TWI353852B - Methods of administering anti-tnfα antibodies - Google Patents

Description

1353852 VI. Description of the Invention: [Technical Field of the Invention] Tumor necrosis factor a (TNFot) is a cytokine produced by several cell types (including single cells and macrophages), which is initially based on its ability to induce tumors in certain mice. Identification (see for example Old, L. (1985) Science 230.: 630-632). Subsequently, a factor called malignant protein associated with dysentery shows the same molecule as TNFoc. TNFa is associated with the regulation of shock (see, for example, Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 5 7 · 505-5 18; Beulter, B. and Cerami, A. (1989) Annu_ Rev. Immunol. Z: 625-655). Furthermore, TNFoc has been linked to various human diseases and disorders, including sepsis, infection, autoimmune diseases, transplant rejection, and limb-to-host disease (see Vasilli, P. (1992) Annu_ Rev. Immunol. l〇_ 411-452 ; Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45_ · 491-503) o [Prior Art] Due to the harmful role of human TNFoc (hTNFoc) in various human disorders, the therapeutic target has Designed to inhibit or counteract hTNFoc activity. In particular, antibodies that bind to and neutralize hTNFoc have been recognized as a means of inhibiting hTNFoc activity. Some of the earliest antibodies were mouse monoclonal antibodies (mAbs) secreted by fusion tumors prepared from lymphocytes of mice immunized with hTNFoc (see, for example, Hahn T., et al. (1985) Proc Natl Acad Sci USA to: 3814 -3818; Liang, CM, et al. (1986) Biochem. Biophys. Res. Commun. 137: 847-854; Hirai, M., et al. (1987) J. Immunol. Methods 96_ ' 57-62 ; Fendly, BM, et al. (1987) [forgot 149555.doc 1353852

Hybridoma 6 : 359-370 ; Moller, A., et al. (199 〇) Cytokine 2 : 162-169; Moeller et al. USP 5, 231, 〇 24; Wallach, D.

European Patent Application 186 833 B1; European Patent Application 218 868 Al to Old et al., Moeller, A. et al., European Patent Application No. 26, 61, B1). Although these mouse anti-hTNFot antibodies often exhibit high affinity for hTNFa (such as KdS 1〇·9 M) and can neutralize hTNFa activity, their in vivo use is limited by the problem of human antibody administration to mice. Such as short serum half-life, the method of restricting certain human effector functions and the undesired immune response to mouse antibodies in humans ("human anti-mouse antibody, (HAMA) response). In order to try to overcome in humans Using whole mouse antibodies, mouse anti-hTNFa antibodies are genetically engineered to be more "human-like,". For example, a variable region in which an antibody chain has been prepared is derived from a constant region of a mouse and an antibody chain, and is known to be a chimeric antibody derived from humans (Knight, D M, et al. (i993)

Immunol. Μ: 1443-1453; Dadd〇na, pE, et al. pcT application WO 92/16553). In addition, highly variable regions in which the variable region of the antibody has been prepared have been derived from mice, but other variable regions and antibody-definite regions are humanized antibodies derived from humans (Adair, JR, et al., pcT application number WO 92 /11383). 'However, due to the fact that the antibody and the humanized antibody still leave a few mouse sequences, it is still possible to induce an unwanted immune response (human anti-chimeric antibody (HACA) response), especially for chronic indications such as rheumatoid arthritis. For long-term administration (see for example Elli〇tt, MJ, et al. (1"4) 344: 1125-1127; Elloit, MJ, et al. (1994) Lancet 344: 1105-1110) 〇149555.doc 1353852 on mouse mAbs A preferred hTNFa inhibitor (or a hostile or humanized antibody) of a derivative thereof or a derivative thereof is required to be an entire human anti-hTNFa antibody, since the agent should not induce the HAMA reaction even for prolonged use. Human monoclonal autoantibodies against hTNFcc have been prepared using human fusion tumor technology (Boyle, P., et al. (1993) Cell. Immunol. 152: 556-568; Boyle, P., et al. (1993) Cell. Immunol. 152· 569-5 8 1 ; European Patent Gazette 614 984 A2) by Boyle et al. However, these monoclonal antibodies derived from fusion tumors have been reported to have too low affinity for hTNFa to be calculated by conventional methods, unable to bind soluble hTNFa and are unable to neutralize hTNFa-induced cytotoxicity (see Boyle et al., supra). literature). Furthermore, the success of human fusion tumor technology relies on the presence of lymphocytes in the peripheral blood of humans that produce autoantibodies specific for hTNFa. Some studies have detected anti-hTNFa serum autoantibodies in human individuals (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30.: 219-223; Bendtzen, K., et al. (1990). Prog. Leukocyte Biol. 10B: 447-452), while others are absent (Leusch, HG., et al. (1 99 1) J. Immunol. Methods 139: 1 45-1 47) o Another natural human anti- The hTNFa antibody is a recombinant hTNFa antibody. Recombinant human antibodies have been described that bind with relatively low affinity (e.g., Kd about 7 Μ) and hTNFa has a rapid dissociation rate (i.e., about 10 '2 seconds' (Griffiths, AD, et al. (1993) EMBO J. 11: 725-734). However, due to their relatively rapid dissociation kinetics, these antibodies are not suitable for therapeutic use. Furthermore, recombinant human anti-hTNFa has been described to be unable to neutralize hTNFa activity but enhance hTNFa binding to Cell surface and enhance the integration of hTNFa (Lidburg, A., et al. (1994) Biotechnol. [ £ 】 149555.doc Ϊ 353852

