitor of GalTI) are also shown (see, e.g., Biotechnol Bioeng. 2007 Feb 15;96(3):538-49; Curr Drug Targets. 2008 Apr;9(4):292-309; J Biochem Mol Biol. 2002 May 31;35(3):330-6). Note that the species containing terminal sialic acid (S1 and S2) are charged species and those without terminal sialic acid (G0F, G1F, and G2F) are neutral species, but the generation of charged species depends on the presence of galactose in G1F and G2F added by the GalT1 enzyme. [Diagram 25] Representative HPLC chromatograms of the oligosaccharide profiles of golimumab expressed in Sp2 / 0 cells and CHO cells are shown. Elution peaks of anthranilic acid-labeled N-glycans are identified with hash marks for all peaks above baseline. Brackets and / or labels are used to indicate groups of peaks corresponding to total neutral, total charged, monosialylated, and disialylated oligosaccharide species. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The present invention comprises a heavy chain (HC) comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37. Compositions comprising anti-TNF antibodies, including provide.

[0057] 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 optionally further affect specific ligands and Without being limited thereto, such antibodies may be used in vitro, in situ, and / or in vivo. Modulate or reduce at least one TNF activity or binding, or TNF receptor activity or binding. Reduce, increase, antagonize, agonize, reduce, mitigate, block, inhibit, suppress, and / or interfere. By way of example, a preferred anti-TNF antibody, specified portion or variant of the invention comprises at least one The antibodies can bind to one TNF or a specified part, variant or domain thereof. Suitable anti-TNF antibodies, specified portions or variants may also be expressed as RNA, DNA, or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TN TNF activity or TNF production and / or synthesis, including, but not limited to, TNF activity, TNF production and / or synthesis, The term "antibody" can also refer to an antibody that is capable of affecting at least one of the functions of the antibody. The term "antibody mimetics" is intended to encompass any of the above-mentioned polypeptides, their digests, fragments, specified portions and variants, including antibody mimetics. or portions of antibodies or their characteristics that mimic the structure and / or function of an antibody. Functional fragments include certain fragments or parts of antibodies, such as single chain antibodies and fragments thereof. Examples of antigen-binding fragments that bind to mammalian TNF include Fab (e.g., papain). digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab' ) 2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), p Fc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, by partial reduction and reassembly), Fv or scFv (e.g., by molecular biology techniques) and fragments thereof, which are capable of binding to TNF or portions thereof. Antibody fragments are encompassed by the invention (see, e.g., Colligan, Immunolo, supra). gy).

[0058] 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 an antibody gene in which one or more stop codons have been introduced upstream of the stop site of Also fragments such as F(ab') 2 Combination of genes encoding the heavy chain The CH of the heavy chain 1 The DNA sequences encoding the domain and / or hinge region are The various portions of the antibody can be conjugated chemically by conventional techniques, or can be genetically conjugated. It can be prepared as a contiguous protein using molecular engineering techniques.

[0059] 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, and C H 3), hinge (VL, V H )) is substantially non-immunogenic in humans and has a sequence Similarly, antibodies from primates (monkeys, baboons, Pansies, etc.), Rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.), and other mammalian directed antibodies specific for such species, subgenera, genus, subfamily, and family. Furthermore, chimeric antibodies include any combination of the above. The mutations optionally and preferably have a greater ability to inhibit the activity of the antibody in humans or other species compared to the unmodified antibody. Thus, a human antibody is a chimeric or human antibody. Human antibodies are distinct from humanized antibodies. Human antibodies are functionally rearranged human immunoglobulins (e.g., A non-human animal, prokaryotic or otherwise capable of expressing the mitochondrial heavy and / or light chain genes. It is noted that human antibodies can be produced by eukaryotic cells. Moreover, when a human antibody is a single chain antibody, In some cases, the heavy chain may contain a linker peptide that is not found in natural human antibodies. For example, an Fv may contain a heavy chain 2 to about 8 glycine or other amino acid residues connecting the first and second variable regions of the light chain Such linker peptides are considered to be of human origin. do.

[0060] Also, monoclonal antibodies having binding specificities for at least two different antigens, Bispecific antibodies (e.g., DuoBody ( A combination of antibodies, heterospecific antibodies, heterobinding antibodies, or similar antibodies may be used. In the present case, one of the binding specificities is for at least one TNF protein. The method for producing a bispecific antibody is described in detail below. Recombinant production of bispecific antibodies is known in the art. It is based on the co-expression of a globulin heavy-light chain pair, where the two heavy chains have different specificities ( Milstein and Cuello, Nature, 305:537 (1983) Due to the random assortment of immunoglobulin heavy and light chains, these hybrids Each quadroma produces a possible mixture of 10 different antibody molecules, of which Only one has the correct bispecific structure, usually by an affinity chromatography step. Purification of the correct molecule performed is laborious and often results in low product yields, making it difficult to perform the purification steps required for successful completion of the procedure. Different approaches have been developed to enhance the production of

[0061] Full-length bispecific antibodies can be produced, for example, in a cell-free environment in vitro or using co-expression. to favor the heterodimerization of two antibody half molecules with different specificities. By introducing substitutions at the heavy chain CH3 interface in each half molecule, two monospecific bivalent Fab arms can be generated using Fab arm exchange (or half molecule exchange) between antibodies. The exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domain. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies is linked to a cysteine ​​of a second parent monospecific antibody molecule. At the same time, the CH3 domain of the parent antibody dissociates. The CH3 domain of the Fab arm is released and reformed by homodimerization. The resulting products may be engineered to favor heterodimer formation. A bispecific antibody having two Fab arms or half molecules capable of binding to a target antigen. It is a body.

[0062] As used herein, "homodimerization" refers to formation of a homodimer having identical CH3 amino acid sequences. As used herein, "homodimer" refers to the interaction of two heavy chains in a homodimer. It refers to an antibody having two heavy chains with the same CH3 amino acid sequence.

[0063] As used herein, "heterodimerization" refers to formation of heterodimers having non-identical CH3 amino acid sequences. As used herein, a "heterodimer" refers to the interaction of two heavy chains having " refers to an antibody having two heavy chains that have non-identical CH3 amino acid sequences.

[0064] "Knob-in-hole" strategies (e.g., WO 2006 / 02893 6) can be used to generate full-length bispecific antibodies. The selected amino acids that form the interface of the CH3 domain in human IgG are heterodimers. To promote merization, the ribozyme was mutated at positions that affect CH3 domain interactions. An antibody in which an amino acid with a small side chain (hole) specifically binds to a first antigen The heavy chain of the antibody is introduced with an amino acid with a large side chain (knob) that specifically binds to a second antigen. After co-expression of the two antibodies, the heterodimer is called the "hole It is formed as a result of preferential interaction between a heavy chain bearing the "end" and a heavy chain bearing the "knob". Exemplary CH3 substitution pairs that form knobs and holes are T366Y / F405A, T36 6W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y 407A, T366W / T394S, F405W / T394S, and T366W / T36 6S_L368A_Y407V (modification in the first CH3 domain of the first heavy chain The results are expressed as modified position / modified position in the second CH3 domain of the second heavy chain.

[0065] Other strategies, such as the use of positively charged residues on one CH3 surface and Heavy chain heterodimerization using electrostatic interactions by replacing negatively charged residues in The promotion of merization is described in U.S. Patent Application Publication Nos. 2010 / 0015133 and 2009 / 0 182127, 2010 / 028637 or 2011 / 0123532 In another strategy, heterodimer formation may be used as described in U.S. Pat. As described in Publication No. 2012 / 0149876 or Publication No. 2013 / 0195849 Replaces: L351Y_F405A_Y407V / T394W, T366I_K3 92M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351 Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392 L_T394W (modified position in the first CH3 domain of the first heavy chain / modified position in the second heavy chain This can be enhanced by the addition of modified positions in the second CH3 domain of the Cut.

[0066] In addition to the methods described above, bispecific antibodies can be produced using the methods described in WO 2011 / 131746. According to the method described, two monospecific homodimers were generated in a cell-free environment in vitro. Asymmetric mutations are introduced into the CH3 region of a dimeric antibody to isomerize disulfide bonds Under reducing conditions, two parent monospecific homodimeric antibodies are synthesized into bispecific heterodimeric antibodies. In this method, a first monospecific bivalent antibody and and a second monospecific bivalent antibody that binds to the CH3 domain, promoting heterodimer stability. These antibodies are engineered to have certain substitutions in the hinge region. The stain is then mixed with the ink under reducing conditions sufficient to isomerize the disulfide bonds. The antibodies are then incubated with Fab arms to generate bispecific antibodies. Incubation conditions can optimally be returned to non-reducing conditions. Exemplary reducing conditions that can be used The agents were 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), di Thioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosph and β-mercaptoethanol, preferably TCEP, L-cysteine, and beta-mercaptoethanol. For example, 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxylate) For example, the reducing agent is selected from the group consisting of ethyl phosphine. At a temperature of In the presence of dithiothreitol, at pH 5 to 8, for example, pH 7.0 or pH 7.4 For this purpose, an incubation period of at least 90 minutes may be used.

[0067] 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 antibody of the present invention, a specified fragment or variant thereof, The antibodies that can be used in the present invention are useful in providing measurable relief of symptoms as well as reduced risk of infection. Optionally, select candidates based on their ability to treat patients for extended periods with acceptable toxicity and / or acceptable toxicity. Low or acceptable immunogenicity and / or high affinity, as well as other favorable characteristics may be considered. These properties can contribute to the therapeutic outcome obtained. is significantly HAHA in less than about 75%, or preferably less than about 50%, of treated patients. , increased HACA or HAMA response, and / or reduced potency in treated patients. Titer (less than about 300, preferably less than about 100 as measured by double antigen enzyme immunoassay) ) (Elliott et al., Lancet 3 44:1125-1127 (1994), incorporated herein by reference in its entirety.

[0068] 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.

[0069] 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 information in any form of media, including electronic or printed matter. Refers to any scientific publications or patents or any other information available. is incorporated herein by reference in its entirety: Ausubel, et al., eds. ,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001) Sambrook, et al., Molecular Cloning: A Labo ratory Manual,2nd Edition,Cold Spring Ha rbor, NY (1989), Harlow and Lane, antibodies. ,a Laboratory Manual,Cold Spring Harbor, NY (1989), Colligan, et al., eds., Current Pr. otocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001), Colligan et al., Curre nt Protocols in Protein Science,John Wil ey & Sons, NY, NY, (1997-2001).

[0070] 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 anti-TN antibody of the present invention comprising all of the light chain variable CDR regions of numbers 4, 5 and 6. The F antibody can be optionally produced from a cell line, mixed cell line, or recombinant cell line, as is well known in the art. It may be produced by immortalized cells or clonal populations of immortalized cells. ,et al.,ed.,Current Protocols in Molecule ar 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 e t al.,Current Protocols in Protein Science ce, John Wiley & Sons, NY, NY, (1997-2001). No. 6,399,433, each of which is incorporated herein by reference in its entirety.

[0071] 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 accomplished by any suitable technique. This can be done using techniques.

[0072] 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, fusion products thereof, or any derived therefrom. or any other suitable cell line known in the art. For example, see www.atcc.org, www.life See, for example, tech.com. Isolated or cloned spleen, peripheral blood, Such as, but not limited to, lymphoid, tonsillar, or other immune or B cell containing cells. antibody-producing cells, or recombinant or endogenous, viral, bacterial, algal, prokaryotic, Amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep, goats, lambs, primates Eukaryotes, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA A, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded, or triple-stranded, hybridized, etc., or combinations thereof. As heterologous nucleic acid, either heavy or light chain constant or variable or framework For example, the Ausu bel, and Colligan, Immunology, supra, Chapter 2. Both documents are incorporated herein by reference in their entireties.

[0073] Antibody producing cells can also be isolated from the peripheral blood of humans or other suitable animals immunized with an antigen of interest, or Preferably, the host cell may be obtained from the spleen or lymph nodes. Any other suitable host cell may be used. and the like, to produce a heterologous nucleic acid encoding an antibody, specified fragment, or variant thereof of the present invention. It is also possible to express the nucleic acid or endogenous nucleic acid. The cells may be isolated using selective culture conditions or other suitable known methods and isolated by limiting dilution or The desired specificity can be obtained by cloning using cell sorting or other known methods. Cells producing antibodies that bind to the antibody can be selected by a suitable assay (e.g., ELISA). can be done.

[0074] Examples of such methods include selecting recombinant antibodies from peptide or protein libraries. Any suitable method for producing or isolating antibodies having the required specificity, including, but not limited to, Other methods can be used (e.g., bacteriophage, ribosome, oligonucleotide Display libraries include, but are not limited to, nucleotides, RNA, cDNA, etc. For example, Cambridge Antibody Technologies, mbridgeshire, UK, MorphoSys, Martinsreid / Pl anegg, DE, Biovation, Aberdeen, Scotland, UK, BioInvent, Lund, Sweden, Dyax Corp., Enzon, A from ffymax / Biosite, Xoma, Berkeley, CA, and Ixsys. (Available from the European Patent Office, see, for example, European Patent No. 368,684, International Application No. PCT / GB91 / 01134, International Application No. PCT / GB92 / 01755, International Application No. PCT / GB92 / No. 002240, International Application No. PCT / GB92 / 00883, International Application No. PCT / GB93 / 00605, U.S. Patent Application No. 08 / 350260 (5 / 12 / 94), International Application P CT / GB94 / 01422, International Application PCT / GB94 / 02662, International Application P CT / GB97 / 01835, (CAT / MRC), International Publication No. 90 / 14443, International Publication No. WO 90 / 14424, International Publication No. WO 90 / 14430, International Application PCT / US 94 / 1234, WO 92 / 18619, WO 96 / 07754 (S cripps), European Patent No. 614 989 (MorphoSys), International Publication No. 95 / 16027 (BioInvent), International Publication No. 88 / 06630, International Publication No. 9 No. 0 / 3809 (Dyax), U.S. Pat. No. 4,704,692 (Enzon), International Patent Application No. PCT / US91 / 02989 (Affymax), International Publication No. WO 88 / 06283 , European Patent No. 371998, European Patent No. 550400, (Xoma), European Patent No. No. 29046, International Application PCT / US91 / 07149 (Ixsys), or probabilistic Peptides or proteins produced by the method according to the present invention - U.S. Patent Nos. 5,723,323 and 5,763 No. 192, No. 5814476, No. 5817483, No. 5824514, No. No. 5976862, International Publication No. 86 / 05803, European Patent No. 590689 (Ixs ys, now Applied Molecular Evolution (AME), For more information, see the following technical papers, which are incorporated herein by reference in their entireties: To generate a repertoire of human antibodies, the methods are known in the art and / or described herein. The immune system relies on the immunization of transgenic animals that can guyen et al.,Microbiol.Immunol.41:901-90 7(1997), Sandhu et al.,Crit.Rev.Biotechno l.16:95-118(1996), Eren et al., Immunol.93 :154-161 (1998) (each of which is incorporated by reference in its entirety). For this purpose, ribosome display (Hanes et al., Proc. Natl. Ac ad.Sci.USA,94:4937-4942(May 1997), Hanes et al.,Proc.Natl.Acad.Sci.USA,95:14130-1 4135 (Nov. 1998)), single cell antibody production technology (e.g., selected lymphocyte antibody method SLAM (U.S. Patent No. 5,627,052, Wen et al., J.I. mmunol.17:887-892(1987), Babcook et al.,P roc.Natl.Acad.Sci.USA 93:7843-7848(1996) ), gel microdroplets 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 (Steenbakker s et al.,Molec.Biol.Reports 19:125-134(1 994), Jonak et al., Progress Biotech, Vol.5 ,In Vitro Immunization in Hybridoma Tech nology,Borrebaeck,ed.,Elsevier Science P publishers BV,Amsterdam,Netherlands(198 8)) but are not limited to these.

[0075] Methods for modifying or humanizing non-human or human antibodies may also be used, and are within the skill of the art. Generally, humanized or modified antibodies are well known in the art. These include, but are not limited to, rabbits, non-human primates, or other mammals. , and one or more amino acid residues from a source other than human. are often referred to as "import" residues and are typically the "import" residues of known human sequences. It may be taken from a variable region, a constant region or other domain.

[0076] Known human Ig sequences are disclosed in numerous publications and websites, e.g., www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / ; www.abcam.com / ; www.antibodyresource.com / onlinecomp.htm l; www.public.iastate.edu / ~pedro / research_ tools.html; www.mgen.uni-heidelberg.de / SD / IT / IT.htm l; www.whfreeman.com / immunology / CH05 / kuby0 5.htm; www.library.thinkquest.org / 12429 / Immune / Antibody.html; www.hhmi.org / grants / lectures / 1996 / vlab; www.path.cam.ac.uk / ~mrc7 / mikeimages.htm l; www.antibodyresource.com / ; www.mcb.harvard.edu / BioLinks / Immunology .html. www.immunologylink.com / ; www.pathbox.wustl.edu / ~hcenter / index.ht ml; www.biotech.ufl.edu / ~hcl / ; www.pebio.com / pa / 340913 / 340913.html; www.nal.usda.gov / awic / pubs / antibody / ; www.m.ehime-u.ac.jp / ~yasuhito / Elisa.htm l; www.biodesign.com / table.asp; www.icnet.uk / axp / facs / davies / links.html ; www.biotech.ufl.edu / ~fccl / protocol.html ; www.isac-net.org / sites_geo.html; www.aximt1.imt.uni-marburg.de / ~rek / AEPS tart.html; www.baserv.uci.kun.nl / ~jraats / links1.ht ml; www.recab.uni-hd.de / immuno.bme.nwu.edu / ; www.mrc-cpe.cam.ac.uk / imt-doc / public / IN TRO.html; www.ibt.unam.mx / vir / V_mice.html;imgt.cn usc.fr:8104 / 、 www.biochem.ucl.ac.uk / ~martin / abs / index .html;antibody.bath.ac.uk / ; www.abgen.cvm.tamu.edu / lab / www.abgen.html; www.unizh.ch / ~honegger / AHOseminar / Slide 01.html; www.cryst.bbk.ac.uk / ~ubcg07s / ; www.nimr.mrc.ac.uk / CC / ccaewg / ccaewg.htm ; www.path.cam.ac.uk / ~mrc7 / humanisation / T AHHP.html; www.ibt.unam.mx / vir / structure / stat_aim. html; www.biosci.missouri.edu / smithgp / index.h tml; www.cryst.bioc.cam.ac.uk / ~fmolina / Web-p ages / Pept / spottech.html; www.jerini.de / frproducts.html; www.patents.ibm.com / ibm.html.Kabat et a l.,

[0077] Sequences of Proteins of Immunology Interest, USDept. Health (1983), each of which is referenced 6,311,421, all of which are incorporated herein by reference in their entirety.

[0078] Such imported sequences may be used to reduce immunogenicity or to enhance the expression of polypeptides, as described in the art. As known in the art, the term "binding," "affinity," "association rate constant," "dissociation rate constant," "binding activity," "specificity," The compounds may be used to reduce, enhance or modify the half-life, half-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. and are well known to those skilled in the art. Computer programs are available that map and display highly three-dimensional structures. By examining these representations, the potential of residues in the function of the candidate immunoglobulin sequence can be determined. This allows the analysis of the residues that affect the antigen-binding ability of immunoglobulin candidates. In this way, desirable antibody effects, such as increased affinity for the target antigen(s), can be achieved. FR residues are selected from the consensus and import sequences such that a conformational characteristic is achieved. Generally, the CDR residues are directly and mostly involved in antigen binding. The humanization or modification of the antibody of the present invention is carried out according to the method described by Winter (Jones et al. t al., Nature 321:522 (1986), Riechmann et al. al., Nature 332:323 (1988), Verhoeyen et al. .,Science 239:1534(1988)), Sims et al.,J. Immunol.151:2296(1993), Chothia and Lesk, J.Mol.Biol.196:901(1987), Carter et al.,P roc.Natl.Acad.SCi.USA89:4285(1992), Pr esta et al., J. Immunol. 151:2623 (1993), Permit No. 5723323, No. 5976862, No. 5824514, No. 58174 No. 83, No. 5814476, No. 5763192, No. 5723323, No. 5 ,766886, 5714352, 6204023, 6180370 No. 5693762, No. 5530101, No. 5585089, No. 522 No. 5539, No. 4816567, International Application No. PCT / :US98 / 16280, US 96 / 18978, US91 / 09630, US91 / 05939, US94 / 0 No. 1234, No. GB89 / 01334, No. GB91 / 01134, No. GB92 / 01755 No. 90 / 14443, No. 90 / 14424, No. 90 / 14430 and European Patent No. 229246 (each of which is incorporated herein by reference in its entirety). Any known method, including but not limited to those described in the literature (including those described in the literature used in the literature) may be used. This can be done using the method of

[0079] 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 Generated by immunization of animals (e.g., mice, rats, hamsters, non-human primates, etc.) Cells producing 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 that described in

[0080] Transgenic mice capable of producing a repertoire of human antibodies that bind to human antigens The mice can be made 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. 0463151(B1) to Kucherlapate et al. European Patent No. 0710719(A1) to Pate et al., U.S. Patent No. 5,500,000 to Surani et al. 45,807, Bruggemann et al., International Publication No. 90 / 04036, Brugg Emann et al., European Patent No. 0438474(B1); Lonberg et al., European Patent No. No. 814259(A2), Lonberg et al., UK Patent No. 2272440(A), Lonberg et al. Nature 368:856-859 (1994), T aylor et al., Int. Immunol. 6(4)579-591 (19 94), Green et al., Nature Genetics 7:13-21( 1994), Mendez et al., Nature Genetics 15:1 46-156(1997), Taylor et al., Nucleic Acids Research 20(23):6287-6295(1992), Tuaillo n et al., Proc Natl Acad Sci USA 90(8)372 0-3724(1993), Lonberg et al., Int Rev Immu nol 13(1):65-93(1995), and Fishwald et al., Nat Biotechnol 14(7):845-851(1996), these are (Each of which is incorporated herein by reference in its entirety.) In general, these mice are functionally At least one human immunoglobulin capable of undergoing constructed or functional rearrangement. The mouse contains at least one transgene that includes DNA from the phosphoryl locus. The endogenous immunoglobulin locus of the mouse is disrupted or deleted to obtain the endogenous gene of the mouse. The ability to produce antibodies encoded by the vector can be eliminated.

[0081] Screening antibodies for specific binding to similar proteins or fragments This can be successfully achieved using peptide display libraries. The method involves scanning a large sample of peptides for individual members with a desired function or structure. Antibody screening of peptide display libraries involves screening. The length of the displayed peptide sequence is 3 to 5 000 or more amino acids, frequently 5-100 amino acids long, most often about 8-25 In addition to direct chemical synthesis methods for generating peptide libraries, Several recombinant DNA methods have also been described. One type uses bacteriophages or Each bacteriophage or cell contains a nucleotide sequence that codes for a particular displayed peptide sequence. Such methods are described in International Patent Application Publication Nos. 91 / 17271, 91 / 18980 and 91 / 19063. The peptide library is described in US Pat. No. 6,399,413 and US Pat. No. 6,399,413. Other systems for making ribozymes include aspects of both in vitro chemical synthesis and recombinant methods. International Patent Publication Nos. 92 / 05258, 92 / 14843 and 96 / 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, U.S. Patent Nos. 5,223,409, 5,403,484, and 557, assigned to Dyax 1698, 5837500, and U.S. Patent No. 54279 assigned to Affymax No. 08, No. 5580717, Cambridge antibody Techno U.S. Patent No. 5,885,793 assigned to Logies and U.S. Patent No. 5,885,793 assigned to Genentech No. 5,750,373 assigned to Samsung Electronics Co., Ltd.; U.S. Patent No. 5,618,920 assigned to Xoma; No. 5595898, No. 5576195, No. 5698435, No. 569349 No. 3, No. 5,698,417, Colligan, supra, Ausubel, supra, or See Sambrook, supra, each of the above patents and publications is incorporated by reference. The entirety of which is incorporated herein.

[0082] 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 also be prepared using anti-TNF antibodies of the same type as those described above. For example, but not limited to, U.S. Pat. No. 0, No. 5,849,992, No. 4,873,316, No. 5,849,992 No. 5,994,616, No. 5,565,362, No. 5,304,489 etc., each of which is incorporated herein by reference in its entirety.

[0083] 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, transgenic vectors expressing recombinant proteins using inducible promoters can be used. Tobacco leaves have been successfully used to provide large amounts of recombinant proteins. For example, Cr amer et al.,Curr.Top.Microbol.Immunol.24 0:95-118 (1999) and references cited therein. Transgenic maize is a plant-derived plant that is genetically engineered to contain proteins or natural resources produced in other recombinant systems. A mammalian protein having biological activity equivalent to that of the protein purified from a commercial source is prepared. For example, Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references therein. Antibodies have also been isolated from single chain antibodies (sBAs), including from tobacco seeds and potato tubers. Antibody fragments such as cFv (antibody fusion protein) have also been produced in large quantities from transgenic plant seeds. For example, Conrad et al., Plant Mol. Biol. 38:101 109 (1998) and the references cited therein. Antibodies can also be produced using transgenic plants, by known methods. For example, Fischer et al., Biotechnol. Appl. Biol. chem.30:99-108(Oct.,1999), Ma et al., Tren ds Biotechnol.13:522-7(1995), Ma et al.,P lant Physiol.109:341-6(1995), Whitelam et. al.,Biochem.Soc.Trans.22:940-944(1994), See also the references cited therein. Each of the above references is incorporated herein by reference in its entirety, However, this is not limited to these.

[0084] 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, for example, 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 K, such as, but not limited to, a range or value of D can be combined with

[0085] 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 supplement.) Parent antibodies that are measured for specific antibody-antigen interactions. Compatibility may differ when measured under different conditions (e.g., salt concentration, pH). Therefore, affinity and other antigen binding parameters (e.g., K D、 K a, K d ) is preferred. or standard solutions of antibodies and antigens, and standard buffers, such as those described herein. This is done using

[0086] A nucleic acid molecule comprising at least one contiguous nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. Nucleotide sequences, specified fragments, variants, or sequences that encode at least 70-100% of the amino acids or a consensus sequence thereof, or a sequence containing at least one of these sequences. Using the information provided herein, including the deposited vector, overlapping sequences of SEQ ID NOs: 1, 2 and 3 can be obtained. All of the light chain variable CDR regions and / or all of the light chain variable CDR regions of SEQ ID NOs: 4, 5 and 6 The nucleic acid molecule of the present invention encoding at least one anti-TNF antibody comprising the These can be prepared or obtained using methods known in the art.

[0087] 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 a combination of these. Any combination of any portion of at least one strand of DNA or RNA. The coding strand, also known as the sense strand, may be the coding strand, or may be called the antisense strand. It may also be the non-coding strand.

[0088] 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 containing the gene code (numbers 7 and 8) as well as nucleic acid molecules substantially different from the above-mentioned nucleic acid molecules. Due to the degeneracy of the code, at least some of the The nucleic acid molecule may comprise a nucleic acid molecule comprising a nucleotide sequence still encoding one or more anti-TNF antibodies. Of course, the genetic code is well known in the art. It is within the skill of the art to generate such degenerate nucleic acid variants that encode the specific anti-TNF antibodies of the 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.

[0089] As provided herein, the nucleic acid molecules of the invention, including nucleic acids encoding anti-TNF antibodies, , which itself encodes the amino acid sequence of an antibody fragment, a full-length antibody or a portion of an antibody The coding sequence for the antibody, fragment or portion thereof, as well as additional sequences, e.g. Non-coding sequences, with or without the additional coding sequences described above, such as at least one intron. Coding 5' and 3' sequences, e.g., splicing and polyadenylation signals (e.g. In transcription, mRNA processing, including ribosome binding and stability of mRNA and additional non-coding sequences, including but not limited to transcribed and non-translated sequences that play a role in In addition, at least one coding sequence for a signal leader or fusion peptide, an additional Additional coding sequences that code for amino acids that provide additional functions, e.g. Thus, the antibody coding sequence can be a marker. For example, the marker sequence can be fused to an antibody fragment having the marker sequence fused thereto. or a sequence encoding a peptide which facilitates purification of the antibody comprising the polypeptide.

[0090] 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 quantify nucleic acids that contain such polynucleotides. For example, the polynucleotides of the present invention can be used to The present invention allows the identification, isolation, or amplification of partial or full-length clones in a library containing the desired sequence. In some embodiments, the polynucleotides are isolated or otherwise. If not, a genomic sequence complementary to a cDNA from a human or mammalian nucleic acid library or This is a cDNA sequence.

[0091] 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. This cDNA library was normalized to increase the representation of rare sequences. Low or medium stringency, which uses sequences with low sequence identity to the complementary sequence, can be used. The hybridization conditions of the present invention are typical, but not limited to, those of the For more highly identical sequences, medium and high stringency conditions are optionally used. Low stringency conditions are preferred for sequences with approximately 70% sequence identity. To allow hybridization and identify orthologous or paralogous sequences Available.

[0092] Optionally, the polynucleotide of the present invention can be any of the polynucleotides described herein. The polypeptides of the present invention will encode at least a portion of an antibody encoded by the polypeptide of the present invention. The nucleotides are capable of selectively hybridizing to the polynucleotides encoding the antibodies of the present invention. For example, Ausubel, supra, includes nucleic acid sequences available for synthesis. See Colligan, supra, each of which is incorporated herein by reference in its entirety. can be.

[0093] Construction of Nucleic Acids. The isolated nucleic acids of the invention can be produced by (a) recombinant methods, as known in the art. (b) synthetic techniques; (c) purification techniques, or a combination thereof. can.

[0094] The nucleic acid can conveniently include sequences in addition to the polynucleotides of the present invention. For example, a multiple cloning site containing one or more endonuclease restriction sites may be inserted into the nucleobase. The nucleic acid can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. can be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of the invention. The nucleic acid of the invention (excluding the coding sequence) can be optionally prepared as a polynucleotide of the invention. The present invention is directed to a vector, adapter or linker for the cloning and / or expression of a gene.

[0095] Additional sequences may be added to such cloning and / or expression sequences to facilitate cloning and and / or optimizing their function in expression, aiding in the isolation of polynucleotides. It may be possible to improve the transfer 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).

[0096] 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. A oligonucleotide probe is used to identify a desired sequence 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. (This refers to.

[0097] 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 probe genomic or cDNA sequences. By hybridization, homologous genes in the same or different organisms can be isolated. If so, use varying degrees of hybridization stringency in the assay. and either the hybridization or the washing medium can be made stringent. It will be clear that the hybridization conditions can be made more stringent. The greater the degree of complementarity between the probe and target when duplex formation occurs, the greater the degree of complementarity between the probe and target. The degree of stringency can be determined by a variety of factors, such as temperature, ionic strength, pH, and formamide. and the presence of such partially denaturing solvents. The stringency of hybridization can be, for example, within the range of 0% to 50% hormonal concentration. This can be successfully altered by manipulating the amide concentration to change the polarity of the reaction solution. The degree of complementarity (sequence identity) required for possible binding depends on the hybridization medium. and / or the stringency of the washing medium. The degree of complementarity is optimally 1 00%, or 70-100%, or any range or value therein. Small differences in sequence in the probes and primers may affect hybridization and / or It is understood that this can be compensated for by reducing the stringency of the wash medium. It should be understood.

[0098] 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.

[0099] Known methods for amplifying DNA or RNA include the polymerase chain reaction (PCR) and related Related amplification processes (e.g., Mullis et al., U.S. Pat. No. 4,683,195; No. 4,683,202, No. 4,800,159, No. 4,965,188, Tab U.S. Pat. Nos. 4,795,699 and 4,921,794 to Orr et al.; U.S. Patent No. 5,142,033 to Wilson et al., U.S. Patent No. 5,122,464 to Wilson et al. U.S. Patent No. 5,091,310 to Innis and U.S. Patent No. 5,091,310 to Gyllensten et al. ,066,584, Gelfand et al., U.S. Pat. No. 4,889,818, Silve U.S. Patent No. 4,994,370 to R. et al. and U.S. Patent No. 4,766,067 to Biswas. (see U.S. Pat. No. 4,656,134 to Ringold), and double-stranded D Using antisense RNA against a target sequence as a template for RNA synthesis NA-mediated amplification (U.S. Patent No. 5,130,238 to Malek et al., which has the trade name NASBA) These include, but are not limited to, the following documents (the entire contents of which are incorporated herein by reference): (See, e.g., Ausubel, supra, or Sambrook, supra.) See.)

[0100] 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 the protein to be expressed, e.g. cloning a nucleic acid sequence encoding the desired mRNA; detecting the presence of the desired mRNA in a sample; To generate nucleic acids to be used as probes for nucleic acid sequencing or for other purposes. It will be useful in guiding those skilled in the art through in vitro amplification methods. Examples of suitable techniques include Berger, supra; Sambrook, supra; and Ausub, supra. el, and Mullis et al., U.S. Pat. No. 4,683,202 (1987), and In nis, et al., PCR Protocols A Guide to Meth ods and Applications,Eds.,Academic Press Inc, San Diego, CA (1990). Commercially available kits are known in the art. See, for example, the T 4 gene 32 protein (Boehringer Mannheim) was used to identify long P The yield of the CR product can be improved.

[0101] 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.

[0102] Recombinant Expression Cassettes. The present invention further provides recombinant expression cassettes comprising a nucleic acid of the invention. The nucleic acid sequences of the invention, e.g., cDNA or genomic sequences encoding the antibodies of the invention, can be used to Thus, a recombinant expression cassette can be constructed that can be introduced into at least one desired host cell. The recombinant expression cassette typically is a polynucleotide that encodes a polypeptide 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 gene. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to express the nucleic acids of the invention. This can lead to the expression of

[0103] 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.

[0104] 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 modified using 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.

[0105] 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 the vector is a recombinant virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell. Can be transduced.

[0106] 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., , UAA, UGA or UAG) will be appropriately positioned. For expression, UAA and UAG are preferred.

[0107] 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. ), dihydrofolate reductase (DHFR, U.S. Pat. No. 4,399,216, U.S. Pat. No. 4, No. 634,665, No. 4,656,134, No. 4,956,288, No. 5,1 No. 49,636, No. 5,179,017, ampicillin, neomycin (G418) , mycophenolic acid or glutamine synthetase (GS) (U.S. Patent No. 5,122,4 64; 5,770,359, 5,827,739) resistance genes, and Tetracycline or ampicillin for culture in E. coli and other bacteria or prokaryotes These include, but are not limited to, methionine resistance genes (the above patents are incorporated herein by reference in their entirety). Appropriate culture media and conditions for the above host cells are described in the art. Suitable vectors will be readily apparent to those skilled in the art. The vector constructs were introduced into the host cells by calcium phosphate transfection. DEAE-dextran mediated transfection, cationic lipid mediated transfection The cells may be affected by injection, electroporation, transduction, infection or other known methods. Such methods are described in Sambrook, supra, Chapters 1-4 and 16-18. , Ausubel, supra, Chapters 1, 9, 13, 15, and 16, among others, It is listed.

[0108] 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 specific antibody, the antibody or at least one of its fragments may be used to facilitate purification. Such regions can be removed prior to final preparation of the segment. ambrook, Chapters 17.29-17.42 and 18.1-18.74, supra, A It is described in many standard laboratory manuals, such as Chapters 16, 17, and 18 of the USUBEL. It is being done.

[0109] Those skilled in the art will appreciate that there are numerous expression vectors available for expressing nucleic acids encoding the proteins of the present invention. I am familiar with the system.

[0110] Alternatively, the nucleic acid of the invention can be derived from a host cell containing endogenous DNA encoding an antibody of the invention. It can be expressed in the host cell by being switched on (engineering) in the host cell. Such methods are described in U.S. Patents 5,580,734, 5,641,670, As described in US Pat. Nos. 5,733,746 and 5,733,761, These are well known in the art and are incorporated herein by reference in their entirety.

[0111] Mammalian cells are useful cells for the production of antibodies, specific portions or variants thereof. Mammalian cell lines are often cultured in the form of cell monolayers, but cells may also be cultured in, e.g. It can also be adapted to grow in suspension in shake flasks or bioreactors. Many suitable host cell lines capable of expressing intact glycosylated proteins are available. Various strains have been developed in the art, including COS-1 (e.g., ATCC® CRL -1650), COS-7 (e.g., ATCC® CRL-1651), HEK 293, BHK21 (e.g., ATCC® CCL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL -26) Cell lines, Chinese hamster ovary (CHO), Hep G2, P3X63A g8.653, Sp2 / 0-Ag14, and HeLa cells. American Type Culture Collection,Manassa In certain embodiments, the host cells are CHO cells, as well as and cells of lymphoid origin, such as myeloma and lymphoma cells, e.g., CHO-K1 cells, P 3X63Ag8.653 cells (ATCC 0CRL-1580, and Sp2 / 0-Ag14 cells (ATCC® CRL-1581).

[0112] CHO cell line Despite the availability of several other mammalian cell lines, recombinant The majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells. (Jayapal KP et al., Recombinant protein therapy eutics from CHO cells-20 years and count ing.Chem.Eng.Prog.2007;103:40-47, Kunert R, Reinhart D.Advances in recombinant an tibody manufacturing.Appl Microbiol Biot Echnol. 2016;100(8):3451-61). Their strengths include, for example: , robust growth in adherent cells or in suspension, adaptability to serum-free and chemically defined media With a proven history of high productivity and regulatory approval for therapeutic recombinant protein production, They are also highly amenable to genetic modification and can be easily used for cell transfection. Methods for replication, recombinant protein expression, and clonal selection are well characterized. CHO cells can also provide post-translational modifications that are compatible with humans. Examples of "CHO ​​cells" include CHO-DG44, CHO-Kl, CHO-M, and CH OS, CHO GS knockout, and modifications and derivatives thereof. The present invention is not limited to these.

[0113] Expression vectors for these cells include an origin of replication, a promoter (e.g., late or early SV 40 promoter, CMV promoter (U.S. Patent No. 5,168,062; U.S. Patent 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 suitable cells are known and / or may be used in the art, e.g., cell lines and hybridomas. Obtained from canned culture collection catalogs or other known or commercial sources. It is possible.

[0114] 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.

[0115] 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, hydro Examples of such methods include xylapatite chromatography and lectin chromatography. The antibody can be recovered and purified from recombinant cell culture by well-known methods, including but not limited to the following: High performance liquid chromatography (HPLC) For example, purification using HPLC (high performance liquid chromatography) can be used. , Colligan, Current Protocols in Immunolog or Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001), e.g. See Chapters 1, 4, 6, 8, 9 and 10, each of which is incorporated herein by reference in its entirety. Be absorbed.

[0116] 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 The glycosylated nucleotide may be glycosylated or unglycosylated, but is preferably glycosylated. The method for doing so is described in Sambrook, supra, sections 17.37-17.42, and Aus ubel, Chapters 10, 12, 13, 16, 18, and 20, Colligan, P. Many standard laboratory manuals, such as Protein Science, Chapters 12-14 all of which are incorporated herein by reference in their entireties.

[0117] Anti-TNF antibody All of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2 and 3 and / or SEQ ID NOs: 4, 5 and 6 The isolated antibody of the present invention comprising all of the light chain variable CDR regions of The amino acid sequence of the antibody disclosed herein encoded by the nucleotide sequence of any isolated Preferably, the human antibody or antigen-binding fragment binds to human TNF. thereby partially or substantially neutralizing at least one biological activity of the protein. At least one biological activity of at least one TNF protein or fragment is maintained. Partially or preferably substantially neutralizing antibodies or specified portions or variants thereof are , by binding to the protein or fragment, thereby binding TNF to the TNF receptor, can inhibit activities mediated through other TNF-dependent or -mediated mechanisms. As used herein, the term "neutralizing antibody" refers to an antibody that is capable of inhibiting a given antibody by approximately 20-120% depending on the assay. , preferably at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 7 5, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10 It refers to an antibody that can inhibit TNF-dependent activity by 0% or more. The ability of the anti-TNF antibody to inhibit TNF-α is preferably as described herein and / or in the art. The TNF protein or receptor assays are performed using at least one suitable TNF protein or receptor assay known in the art. The human antibodies of the present invention can be of any class (IgG, IgA, IgM, IgE, IgA ... D, etc.) or isotype and may contain κ or λ light chains. In the present invention, the human antibody is an IgG heavy chain or a defined fragment, e.g., IgG1, IgG2, The antibody contains at least one of the following isotypes: IgG3 or IgG4. is at least one human Light chain (e.g., IgG, IgA) and IgM (e.g., γ1, γ2, γ3, γ4) transgenes Using a transgenic mouse or other transgenic non-human mammal containing the gene In another embodiment, the human anti-human TNF antibody can be prepared by comprises an IgG1 heavy chain and an IgG1 light chain.

[0118] As used herein, the term "antibody" or "antibodies" refers to the antibody or antibodies of the 2009 Biol. The BPCI Act and similar global laws and regulations Under the BPCI Act, antibodies are classified as clinically approved biosimilar antibody molecules. Despite slight differences in chemically inactive ingredients, it is "very similar" to the reference product. The data indicates that the product is "expected" to produce clinical results equivalent to those of the reference product in terms of safety, purity, and potency. If the data show that it is a biosimilar, it can be demonstrated (Endocrine Prophylaxis). actice:February 2018,Vol.24,No.2,pp.195- 204) These biosimilar antibody molecules offer an abbreviated approval pathway, thereby Applicants must rely on clinical data from the innovator's reference product to secure regulatory approval. Original innovator criteria approved by the FDA based on successful clinical trials In comparison to antibodies, biosimilar antibody molecules are referred to herein as "biosimilars." As presented herein, SIMPONI® (golimumab) has The original innovator reference anti-TNF antibody approved by the FDA based on successful trials Yes. Golimumab has been available in the United States since 2009.

[0119] 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.

[0120] 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 Non-limiting examples include antibodies 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 comprises corresponding CDR1, 2 and / or 3. at least one heavy chain having an amino acid sequence of (e.g., SEQ ID NO: 1, 2 and / or 3); An antigen-binding fragment comprising at least a portion of a CDR (i.e., CDR1, CDR2 and / or CDR3). In another specific embodiment, the antibody or antigen-binding portion or or variants thereof, such as those shown in SEQ ID NO:4, At least one light chain CDR (i.e., CDR1, CDR2, 3, 4, 5, and / or 6) and / or CDR3) of the antigen-binding region. In a preferred embodiment, the three heavy chain CDRs and the three light chain CDRs of the antibody or antigen-binding fragment are the mAbs TNV148, TNV14, TNV15, TNV1 96, TNV118, TNV32, and TNV86 Such antibodies can be produced using conventional techniques involving recombinant DNA technology. and preparing and expressing a nucleic acid molecule (i.e., one or more) encoding an antibody. The antibody can be isolated using conventional techniques, for example by using a nucleic acid sequence similar to that described above, or by using any other suitable method. It is prepared by chemically linking various parts (e.g., CDR, framework) together. It can be manufactured.

[0121] 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 The production of antibodies can be induced by immunization with TNF or a fragment thereof. The producing cells can be isolated and used as described herein and / or in the art. As is known in the art, hybridomas or other immortalized antibody-producing cells can be prepared by Alternatively, the antibody, specified portion or variant can be expressed in a suitable host cell. and can be expressed using the encoding nucleic acid or a portion thereof.

[0122] The present invention also relates to amino acids in 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. Alternatively, such antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs may be highly homologous. Harmonic (e.g., 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 described in the specification include conservative amino acid sequences. Conservative amino acid substitutions include sequences containing substitutions as well as amino acid deletions and / or insertions. , by isolating the first amino acid from a nucleic acid sequence having chemical and / or physical properties similar to those of the first amino acid (e.g. For example, the substitution of a second amino acid with a different amino acid sequence (e.g., charge, structure, polarity, hydrophobicity / hydrophilicity) is used. Conservative substitutions include replacing one amino acid with another amino acid within the following group: Lysine (K), Arginine (R) and Histidine (H); Aspartate (D) and and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), onine (T), tyrosine (Y), K, R, H, D, and E; alanine (A), valine (V ), leucine (L), isoleucine (I), proline (P), phenylalanine (F), Tryptophan (W), methionine (M), cysteine ​​(C), and glycine (G); F , W, and Y; C, S, and T.

[0123] 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. and the codon or codons of the amino acid sequence may be used to denote the amino acid sequence. is well understood in the r Biology of The Cell,Third Ed.,Garland (See, e.g., IEEE Publishing, Inc., New York, 1994).

[0124] [Table 1]

[0125] 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.

[0126] 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 .

[0127] 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 involves the introduction of single alanine mutations at every residue in the molecule. and the resulting mutant molecule has at least one TNF neutralizing activity, e.g. The antibodies are tested for biological activity, including but not limited to: The sites that are important for the synthesis of the nucleotides have also been identified by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling. (Smith, et al., J. Mol. Biol. 224:8 99-904 (1992) and de Vos, et al., Science 255: 306-312(1992)).

[0128] 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.

[0129] 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.

[0130] 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, 9 2, 93, 94, 95, 96, 97, 98, 99, 100, or any range therein The values ​​can be calculated using suitable computer algorithms, as known in the art. is determined using

[0131] Exemplary heavy and light chain variable region sequences 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.

[0132] As will be apparent to one of skill in the art, the present invention includes at least one biologically active compound of the present invention. Biologically active antibodies include antibodies that are naturally occurring (non-synthetic), endogenous or related At least 20%, 30% or 40% of that of the known antibody, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95% It has a specific activity of 1000%. Methods for assay and quantification of enzyme activity and substrate specificity is well known to those skilled in the art.

[0133] 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. The present invention relates to human antibodies and antigen-binding fragments that have been modified to have improved pharmacokinetic properties. (e.g., increased serum half-life in vivo) to produce antibodies or antigen-binding fragments. The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester. In certain embodiments, the hydrophilic polymeric group can be a tertiary ether group. 0 to about 120,000 daltons, and polyalkane glycols (e.g., polyethylene glycols). Polyethylene glycol (PEG), polypropylene glycol (PPG), carbohydrate polymers, It may be an amino acid polymer or polyvinylpyrrolidone, and may also be a fatty acid group or a fatty acid ester. The alkyl group can contain from about 8 to about 40 carbon atoms.

[0134] The modified antibodies and antigen-binding fragments of the invention are directly or indirectly covalently attached to the antibody. The antibody or antigen-binding fragment of the present invention may contain one or more organic moieties that are attached to the antibody or antigen-binding fragment of the present invention. Each organic moiety is independently a hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" refers to mono- and di-carboxylic acids. As used herein, the term "hydrophilic polymeric group" includes octane, It refers to an organic polymer that is more soluble in water than in water. For example, polylysine is Therefore, antibodies modified with covalent polylysine have a higher solubility in water than Suitable hydrophilic polymers for modifying the antibodies of the invention include linear or branched hydrophilic polymers. They may be branched, for example polyalkane glycols (e.g., PEG, monomethoxy-poly ethylene glycol (mPEG, PPG, etc.), carbohydrates (e.g., dextran, sugars, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, poly realginine, polyaspartic acid, etc.), polyalkane oxide (e.g., polyethylene Polyvinylpyrrolidone, polyvinyl oxide, polypropylene oxide, etc. For example, the hydrophilic polymer that modifies the antibody of the present invention has about 800 to about 1000 individual molecular entities. It has a molecular weight of 50,000 daltons. For example, PEG 5000 and PEG 20,00 0 can be used, where the subscript is the average molecular weight of the polymer in Daltons. The water-soluble polymeric group is 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 are preferred. For example, a polymer containing amine groups can be prepared by It can be linked to the carboxylate of a fatty acid or a fatty acid ester, Activated carboxylates on esters (e.g., activated with N,N-carbonyldiimidazole) The hydroxyl groups on the polymer can be linked to the hydroxyl groups on the polymer.

[0135] 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.

[0136] The modified human antibodies and antigen-binding fragments can be subjected to any suitable treatment, such as by reacting them with one or more modifying agents. As used herein, the term "modifying agent" means , a suitable organic group (e.g., a hydrophilic polymer, a fatty acid, a fatty acid ester) containing an activating group. An "activating group" means a group that, under appropriate conditions, reacts with a second chemical group, thereby forming a modifying agent. A chemical moiety or functional group capable of forming a covalent bond between a first chemical group and a second chemical group. For example, amine-reactive activating groups include tosylate, mesylate, halo (chloro, bromo, fluoro, Electrophilic groups such as fluoro, iodo, and N-hydroxysuccinimidyl ester (NHS) Examples of activating groups capable of reacting with thiols include maleimide, iodopropanol, etc. Cetyl, acryloyl, pyridyl disulfide, thio 5-thiol-2-nitrobenzoate The aldehyde functional group can be an amine or hydrathiol. 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 phospho It is possible to form phosphoramidate or phosphorimide bonds, introducing activating groups into the molecule. Suitable methods for doing so are known in the art (e.g., Hermanso et al., n,GT,Bioconjugate Techniques,Academic Press: San Diego, CA (1996). Directly to the functional group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) or via a linker moiety minutes, e.g., divalent C 1 ~C 12 A group in which one or more carbon atoms are oxygen, nitrogen, or sulfur. The linker may be a heteroatom such as 1,4-dihydro-1,4-triazolyl group, which may be substituted with a heteroatom such as 1,4-diazolyl group. The moiety can be, for example, tetraethylene glycol, -(CH 2 ) 3 -, -NH-(CH 2 ) 6 -NH-, -(CH 2 )2 -NH- and -CH 2 -O-CH 2 -CH 2 -O-CH 2 -C H 2 The linker moiety-containing modifying agent includes, for example, 1-ethyl-3- In the presence of (3-dimethylaminopropyl)carbodiimide (EDC), mono-Boc- Alkyl diamines (e.g., mono-Boc-ethylenediamine, mono-Boc-diamino By reacting hexane with fatty acids, free amines and fatty acid carboxylates are produced. The Boc protecting group can be converted to a trifluoro The product was removed by treatment with acetic acid (TFA) and converted to another carboxylate as described. This exposes a primary amine that can be coupled with maleic anhydride or reacted with maleic anhydride. and the resulting product can be cyclized to produce an activated maleimide derivative of the fatty acid. (See, e.g., Thompson et al., the teachings of which are incorporated herein by reference in their entirety. (See WO 92 / 16221.)

[0137] 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. NHS esters of the compounds can be used to conjugate to antibodies in a non-site-specific manner. Disulfide bonds (e.g., intrachain disulfide bonds) of antibodies or antigen-binding fragments can be (a) preparing a modified human antibody or antigen-binding fragment thereof; In this case, the reduced antibody or antigen-binding fragment can be subjected to thiol-reactive modification. The antibody of the present invention can be reacted with a decorating agent to produce a modified antibody of the present invention. Modified human antibodies and antigen-binding fragments containing organic moieties attached to specific sites of the antibody are known as reverse transcription antibodies. Protein degradation (Fisch et al., Bioconjugate Chem., 3 :147-153(1992), Werlen et al.,Bioconjugat e Chem.,5:411-417(1994), Kumaran et al.,P rotein Sci.6(10):2233-2241(1997), Itoh et al. al.,Bioorg.Chem.,24(1):59-68(1996), Cape llas et al.,Biotechnol.Bioeng.,56(4):456 -463 (1997)) and Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA( The compounds can be prepared using any suitable method, such as that described in (1996).

[0138] 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 immunized strain (such as a human immunized strain) with the antibody or a CDR-containing region thereof. The immunized animal recognizes and responds to the idiotypic determinants of the immunizing antibody, producing anti-Id The anti-Id antibody can also be used as an "immunogen" to induce an immune response in yet another animal. to generate so-called anti-anti-Id antibodies.

[0139] 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.

[0140] The anti-TNF antibody compositions of the present invention further comprise a compound that is capable of inhibiting the proliferation, proliferation, and / or proliferation of cells in need of such modulation, treatment or therapy. , a tissue, an organ, an animal or a patient, and optionally at least one anti-TNF antibody. , at least one TNF antagonist (e.g., a TNF antibody or fragment, a soluble TNF receptor The present invention relates to a TNF antagonist or a fragment thereof, a fusion protein thereof, or a small molecule TNF antagonist. and the like), antirheumatic drugs (e.g., methotrexate, auranofin, auranofin, etc.), Lotioglucose, azathioprine, etanercept, sodium gold thiomalate, sulfate Hydroxychloroquine, leflunomide, sulfasalidine), muscle relaxants, anesthetics (nar cotic, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics (anesth etics), sedatives, local anesthetics, neuromuscular blockers, antibacterial agents (e.g., aminoglycosides), Antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinols lones, macrolides, penicillins, sulfonamides, tetracyclines, and other antibacterial agents ), antipsoriatic drugs, corticosteroids, anabolic steroids, diabetes-related drugs, minerals , nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, anti Ulcer drugs, laxatives, anticoagulants, erythropoietic drugs (e.g., epoetin alfa), filgras sargramostim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Le ukine) vaccinations, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine) sporin, daclizumab), growth hormone, hormone replacement drugs, estrogen receptor modulators , mydriatics, cyclomyorheological agents, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, Depressants, antimanics, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil Medications for asthma, beta-agonists, inhaled steroids, leukotriene inhibitors, methyloxalate cephalosporin, cromolyn, epinephrine or similar drugs, dornase alfa (Pulmozyme), any of the above, further comprising at least one selected from a cytokine or a cytokine antagonist; The composition may include at least one of a suitable and effective amount of a composition or a pharmaceutical composition. Non-limiting examples of cytokines include IL-1 to IL-23. Suitable dosages are well known in the art. For example, Wells et al., eds., Pharmacotherapy Handbo ok,2 nd Edition,Appleton and Lange,Stamfo rd, CT(2000), PDR Pharmacopoeia, Tarascon P ocket Pharmacopoeia 2000,Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000) No. 6,399,433, each of which is incorporated herein by reference in its entirety.

[0141] 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. 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).

[0142] 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 comprise 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 mutant composition is selected. , can be selected on a daily basis.

[0143] Pharmaceutical additives and excipients useful in the present compositions include, but are not limited to, tannins, 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, at 1 to 99.99% by weight or volume. Protein additives 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.

[0144] Carbohydrate additives suitable for use in the present invention include, for example, fructose, maltose, and the like. Monosaccharides such as lactose, galactose, glucose, D-mannose, and sorbose; Disaccharides such as sucrose, trehalose, and cellobiose, raffinose, melezitol, and Polysaccharides such as maltodextrin, dextran, and starches, mannitol, xylitol, and glyceryl stearate. Litol, maltitol, lactitol, xylitol sorbitol (glucitol), Preferred carbohydrates for use in the present invention include alditols such as myo-inositol. The additives are mannitol, trehalose, and raffinose.

[0145] 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.

[0146] 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 such as glycerides, lipids (e.g., phospholipids, fatty acids), steroids (e.g., co Additives / additives such as esterol, chelating agents (e.g., EDTA) It may include.

[0147] These and further compounds suitable for use in the anti-TNF antibody, portion or variant compositions according to the invention. Additional known pharmaceutical additives and / or excipients are known in the art, e.g. ``Remington: The Science & Practice of P. "Harmacy," 19th ed., Williams & Williams, (1 995), and Physician's Desk Reference, 52 nd ed, Medical Economics, Montvale, NJ (1998). and the disclosures of which are incorporated herein by reference in their entireties. Carrier or excipient materials include carbohydrates (e.g., monosaccharides and alditols) and buffers (e.g., citric acid) or a polymeric agent.

[0148] 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 US201002326333A1 - Pharmaceutical or veterinary uses comprising at least one anti-TNF antibody in a therapeutically acceptable formulation - Google Patents The present invention provides a multi-use preserved formulation suitable for use in a variety of applications. The preserved formulation comprises at least one pre-existing known, i.e., at least one of phenol, m-cresol, p-cresol, o-cresol, Resole, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol Alcohol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate) , alkylparaben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride , benzethonium chloride, sodium dehydroacetate, and thimerosal, or a mixture thereof The composition further comprises a preservative, optionally selected from the group consisting of those known in the art. As in, 0.001 to 5%, or 0.001, 0.003, 0.005, 0.00 9, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0 .4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, Such as, but not limited to, 4.7, 4.8, 4.9, or any range or value therein. Any suitable concentration or mixture may be used, including any range or value therein. Non-limiting examples include 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., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.00 Thimerosal from 1 to 0.5% (e.g., 0.005, 0.01), 0.001 to 2.0% of phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% 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%).

[0149] As mentioned above, the present invention provides a packaging material and, optionally, a buffer formulated in an aqueous diluent and 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.

[0150] At least one anti-TNF antibody used according to the present invention may be derived from a mammalian cell or a transgenic mammalian cell. It may be produced by recombinant means, including from a transgenic preparation, or as described herein. or may be purified from other biological sources as known in the art.

[0151] 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.

[0152] 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. The amount of preservative used will vary depending on the preservative used and can be readily determined by one of skill in the art.

[0153] Other additives, 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 pH. The formulation provides a pH control of about pH 4 to about pH 10, and preferably 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).

[0154] Other additives, such as Tween 20 (polyoxyethylene (20) sorbitan monolayer) urate), Tween 40 (Polyoxyethylene (20) Sorbitan Monopalmitate Tween 80 (Polyoxyethylene (20) Sorbitan Monooleate), P LURONIC F68 (Polyoxyethylene polyoxypropylene block copolymer) ), and a pharma- ceutically acceptable solubilizer, such as PEG (polyethylene glycol), or Polysorbate 20 or 80 or Poloxamer 184 or 188, Pluron Nonionic surfactants such as ic® polyols and other block copolymers and optionally adding chelating agents such as EDTA and EGTA to the formulation or composition. These additives can be used in pumps or other devices to administer the formulation. is particularly useful when plastic containers are used. The presence of reduces the tendency of the protein to aggregate.

[0155] 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

[0156] The claimed formulations may be formulated as clear solutions or in the form of a mixture of water, preservatives and / or additives, 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. The drug may be provided to the patient as a dual vial containing a vial of Either single solution vials or dual vials requiring reconstitution are reused multiple times. can be used to fulfill single or multiple patient treatment cycles and therefore are This can provide a more convenient treatment regimen than is currently available.

[0157] 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 optionally be safely stored at a temperature between about 2 and about 40° C., and the biological properties of the protein may be maintained. The activity can be maintained for a long period of time, and therefore the solution can be labeled with 6, 12, 18, 24, 36 It may be indicated that the product may be retained and / or used for 48, 72, or 96 hours or more. If a diluent that is stored in the container is used, the label may state that the maximum period is 1 to 12 months, 6 months, or 1 year. May include up to half and / or two years of use.

[0158] 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 which includes 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.

[0159] The claimed product is available as a clear solution or in a second vial containing an aqueous diluent. A dual vial containing at least one lyophilized anti-TNF antibody that is reconstituted in a lyophilized solution. It can be provided to patients as a single solution vial or as a vial requiring reconstitution. Each dual vial can be reused multiple times and can be used for single or multi-patient treatment. The present invention provides a method for treating pulmonary circulation disorders that can meet the patient's daily life expectancy and thus provide a more convenient treatment regimen than is currently available. Provide.

[0160] The claimed product is a clear solution or a second vial containing an aqueous diluent. A dual vial containing at least one lyophilized vial of an anti-TNF antibody, to pharmacies, clinics, or other such institutions and facilities, In this case, the clear solution can be provided indirectly to the patient up to 1 liter or even more. The larger container may contain at least one smaller volume of antibody solution. The vial is then dispensed one or more times into a smaller vial and delivered to the customer by the pharmacy or clinic. and / or can be provided to the patient.

[0161] The approved devices that contain these single-vial systems are BD Pens, BD Auto jector(registered trademark), Humaject(registered trademark), NovoPen(registered trademark) ), BD® Pen, AutoPen®, and OptiPen® GenotropinPen (registered trademark), Genotronorm Pen (registered trademark), Humatro Pen (registered trademark), Reco-Pen (registered trademark), Roferon Pen®, Biojector®, iject® Registered trademark), J-tip Needle-Free Injector (registered trademark), I Delivering solutions such as Intraject®, Medi-Ject®, etc. For example, they include pen-type injector devices for Becton Dickensen (Franklin Lakes, NJ, www. bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.dis etronic.com, Bioject, Portland, Oregon (www.bioject.com ), Weston Medical (Peterborough, UK, www.west on-medical.com), Medi-Ject Corp (Minneapolis, MN, www.medij ect.com).

[0162] Approved devices that contain dual vial systems include HumatroPen (registered trademark) The reconstituted drug is reconstituted in a cartridge for delivering the reconstituted solution, such as a lyophilized drug cartridge (e.g., Examples include pen-type injector systems for injecting blood.

[0163] 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 also provides the conditions under which the product can be used. At least one anti-TNF antibody is reconstituted in an aqueous diluent to form a solution, and incubated for a period of 2 to 24 hours or more. Over the course of the period, the instructions stated that this solution should be used for two vials of wet / dry products. For single vial solution products, the label shall state that the solution is The products claimed herein are intended to be used in human medicine for a period of time longer than that required by the present invention. It is useful for product applications.

[0164] 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.

[0165] The claimed stable or preserved formulations are either present as clear solutions or in aqueous diluents containing preservatives or or a second vial containing buffer and additives. The two drugs should be provided to patients as dual vials, each containing one vial of anti-TNF antibody. Either the single solution vial or the dual vial requiring reconstitution can be used multiple times. It can be reused and can fulfill single or multiple patient treatment cycles, thus This provides a more convenient treatment regimen than is currently available.

[0166] 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. The present invention can be administered via a variety of delivery methods, including other means well known in the art and understood by those of skill in the art. The composition can be administered to a subject by any method.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] The present invention also relates to leukemia, acute leukemia, acute lymphocytic leukemia (ALL), B-cell, T-cell cells or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma tumor, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, malignant paraneoplastic syndromes Symptoms / hypercalcemia, solid tumors, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer and / or cancer-related bone pain. To modulate or treat at least one malignant disease in a cell, tissue, organ, animal or patient. The present invention also provides a method for

[0173] The present invention also relates to the treatment of neurodegenerative diseases, multiple sclerosis, migraine headaches, AIDS dementia syndrome, demyelinating diseases, For example, multiple sclerosis and acute transverse myelitis, extrapyramidal and cerebellar disorders, e.g., corticospinal Lesions of the cerebral cortex, disorders of the basal ganglia or cerebellar disorders, hyperkinetic movement disorders, e.g., Huntington's disease Chorea and senile chorea, drug-induced movement disorders, e.g., blocking CNS dopamine receptors Drug-induced hypokinetic movement disorders, e.g., Parkinson's disease, progressive Supranuclear palsy, structural lesions of the cerebellum, spinocerebellar degeneration, e.g., spinal ataxia, free-drying Ataxia, cerebellar cortical degeneration, multiple system degeneration (Mencel, Dejerine-Thom as, Shi-Drager and Machado-Joseph), systemic diseases (Refs Disease, abetalipoproteinemia, ataxia, telangiectasia, and mitochondrial multilineage syndrome Disorders), demyelinating core disorders, e.g., multiple sclerosis, acute myelopathy, 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, Marr's disease, Down's syndrome in middle-aged people, diffuse Lewy body disease, senile dementia with Lewy bodies Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakob disease , subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and dementia pugilistica. at least one of, but not limited to, Also provided is a method 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 a specified portion or variant thereof. or the pharmaceutical composition is administered to cells, tissues, organs, The method may include administering the compound to an animal or a patient. l,16 th Edition, Merck & Company, Rahway, NJ (1992).

[0174] 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 at least one anti-TNF antibody, The administration of the selected portion or variant thereof may be in combination with at least one TNF antagonist (e.g., a TNF antibody). or fragments, soluble TNF receptors or fragments, fusion proteins thereof, or small molecule TNF F antagonists, etc., but are not limited to, antirheumatic drugs (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold Sodium oleate, hydroxychloroquine sulfate, leflunomide, sulfasalazine) , muscle relaxants, anesthetics (narcotics), nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics Anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials (e.g., aminoglycosides, Antifungals, anthelmintics, antivirals, carbapenems, cephalosporins, fluoroquinolones, Macrolides, penicillins, sulfonamides, tetracyclines, other antibacterial agents), antipsoriatic drugs, corticosteroids, anabolic steroids, diabetes-related drugs, minerals, nutritional drugs, Thyroid medications, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcer drugs, palliatives Laxatives, anticoagulants, erythropoietin (e.g., epoetin alfa), filgrastim (e.g., For example, G-CSF, Neupogen, sargramostim (GM-CSF, Leukin e), vaccinations, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine daclizumab), growth hormone, hormone replacement drugs, estrogen receptor modulators, mydriasis Drugs, cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants , antimanic drugs, antipsychotic drugs, anxiolytic drugs, hypnotics, sympathomimetics, stimulants, donepezil, Clin, asthma medications, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines , cromolyn, epinephrine or analogues, dornase alpha (Pulmozyme), cytochrome P450 and / or 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.

[0175] 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, thalidoma id, tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline and lorlatinib Plum), A2b adenosine receptor agonist and A2b adenosine receptor enhancer Preventing and / or inhibiting TNF synthesis, TNF release or its action on target cells, such as compounds that inhibit TNF receptors, such as mitogen-activated protein (MAP) kinase inhibitors; Compounds that prevent and / or inhibit signal transduction, block and / or inhibit membrane TNF cleavage Compounds, e.g., metalloproteinase inhibitors, compounds that block and / or inhibit TNF activity. compounds such as angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril); and compounds that block and / or inhibit TNF production and / or synthesis, such as MAP kinase. These include, but are not limited to, enzyme inhibitors.

[0176] As used herein, "tumor necrosis factor antibody," "TNF antibody," "TNFα antibody" " or "fragment" etc., can be used to describe the synthesis of 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

[0177] 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.

[0178] 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 × l0 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.

[0179] 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 born.

[0180] 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).

[0181] 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.

[0182] 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.

[0183] 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 expressed 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: 215-219 (1994), Butler et al., Cytokine 6( 6):616-623(1994), Baker et al., Eur. J. Immu Nol. 24:2040-2048 (1994), Beutler et al., U.S. Pat. No. 47,851 and U.S. Application No. 08 / 442,133 (filed May 16, 1995), Each of these references is incorporated herein by reference in its entirety. Methods for producing synthetic molecules are described by Capon et al., U.S. Pat. No. 5,116,964; Capon et al., No. 5,225,538 and Capon et al., Nature 337 :525-531 (1989), which references are incorporated by reference in their entireties. is incorporated herein.

[0184] 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 of the TNF receptor molecule sequence that encodes the TNF receptor molecule. Functional equivalents of receptor molecules that function in the TNF receptor molecules can also be used in the present invention. modified polypeptides 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 the "SILEN A "T" codon, or one or more amino acid substitutions, deletions, or additions (e.g., one acidic amino acid amino acid may be substituted for another acidic amino acid, or the same or a different hydrophobic amino acid may be substituted. Substitute one codon that codes for a hydrophobic amino acid for another codon that codes for a hydrophobic amino acid. ). Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc.and Wiley-Interscience,New York(19 87-2000).

[0185] 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.

[0186] Therapeutic Treatment. Any of the methods of the invention include administering to a cell in need of such modulation, treatment or therapy, administering to the tissue, organ, animal or patient an effective amount of a composition comprising at least one anti-TNF antibody or The present invention may include a method for treating a TNF-mediated disorder comprising administering the pharmaceutical composition. Such methods optionally further include co-administration or combination therapy for the treatment of such immune disorders. and wherein the administration of the at least one anti-TNF antibody, specified portion or variant thereof comprises: At least one TNF antagonist (e.g., a TNF antibody or fragment, a soluble TNF receptor or fragments thereof, fusion proteins thereof, or small molecule TNF antagonists. (not specified), antirheumatic drugs (e.g., methotrexate, auranofin, aurothiouracil, Glucose, azathioprine, etanercept, sodium gold thiomalate, hydrochloride sulfate oxychloroquine, leflunomide, sulfasalazine), muscle relaxants, anesthetics (narcotics), Nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials (e.g., aminoglycosides, antifungals, anthelmintics, antivirals, Rubapenem, cephalosporin, fluoroquinolone, macrolide, penicillin, sulfo antibacterials, antipsoriatics, corticosteroids, anabolites, 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), Vaccination, Immunoglobulin, Immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormone , hormone replacement drugs, estrogen receptor modulators, mydriatics, cycloplegics, alkylating agents, Anti-metabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanics, antipsychotics, anxiolytics , hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or from analogs, dornase alpha (Pulmozyme), cytokines or cytokine antagonists It further includes administering before, simultaneously with, and / or after at least one selected.

[0187] 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 an effective amount or dose of a TNF antibody composition. Suitable serum concentrations include those between 0.1 and 5000 μg / mL per single or multiple dose. Suitable dosages will be known to medical practitioners and will, of course, vary depending on the particular disease. It depends on the condition, the specific activity of the composition being administered, and the particular patient undergoing treatment. In some cases, repeated administration, i.e., specific monitored doses or may require providing repeated individual doses of a metered amount, where the individual doses are Repeated until the desired daily dose or effect is achieved.

[0188] 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.

[0189] Alternatively, the dose to be administered may be determined based on the pharmacodynamic characteristics of the particular agent and its method and route of administration. , age, health condition and weight of the recipient, nature and severity of symptoms, type of concurrent treatment, treatment The dosage of the active ingredient is usually determined by known factors such as the frequency of administration, and the desired effect. The dosage may be about 0.1 to 100 milligrams per kilogram of body weight. 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram or in sustained release form; It is effective in achieving the desired results.

[0190] 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.

[0191] 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.

[0192] For parenteral administration, the antibody is provided in combination with or separately from a pharma- ceutically acceptable parenteral vehicle. It can be formulated as a solution, suspension, emulsion or lyophilized powder, which is delivered to the patient. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. 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 be sterilized by known or suitable techniques. It will be sterilized.

[0193] 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.

[0194] Alternative Administration. A pharma- ceutical effective amount of at least one anti-TNF antibody according to the invention is administered. Many known and developed methods of administration 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.

[0195] The TNF antibody of the present invention may be present in a carrier as a solution, emulsion, colloid or suspension. or as a dry powder, by inhalation, or by administration 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.

[0196] Parenteral formulations and administration. Parenteral formulations contain sterile water or saline as common excipients. 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.

[0197] 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 composition may be administered parenterally (subcutaneously, intramuscularly or intravenously) or by any other route. In particular for use in the form of a liquid solution or suspension, in particular as creams and suppositories. In a semi-solid form, such as, but not limited to, for use in vaginal or rectal administration. Oral or sublingual administration, such as, but not limited to, tablets or capsules; For oral administration, or as a powder, nasal drops or aerosol, or as certain medications, etc. The present invention can be applied in the form of, but not limited to, intranasal administration, or by modifying the skin structure or by a transdermal patch. Chemical stimulants such as dimethyl sulfoxide can be used to either increase drug concentrations in the blood or Using accelerators (Junginger, et al. In "Drug Permeation n Enhancement”;Hsieh,DS,Eds.,pp.59-90( Marcel Dekker, Inc. New York 1994, incorporated by reference in its entirety. (incorporated herein) or application of protein and peptide-containing formulations to the skin. using an oxidizing agent that allows the oxidation of the oxidizing agent (WO 98 / 53847) or by electroporation for creating a transient delivery pathway, such as iontophoresis, or for skin Application of an electric field to increase the mobility of a charged drug through the skin, or ultrasonics such as sonophoresis Using wave applications (U.S. Patents 4,309,989 and 4,767,402), Such formulations include, but are not limited to, gels, ointments, lotions, suspensions, or patch delivery systems. (The above publications and patents are incorporated herein by reference in their entireties.) (Incorporated herein).

[0198] 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 The inhaler may be comprised of either solid particles. Metered dose inhalers typically use a propellant gas and require actuation during inspiration (e.g., International (See Publication No. 94 / 16970 and WO 98 / 35888). haler™ (Astra), Rotahaler® (Glaxo), Diskus(R) (Glaxo), Spiros(TM) Inhaler (Dura), The Spinhaler (registered trademark) is a device sold by Inhale Therapeutics. Dry powder inhalers, such as the Fisons® Dry Powder Inhaler, use breath actuation of a mixed powder. (U.S. Pat. No. 4,668,218 Astra, European Patent No. 237,507 Astra , WO 97 / 25086 Glaxo, WO 94 / 08552 Dura, US Patent No. 5458135 Inhale, International Publication No. 94 / 06498 Fisons, (All of which are incorporated herein by reference in their entireties). AERx™ Aradigm , Ultravent™ nebulizer (Mallinckrodt), and Aco rnII® Nebulizer (Marquest Medical Products) ts) (U.S. Pat. No. 5,404,871 Aradigm, International Publication No. WO 97 / 22376) Nebulizers, such as , produce aerosols from solutions, while metered-dose inhalers, dry powder inhalers, The above references are incorporated herein by reference in their entireties. These specific examples of commercially available inhalation devices are intended to illustrate the specific devices suitable for the practice of the present invention. It is intended to be representative and not to limit the scope of the invention. Preferably, the composition comprising at least one anti-TNF antibody is administered as a dry powder adjuvant. The method of administering at least one antibody of the present invention is to deliver the same to a patient by inhalation or aerosol delivery. There are several desirable features of an inhalation device. For example, delivery by an inhalation device: Advantageously, the inhalation device is reliable, reproducible and accurate. For example, small dry particles of less than about 10 μm, preferably about 1 to 5 μm, are used so that the particles can be breathed easily. The particles can be delivered.

[0199] 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.

[0200] 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 The amount of the solution may be, but is not limited to, 100 mg / mL or 100 mg / gm, or 1 ml or less. or about 0.1 mg to about 100 mg per 1 mg / gm solution, or any range therein or a concentration of at least one anti-TNF antibody composition protein of the value. The formulation comprises an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and preferably The formulation may contain agents such as a buffer, a reducing agent, bulk proteins or carbohydrates. The antibody composition may also include an excipient or agent for stabilizing the antibody composition protein, such as an antibacterial agent. Bulk proteins useful in formulating the composition protein include albumin, protamine, Exemplary carbohydrates useful in formulating antibody composition proteins include Examples of suitable sugars include sucrose, mannitol, lactose, trehalose, and glucose. Composition protein formulations also have the potential to induce antibody reactions caused by atomization of the solution during aerosol formation. The composition may also include a surfactant that can reduce or prevent surface-induced aggregation of protein. Polyoxyethylene fatty acid esters and alcohols, as well as polyoxyethylene sols Various conventional surfactants such as butyl fatty acid esters can be used. Generally, it will range from 0.001 to 14% by weight of the formulation. The surfactants that are useful are polyoxyethylene sorbitan monooleate, polysorbate 80, Polysorbate 20. Anti-TNF antibodies or specified portions or variants thereof. Formulations of the protein may also include additional agents known in the art. can.

[0201] 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.

[0202] At least one anti-T suitable for use with either a jet or ultrasonic nebulizer Formulations of NF antibodies typically contain at least about 0.1 mg to about 100 mg per mL of solution. The formulations each contain one concentration of anti-TNF antibody protein. The formulations also contain excipients, buffers, isotonicity agents, , preservatives, surfactants, and preferably zinc. , a reducing agent, a bulk protein, or a carbohydrate. It may also contain excipients or agents for stabilization of the protein. At least one anti-TNF antibody. Bulk proteins useful in formulating the composition protein include albumin, protamine, Exemplary carbohydrates useful in the formulation of at least one anti-TNF antibody include: Examples include sucrose, mannitol, lactose, trehalose, glucose, etc. At least one anti-TNF antibody formulation may also be drawn by atomization of the solution upon forming an aerosol. and (b) an interface capable of reducing or preventing surface-induced aggregation of at least one anti-TNF antibody. Active agents may also be included. Polyoxyethylene fatty acid esters and alcohols, as well as polyoxyethylene fatty acid esters and alcohols. A variety of conventional surfactants such as diethylene sorbital fatty acid esters can be used. The amount is generally in the range of 0.001 to 4% by weight of the formulation. The preferred surfactants are polyoxyethylene sorbitan monooleate, polysorbate Polysorbate 80, Polysorbate 20, etc. For preparations of proteins such as antibody proteins, Additional pharmaceutical agents known in the art can also be included in the formulation.

[0203] 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 additive or other additives may include 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

[0204] 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.

[0205] 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.

[0206] 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), parabens, preservatives (sorbitol, acid, ascorbic acid, α-tocopherol, etc.), antioxidants (cysteine, etc.), The composition may contain additives, binders, thickeners, buffers, sweetening agents, flavoring agents, perfumes, and the like.

[0207] 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 US Pat. Nos. 79,681 and 5,5871,753 are biologically Its use for the oral delivery of active agents is known in the art.

[0208] 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. Preparations, such as suppositories, may contain, as additives, for example, polyalkylene glycols, petrolatum, calcium carbonate, etc. The formulation for intranasal administration may be solid and may contain additives such as cocoa butter. For example, it may contain lactose or may be an aqueous or oily solution nasal spray. For oral administration, sugar, calcium stearate, stearic acid magnesium, pregelinatined starch, etc. (U.S. Patent No. 5,333,633). , No. 849, 695).

[0209] 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).

[0210] 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. Pharmacologically acceptable non-toxic salts of the compounds, for example, (a) phosphoric acid, sulfuric acid, citric acid, tartaric acid, 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 appropriate solution for injection, such as sesame oil. It can also be formulated in a gel, for example an aluminum monostearate gel. Particularly preferred salts are zinc salts, zinc tannate, and zinc pamoate. The injectable sustained release depot formulations are, for example, the polysaccharides described in U.S. Pat. No. 3,773,919. In slowly degrading, non-toxic, non-antigenic polymers such as lactic acid / polyglycolic acid polymers The compound or salt is preferably dispersed in the aqueous solution for encapsulation. Relatively insoluble salts such as those mentioned above are useful as cholesterol markers, particularly for use in animals. It can also be formulated in Trix Silastic pellets. Implant formulations, such as gaseous or liquid liposomes, have been described in the literature (U.S. Pat. Nos. 5,713,331 and 5,721,625). No. 70,222 and "Sustained and Controlled Release se Drug Delivery Systems”, JRRobinson e d., Marcel Dekker, Inc., NY, 1978).

[0211] 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.

[0212] 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, long terminal repeats (LTRS) from HIVI, and cytomegalovirus ( This can be achieved using the early promoter of CMV. However, The human actin promoter may also be used in the practice of the present invention. Suitable expression vectors for this purpose include, for example, pIRES1neo, pRetro-Off, pR etro-On, PLXSN or pLNCX (Clonetech Labs, Pa lo Alto, CA), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -) or pcDNA3.1 / Hygro(+ / -) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (A pSV2dhfr (ATCC 37146 ) as well as vectors such as pBC12MI. Mammalian host cells that can be used The cells include human Hela293, H9, and Jurkat cells, mouse NIH3T3, and C12 7 cells, Cos1, Cos7 and CV1, quail QC1-3 cells, mouse L cells and chafer cells. Chinese hamster ovary (CHO) cells are included.

[0213] Alternatively, the gene can be expressed in a stable cell line that contains the gene integrated into a chromosome. It is possible to express dhfr, gpt, neomycin, or hygromycin. Co-transfection with a selectable marker allows identification and characterization of transfected cells. Allows for isolation.

[0214] 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.

[0215] 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.

[0216] Cloning and expression in CHO cells One vector commonly used for expression in CHO cells is pC4. The plasmid pC4 is a clone of the plasmid pSV2-dhfr (ATCC® 37146). This plasmid contains mouse DHFR under the control of the SV40 early promoter. The plasmids transfected contain the gene encoding dihydrofolate activity. Chinese hamster ovary cells or other cells lacking Selective medium (e.g., α-MEM, Life Technologies, G Cells can be selected by growing them in a 20-well plate (Aithersburg, MD). Amplification of the DHFR gene in methotrexate (MTX)-resistant cells is sufficient The results are documented (e.g., FW Alt, et al., J. Biol. Chem. 25 3:1357-1370(1978), J.L.Hamlin and C.Ma,Bi. ochem.et Biophys.Acta 1097:107-143(1990) , and MJ Page and MA Sydenham, Biotechnology gy 9:64-68 (1991). Cells develop DHFR by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. , developing resistance to the drug. If a second gene is linked to the DHFR gene Using this approach, the amplified gene(s) It has been shown that the present invention can develop cell lines that have more than 1,000 copies of the Subsequently, in recovering methotrexate, one or more of the host cells are Cell lines are obtained that contain the amplified gene integrated into the above chromosome(s).

[0217] 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 can be used to express TNF in a regulated manner in mammalian cells. (M. Gossen, and H. Bujard, Proc. Natl. Ac Ad. Sci. USA 89:5547-5551(1992)). Other signals, for example from the human growth hormone or globin genes, can also be used for ylation. Stable cell lines carrying the gene of interest integrated into the chromosome can be produced by Co-transfection with a selection marker such as pt, G418 or hygromycin Alternatively, the selection can be performed by first introducing more than one selectable marker, e.g., G418, in addition to the It is advantageous to use methotrexate in this regard.

[0218] 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.

[0219] 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.

[0220] 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 1μ The cells are seeded in alpha minus MEM supplemented with 100 µg / mL G418. After 2 days, the cells are The cells were then lysed in a hybridoma cloning plate (Greiner, Germany). ) in 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.

[0221] Example 2: Using transgenic mice, a high affinity human TNF-reactive TNF receptor was generated Production 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)F 2 Hybrid mice are immunized with human recombinant TNF (Taylor et al. al., Intl. Immunol. 6:579-591 (1993), Lonber g,et al.,Nature 368:856-859(1994), Neuber Ger, M., Nature Biotech. 14:826 (1996), Fishw. ild,et al.,Nature Biotechnology 14:845-8 51 (1996)). Several fusions were synthesized to produce fully human TNF-reactive IgG monoclonal antibodies. A panel of one or more fully human anti-TNF antibodies have been generated and will be further characterized. IgG1κ. Such antibodies are approximately 1×10 9 ~9×10 12 has an affinity constant of The unexpected high affinity of these fully human monoclonal antibodies allowed These make them good candidates for therapeutic use in TNF-related diseases, conditions or disorders.

[0222] Abbreviations: BSA-bovine serum albumin, Co 2 - Carbon dioxide, DMSO - Dimethylsulfur oxide, EIA-enzyme immunoassay, FBS-fetal bovine serum, H 2 O 2 -hydrogen peroxide, HRP-horseradish peroxidase, ID-interadermal, Ig - immunoglobulin, TNF-tissue necrosis factor alpha, IP-intraperitoneal, IV-intravenous, Mab or mAb-Monoclonal antibody, OD-Optical density, OPD-o-phenylenediamine dihydrochloride , PEG - polyethylene glycol, PSA - penicillin, streptomycin, amphotericin Tericin, RT - room temperature, SQ - subcutaneous, v / v - volume per unit volume, w / v - unit volume Weight per piece.

[0223] 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 the γ3 constant region. Mice from the appropriate genotype strain can be used in the immunization and fusion process. Fully human monoclonal antibodies against TNF can be generated.

[0224] Immunization. One or more immunization schedules can be used to generate anti-TNF human hybridomas. According to the following exemplary immunization protocol, Several fusions can be performed, but other similar known protocols can also be used. Several 14-20 week old female and / or surgically castrated male transgenic mice were Add an equal volume of TITERMAX® 200 to a final volume of 100–400 μL (e.g., 200 mL). or 1-1000 μg of recombinant human TNF emulsified in complete Freund's adjuvant was administered IP. Each mouse is also inoculated with 100 μL of viable vaccine in each of 2 SQ sites, if desired. Mice can then receive 1-10 μg of naphthalene in saline. IP (1-400 μg) and and SQ (1-400μg x 2) with an equal amount of TITERMAX or complete Freund's adjuvant Immunization with TNF emulsified in a bunt. 12-25 and 25-40 without anticoagulant After 2 days, mice can be bled by retro-orbital puncture. The blood can then be incubated at room temperature for 1 h. Allow to clot, collect serum and titer using a TNF EIA assay by known methods. If repeated injections do not result in an increase in titer, fusions are performed. Mice are then injected with 100 μL of Give a final IV booster injection of 1-400 μg of TNF diluted in physiological saline. After 3 days, the mice were euthanized by cervical dislocation and the spleens were aseptically removed. 100 U / mL penicillin, 100 μg / mL streptomycin and 0.25 μg 10 mL of cold phosphate buffered saline containing 1 mL of amphotericin B (PSA) The spleen can be immersed in PSA-PBS (PBS) by sterile perfusion of the spleen with PSA-PBS. 2. Harvest splenocytes. Wash cells once in cold PSA-PBS and perform trypan blue dye exclusion. Count the cells using a 3x counting kit and resuspend in RPMI 1640 medium containing 25 mM Hepes.

[0225] Cell fusion. Cell fusion can be carried out in a ratio of 1:1 to 1:10 according to known methods, for example, conventional methods in the art. The fusion can be performed at a ratio of mouse myeloma cells to live spleen cells. The cells and myeloma cells can be pelleted together. The pellet is then left for 30 seconds. Slowly add 1 mL of 50% (w / v) PEG / PBS solution (PEG molecular weight 1,4 The cells can then be resuspended in 50 mL of 25 mM Hepes for 1 min. The cells were incubated at 37°C for 1 h by slowly adding 10.5 mL of RPMI 1640 medium (37°C) containing Fusion can be stopped by centrifuging the fused cells at 500-1500 rpm for 5 minutes. The cells were then cultured in HAT medium (25 mM Hepes, 10% fetal clonal antibody (Hypertrimesterone I serum)). clone), 1mM sodium pyruvate, 4mM L-glutamine, 10μg / mL of gentamicin, 2.5% Origen culture supplement (Fisher), 10% 653 Adjusted RPMI 1640 / Hepes medium, 50 μM 2-mercaptoethanol, 10 RP containing 0 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine After resuspending in 100 mL of 100% PBS (MI 1640 medium), 200 mL of 100% PBS was plated in 15 96-well flat-bottom tissue culture plates. Plate at 1 μL / well. Then incubate in 5% CO for 7-10 days. 2 and 95% air Place the plate in a humidified 37°C incubator.

[0226] 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 .01% H 2 O 2 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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 was 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. Anti-human IgG 1 or HRP-conjugated goat anti-human IgG 3 Probe for 1 hour at room temperature. The plates are washed again and incubated with substrate solution as above.

[0231] 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.

[0232] 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 ... .

[0233] 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.

[0234] 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.

[0235] 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 only of human heavy and light chains (see All of the human hybridomas are predicted to be IgG1κ.

[0236] 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 and 2 show the results of the relative binding efficiency of these antibodies. The binding activity of TNF to a nucleoside antigen (epitope) is measured by directly binding TNF to an EIA plate. Combination with β-lactams can cause denaturation of the protein, and the apparent binding affinity is lower than that of the native protein. It should be noted that the 50 percent binding cannot reflect the quality of the binding. is seen across a wide range of concentrations.

[0237] 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.

[0238] Conclusion. Some fusions incorporate human variable and constant region antibody transgenes that are immunized with human TNF. This is done 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 12 The 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.

[0239] Example 3: Generation of human IgG monoclonal antibodies reactive to human TNFα. Overview. A human antibody containing both heavy and light chain human variable and constant region transgenes (CBA / JxC57BL / 6J)F 2 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).

[0240] Abbreviations: BSA-bovine serum albumin, Co 2 - Carbon dioxide, DMSO - Dimethylsulfur oxide, EIA-enzyme immunoassay, FBS-fetal bovine serum, H 2 O 2 -hydrogen peroxide, HC-heavy chain, HRP-horseradish peroxidase, ID-intradermal al), Ig-immunoglobulin, TNF-tissue necrosis factor alpha, IP-intraperitoneal, IV-intravenous , Mab-monoclonal antibody, OD-optical density, OPD -o-phenylenediamine disalt Acid salts, PEG-polyethylene glycol, PSA-penicillin, streptomycin, Amphotericin, RT-room temperature, SQ-subcutaneous, TNFα-tumor necrosis factor alpha, v / v-unit volume Volume per unit, w / v - weight per unit volume.

[0241] 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

[0242] As defined herein, the term "half-life" refers to the time period during which the plasma concentration of a drug (e.g., a therapeutic TNFα antibody) is reduced by half after one elimination half-life. With each half-life, less drug is eliminated. After one half-life, the amount of drug remaining in the body is 5 0% after 2 half-lives, 25% after 2 half-lives, etc. The elimination half-life is considered to be independent of the amount of drug in the body. can be done.

[0243] Materials and Methods. Animals. Transgenic mice expressing human immunoglobulins but not mouse IgM or Igκ. The Genic Mouse was developed by GenPharm International. These mice have been shown to exhibit V(D)J joining, heavy chain class switching and somatic mutations. to generate a repertoire of antigen-specific human immunoglobulins (1), The light chain transgene is derived in part from the germline human Vκ locus. It is derived from a yeast artificial chromosome clone that contains approximately half of the heavy chain in addition to several VH genes. (HC) The transgene contains both human μ and human γ1(2), and / or γ3 constant regions. Immunization and fusion to generate the monoclonal antibodies described herein. Mice from the HCo12 / KCo5 genotype strain were used in the mating process.

[0244] 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 adding 1 / 9 of the volume of 10x Dulbecco's PBS ( Mix with PBS (D-PBS) and pass through the column at 4 mL / min at 4°C. Next, wash the column with PBS. Clean and elute TNFα with 0.1 M sodium citrate, pH 3.5, and 2 M Tris-HCl Purified TNFα was diluted in 10 mM Tris, 0.12 M sodium chloride and neutralized at pH 8.5. The solution was buffer exchanged to sodium, pH 7.5, and filtered through a 0.2 um syringe filter. Ta.

[0245] Immunization. Female GenPharm mice, approximately 16 weeks of age, were immunized with equal doses of IgG on days 0, 12, and 28. A total of 100 μg of TNFα (lot JG10) emulsified with Titermax adjuvant 2298 or JG102098), IP (200 μL) and ID (10 at the base of the tail) Mice were immunized with 0.0 μL of IgG1 (0.0 μL) by retroorbital puncture on days 21 and 35 without anticoagulant. Blood was allowed to clot at room temperature for 1 hour, and serum was collected and analyzed by TNFα solid-phase EIA assay. After injection on the 28th day, mice were allowed to rest for 7 weeks before being titrated with GenTN. The fusion, designated V, was then combined with a specific human IgG against TNFα at 1:160. Titered mice were treated with 50 μg of TNFα diluted in 100 μL of physiological saline. Three days later, mice were euthanized by cervical dislocation and the spleen was Aseptically remove the lysate and inoculate with 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 mL of cold lysine containing 0.25 μg / mL amphotericin B (PSA) and 0.25 μg / mL amphotericin B (PSA) was The spleen was then immersed in phosphate-buffered saline (PBS). Spleen cells were harvested by washing once in cold PSA-PBS and then cultured using a Coulter Count using a counter and resuspend in RPMI 1640 medium containing 25 mM Hepes. Ta.

[0246] 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% F Cryopreservation in BS and 5% DMSO (Sigma) was followed by vapor-phase liquid nitrogen freezing of CBS. The cell bank was kept sterile (Quality Control Center). cor, Malvern), Mycoplasma (Bionique Laboratories es). Cells were maintained in logarithmic growth culture until confluence. Prior to confluence, they The cells were washed in PBS, counted, and viability was determined by trypan blue dye exclusion (95% super).

[0247] Human TNFα was produced by a recombinant cell line, designated C237A, from Centococcus cerevisiae. The cell lines were grown at Molecular Biology, Orlando, CA in 5% (v / v) F 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.

[0248] 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 0 μL / well. Then, incubate in 5% CO for 7–10 days. 2 and contains 95% air Plates were placed in a humidified 37° C. incubator.

[0249] 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 Probe the plates with 100 µL of HRP-conjugated goat anti-human IgG for 1 hour at room temperature. Wash the plates again. Wash and add 100 μL / well of citrate-phosphate substrate solution (0.1 M citric acid and 0. 2M sodium phosphate, 0.01% H 2 O 2 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.

[0250] 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.

[0251] 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 IgG diluted 1:4000 in PBS 1 , IgG 2 , Ig G 3 or IgG 4 The plate was probed with 1000 ng / ml Binding Site at room temperature for 1 hour. The plates were washed again and incubated with substrate solution as above.

[0252] 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. The cell line names and their respective C code designations are listed in Table 1. Each was frozen in a 12-vial research cell bank stored in liquid nitrogen.

[0253] 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.

[0254] [Table 2]

[0255] 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.

[0256] 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) .

[0257] Example 4: Cloning and preparation of cell lines expressing human anti-TNFα antibodies. Summary. A panel of eight human monoclonal antibodies (mAbs) expressing TNV 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 the eight m The original panel of Abs was represented by TNV14, TNV15, TNV148, and TNV196. We also found that the mAb contained only four distinct mAbs. Based on the results of the column analysis and the in vitro TNFα neutralization data, mAb TNV148 and and TNV14 were selected for further study.

[0258] 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 considered to be a known reproductive 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 used. A was based on the recently cloned heavy and light chain genes of another human mAb (12B75). The IL-12 Antibody was developed based on the results of a study published in International Publication No. WO 02 / 12500. ies, Compositions, Methods and Uses as the title of the invention No. 60 / 236,827, filed Oct. 7, 2000, which is incorporated herein by reference. The resulting cDNA was cloned into a newly prepared expression vector (which is incorporated herein in its entirety).

[0259] 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, whereas Sp2 / 0 transfectant 1.73-12-122 (C467A) was stable in the spent culture 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.

[0260] 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 some in vitro characterization of the mAbs, TNV148 was selected as the mAb to be characterized.

[0261] The DNA sequence encoding the TNV148 mAb was cloned and the appropriate constant region was encoded. The well-characterized 653 and S p2 / 0 mouse myeloma cells and the resulting transfected cells A subclone was identified that produced 40-fold more mAb than the original hybridoma cell line. Screened up to.

[0262] 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.

[0263] Table 2. TNV mAb genes used to clone, modify, or sequence 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.

[0264] [Table 3]

[0265] 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 frozen material was prepared as described above. The frozen vials were stored in freezer boxes AA and AB of the CBC. These cells were thawed and subjected to all Sp2 / 0 transfections described herein. It was used for the

[0266] 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). 50 in PBS: Add 50 μL of 0.5 μg / ml p55-sf2 to Optiplates and Coat the wells during a 1 hour incubation at 7°C. PBS / 0.1% B Serial dilutions of eight TNV ​​cell supernatants were plated in a 96-well round-bottom plate using SA as the diluent. Cell supernatant containing anti-IL-18 mAb was included as a negative control. cA2 (anti-TNF chimeric antibody, Remicade, U.S. Patent No. 5,770,198) The same anti-IL-18 supernatant spiked with IL-18 (Figure 1A, which is incorporated herein by reference in its entirety) Included as a positive control was a final TNFα concentration of 5ng / ml. 125 I sign TNFα (58:Ci / :g, D. Shealy) was added to 100:l of cell supernatant. The mixture was preincubated at room temperature for 1 hour. s was washed to remove unbound p55-sf2, and 50:l 125 I-TNFα / on cells The supernatant mixture was transferred to Optiplates. After 2 h at room temperature, the plates were washed three times with PBS-Tween. The Optiplates were washed and 100:1 of Microscint-20 was added. The cpm bound was determined using a TopCount gamma counter.

[0267] 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 Centrifuge the samples for 10 minutes at 4°C. Transfer the aqueous phase 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 minutes 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.

[0268] 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 incubated in a water bath at 70°C for 10 minutes. Incubate for 10 min, then chill on ice for 10 min. 2.5 μl 10x reverse transcriptase buffer reagent, 10 μl of 2.5 mM dNTPs, 1 μl of reverse transcriptase (20 units), and 0.4 μl Prepare a separate mixture consisting of 1 ribonuclease inhibitor RNasin (1 unit). 13.5 μl of this mixture was added to 11.5 μl of cold RNA / oligonucleotide mixture. The reaction was incubated at 42°C for 40 minutes. The material is stored in a -20°C freezer until use.

[0269] 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 XHIFI buffer, and 2 mM magnesium sulfate. The program consisted of 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 62°C for 30 s, and followed by a final incubation at 68°C for 10 min. Do the following.

[0270] 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 thermal cycler program was 25 cycles (96°C for 30 s, 50°C for 15 s, 6 0°C for 4 min, followed by overnight at 4°C. The reaction products were then run through a polyacrylamide gel. The proteins were fractionated and detected using an ABI 377 DNA sequencer.

[0271] 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 Prepare a mutagenesis reaction using 1 μl of Pfu DNA polymerase and 400 μl of 10 ... Sterile water was added to bring the total volume to 50 μl. The reaction mixture was then incubated at 95° C. for 3 h. Incubate with the thermal cycler programmed to incubate for 0 seconds. This was followed by incubation at 95°C for 30 seconds, 55°C for 1 minute, 64°C for 1 minute, and 68°C for 7 minutes. Cycle the mixture 14 times with a series of incubations at 30°C for 2 min (1 cycle). These reactions consisted of injecting the mutagenic oligonucleotide into otherwise identical newly synthesized It was designed to integrate into the plasmid that was created to remove the original TNV148 plasmid. Add 1 μl of DpnI endonuclease, which cuts only the original methylated plasmid. Afterwards, the samples were incubated at 37° C. for 1 hour. 1 μl of the reaction was then used to Epicurian Coli XL1-Blue supercells were isolated by standard heat shock methods. -Competent E. coli was transformed and plated on LB-ampicillin agar plates. The transformed bacteria were subsequently identified. Plasmid minipreps were prepared using the Wizard™ column. After elution of the sample, the plasmid DNA was further isolated by precipitating the plasmid DNA with ethanol. The purified fragments were then resuspended in 20 μl of sterile water. DNA sequence analysis was then performed to identify the desired fragments. We identified plasmid clones with base changes and confirmed that other base changes were inadvertently introduced into the TNV148 clone. The same parameters described in Section 4.3 were used to confirm that the Using a cytometer, mix 1 μl of plasmid, 3 μl of BigDye mix, 1 μl of p UC19 forward primer, and cycle sequencing reaction prepared in 10 μl of sterile water. It was served as a gift.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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 transferred into The expression plasmids were p1781 (TNV14), p1782 (TNV148), and p1 The variable region was also designated 12B 783 (TNV148B) (see Table 2). It was also cloned upstream of the IgG1 constant region from the 75 (GenPharm) gene. These expression plasmids encoding IgG1 of the G1m(z) allotype are also listed in Table 3. are listed.

[0277] 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.

[0278] [Table 4]

[0279] 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).

[0280] 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.

[0281] 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 20 mM glutamine and 5% CO 2 Incubate at 37°C in a 200°C incubator. After 2 days, the cells were incubated in an equal volume of IMDM, 5% FBS, 2 mM glutamine, 2×MHX, and Selection (1 × MHX = 0.5:g / mL mycophenolic acid, 2.5:g / mL hypoxic xanthine, 50 μg / mL xanthine) was added and incubated for another 2 h while colonies formed. Plates were incubated for ~3 weeks.

[0282] 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.

[0283] 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 the cells and incubate in 5% CO for 12-20 days until colonies appear. 2 Incubator The wells were incubated at 37° C. for 1 hour. Cell supernatants were collected and analyzed by ELISA as described above. Selected colonies were cultured for 24 h. After passage onto 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 subclone was subjected to a second round of subcloning. The best subclone from the second round was selected as the cell line for development.

[0284] 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. 1 × 10 in MDM, 5% FBS, 2 mM glutamine, and 1 × MHX (or serum-free medium) 5 Aliquots of 300 μl were removed at 24-h intervals and viable cell density was assessed. The viable cell count was measured at 1 × 10 5 Analysis was continued until the total number of cells / ml was less than 1. Aliquots of the collected cell supernatants were assayed for the concentration of antibody present. ELISA assays were performed using rTNV148B or rTNV14 JG92399. The ELISA plate was coated with polyclonal goat anti-human IgG Fc. The samples were incubated on a plate for 1 h, and the bound mAb was then incubated with 1:1000 dilution of alkaline phosphatase Detection was performed with phatase-conjugated goat anti-human IgG (H+L).

[0285] 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 treated with no MHX, 0.2× MHX, or 1× MHX (1× MHX=0.5 :g / mL mycophenolic acid, 2.5:g / mL hypoxanthine, 50:g / mL After 1 day, the cultures were split into three groups containing either 100% T75 or 100% xanthine. Scotch, 1×10 5 Cultures were seeded at a starting density of cells / ml and cells were incubated for 1 week at 24 h. Aliquots were not collected for mAb production. SOP PD32. The doubling times for these samples were calculated using the formula provided in .025.

[0286] 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.

[0287] 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 TNFα binding to the p55 receptor. In fact, TNV mAb effectively blocked the TNV mAb spiked into the negative control hybridoma supernatant. appears to be more effective at inhibiting TNFα binding than the cA2 positive control mAb These results demonstrate that TNV ​​mAbs inhibit the biogenesis of TNFα in cell-based assays and in vivo. This indicates that the activity of the β-lactamase inhibitors is likely to be blocked, and therefore further analysis is required. This was interpreted as:

[0288] 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 mAbs, the seven hybrids that produced these mAbs were 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.

[0289] 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.

[0290] 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. 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 are all J6 exons, and because they all use the J6 exon, they are clearly somatic changes resulting from a single VDJ gene rearrangement event that subsequently renders each mAb unique DNA sequence analysis revealed the presence of only two distinct light chain genes in the four mAbs. The light chain variable region codes for TNV14 and TNV15 were The sequences are identical to each other and are representative of the germline Vg / 38 K family of human kappa chains. The TNV148 and TNV196 light chain coding sequences are identical to each other. However, the germline sequences differ at two nucleotide positions (Fig. 3 ).

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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).

[0295] 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, whereas the Centoc or because the IgG1 expression vector p104 encodes a Lys residue at that position. The JC intron, the complete constant region coding sequence, and the 3' flanking sequence are 12B75 Other heavy chain expression plasmids derived from the heavy chain gene (e.g., p1786, p1788) have been prepared. The cell lines transfected with these genes were selected as production cell lines. The vector was constructed using the subsequent PCR amplified V region that would result in the final expression plasmid. It was carefully designed to allow one-step cloning of the region.

[0296] 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).

[0297] 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.

[0298] Highest producing rTNV148B cell line One of the 653 highest producing parental lines from rTNV148B transfection 2 0 (producing 5–10 μg / mL in spent 24-well cultures) To screen for higher producing cell lines, a more homogenous cell population was prepared. Two of the subclones of the parent strains 2.320, 2.320-17, and 2.320-20 produced approximately 50 μg / ml in spent 24-well cultures, which is higher than their parent strains The subcloned strains 2.320-17 and 2.320 This was the result of a second subcloning of -20.

[0299] The identification numbers of the heavy and light chain plasmids encoding each mAb are shown. For transfections performed with gene inserts, the source of the gpt selection marker The heavy chain constant region was from Remicad The same human IgG1 expression vector ("old") used to encode e or 12B7 5 (GenPharm / Medarex) constant region contained within the heavy chain gene ("novel" H1 / L2 is encoded by either the TNV14 heavy chain or the TNV148 light chain. The plasmids p1783 and p1801 refer to "novel" mAbs composed of two chains. The only difference is how many JC introns the heavy chain genes contain. The transfection numbers, which define the first numbers of the gene names of the transfections, are shown on the right. rTNV148B-producing cell lines C466 (A, B, C, D) and C467 as described in the specification A were derived from transfections no. 2 and 1, respectively. rTNV14-producing cell lines C476A was derived from transfection no. 3.

[0300] [Table 5]

[0301] 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 comparable to 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.

[0302] 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 of Sp2 in well cultures. The / 0 cell line was designated C467A (Table 5).

[0303] 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).

[0304] Table 5. Summary of selected producer 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.

[0305] [Table 6]

[0306] Characterization of subcloned cell lines To more carefully characterize the growth characteristics of the cell lines 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 leaves is 1.0×10 6 ~1.25×10 6 Peak cell density in cells / ml and 11 It was shown that a maximum mAb accumulation level of 0–140 μg / mL was reached (Figure 7). The highest producing Sp2 / 0 subclone, C467A, produced 2.0 × 10 6 Peat of cells / mL The cell density and maximum mAb accumulation level of 25 μg / mL were reached (Figure 7). Growth curve analysis was not performed on the rTNV14-producing cell line C476A....

Claims

1. An anti-TNF antibody, comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and (ii) The light chain comprises the amino acid sequence of SEQ ID NO: 37, and is expressed in Chinese hamster ovary cells (CH An anti-TNF antibody expressed in human TNF-α / TNF-α (H1N1)-dependent IL-1 (IL-0) cells.

2. The oligosaccharide profile of the anti-TNF antibody is >99.0% total neutral oligosaccharide species and >99.0% total carbohydrate. The anti-TNF antibody of claim 1 , which is composed of <1.0% charged oligosaccharide species.

3. The oligosaccharide profile of the anti-TNF antibody is G0 for each neutral oligosaccharide species.

2. The method of claim 1, further comprising: G1F<20.0%; and G2F<5.0%. The anti-TNF antibodies described above.

4. When measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA) The anti-TNF antibody of claim 1 , wherein the anti-TNF antibody does not contain disialylated glycan species. 。

5. The anti-TNF antibody has a longer length compared to the anti-TNF antibody expressed in Sp2 / 0 cells. 1 to 5, which have an improved half-life or antibody-dependent cell-mediated cytotoxicity (ADCC).

5. An anti-TNF antibody according to any one of claims 4 to 4.

6. The anti-TNF antibody of claim 5 , wherein the anti-TNF antibody is a biosimilar.

7. (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an amino acid sequence of SEQ ID NO:

37. A method for producing an anti-TNF antibody comprising a light chain comprising the sequence My body, a. Transfecting a Chinese hamster ovary mouse model with nucleotides encoding the anti-TNF antibody Culturing cells (CHO cells); b. expressing the anti-TNF antibody in the CHO cells; and c. purifying the anti-TNF antibody. method.

8. the oligosaccharide profile of the anti-TNF antibody is >99.0% total neutral oligosaccharide species; The process of claim 7, which is comprised of <1.0% total charged oligosaccharide species.

9. The oligosaccharide profile of the anti-TNF antibody is G0 for each neutral oligosaccharide species.

8. The method of claim 7, further comprising: G1F<20.0%; and G2F<5.0%. The manufacturing method described above.

10. When measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA) The method of claim 7 , wherein the anti-TNF antibody does not contain disialylated glycan species.

11. The anti-TNF antibody has a longer length compared to the anti-TNF antibody expressed in Sp2 / 0 cells. 7 to 10, which have an improved half-life or antibody-dependent cell-mediated cytotoxicity (ADCC). The manufacturing method according to any one of claims 10 to 15.

12. The method of claim 11 , wherein the anti-TNF antibody is a biosimilar.

13. (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 36, and (ii) the light chain comprises the amino acid sequence of SEQ ID NO:

37. an anti-TNF antibody comprising an amino acid sequence of Chinese hamster ovarian hyperplasia; A composition expressed in ovarian cells (CHO cells).

14. the oligosaccharide profile of the anti-TNF antibody is >99.0% total neutral oligosaccharide species; 14. The composition of claim 13, which is comprised of <1.0% total charged oligosaccharide species.

15. The oligosaccharide profile of the anti-TNF antibody is G0 for each neutral oligosaccharide species.

14. The method of claim 13, further comprising: G1F<20.0%; and G2F<5.0%. The composition described.

16. The anti-TNF antibody is dicyaline as measured by high performance liquid chromatography (HPLC). The composition of claim 13, wherein the composition is free of glycan species.

17. The anti-TNF antibody has a longer length compared to the anti-TNF antibody expressed in Sp2 / 0 cells.

13. The antibody of claim 13, having an improved half-life or antibody-dependent cell-mediated cytotoxicity (ADCC).

17. The composition according to any one of claims 1 to 16.

18. The composition of claim 17 , wherein the anti-TNF antibody is a biosimilar.

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