Ther. L. 27-45 ’ Aston, R., et al. PCT Application No. WO 92/03145) 〇

Recombinant human antibodies that bind to soluble hTNFa with high affinity and reduced kinetics and have neutralizing hTNFcx activity (including hTNFa-induced cytotoxin and hTNFa-induced cell activation in vitro and in vivo) are also described ( See USP 6,090,382). The typical mode of administration of antibodies is performed intravenously weekly. Administration of antibodies and/or any drug per week may be costly, inconvenient, and result in several side effects due to the number of administrations. Intravenous administration also has a limitation that administration by a drug trainer is generally performed. [Summary of the Invention]

The present invention provides a biweekly administration regimen for the treatment of TNFa-related disorders, preferably by subcutaneous route. Biweekly administration has many advantages over weekly administration, including (but not limited to) fewer total injections, reduced number of injection site reactions (such as localized pain and swelling), increased patient compliance (due to less Left shots - number of people) and patients and care providers are less expensive. The advantage of subcutaneous administration is that the patient itself can be administered a therapeutic substance f, such as a human being, which is convenient for both the patient and the care provider. The present invention provides a method of treating a disorder in which TNFa activity is detrimental. The method comprises preferably subcutaneously injecting a capsid antibody 1 antibody to an individual that is capable of specifically binding to a human recombinant human antibody. The present invention further provides a method for treating a disease in which TNFa activity is harmful (IV), and the like, wherein the human antibody and other therapeutic agents are administered to an individual, such as one or more other antibodies that can bind to the known antibody (eg, Binding to cytokines or antibodies that bind to cell surface molecules), or a variety of cells 149555.doc 1353852, soluble TNFot receptor (see, for example, PCT Patent Publication WO 94/06476) and/or one or more can inhibit hTNFcx production or activity The chemical agent (such as the cyclohexylene derivative described in PCT Publication WO 93/19751) is preferably ampicillin. Preferably, the antibody is a recombinant human antibody that specifically binds to human TNFa. The antibodies of the invention are characterized by high affinity and reduced kinetics binding to hTNFa and neutralizing hTNFa activity, including hTNFcx-induced cytotoxicity (in vitro and in vivo) and hTNFct-induced cell activation. The antibody may be full length (e. g., IgG1 or IgG4 antibody) or may include only antigen-binding sites (e.g., Fab, F(ab')2, scFv fragments or a single region). The optimal recombinant antibody of the invention is referred to as D2E7, having a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy amino acid sequence comprising SEQ ID NO: 4 (see Appendix B). chain. Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the amino acid sequence of sequence number: 1 and a heavy chain (HCVR) comprising the amino acid sequence of SEQ ID NO: 2. Such antibodies are described in U.S. Patent No. 6,090,382, the disclosure of which is incorporated herein by reference. In one embodiment, the invention provides a method of treating a disorder in which TNFoc activity is detrimental. Such methods include inhibiting human TNFoc activity by subcutaneous administration of an anti-TNFa antibody in two weeks and thus treating the disorder. The disorder may be, for example, septicemia, autoimmune diseases (such as rheumatoid arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, and nephrotic syndrome), infectious diseases, cachexia, transplant rejection, or grafting. Limb-to-host disease, lung disorders, bone disorders, intestinal disorders, or heart disorders. In another embodiment, the invention provides a method of treating a disorder in which TNFot activity is deleterious. Such methods include subcutaneous administration of an anti-[S] 149555.doc TNFa antibody and a methotrexate to the human bifolium to inhibit human TNFa activity and thereby treat the disorder. In the aspect, & 'β 虱曱喋呤 is administered prior to administration of the anti-TNFa antibody. In another aspect, the methotrexate is administered in a preferred embodiment after administration of the anti-TOTcx antibody to treat an anti-TMFCt antibody wherein the TNFa activity is detrimental to the human anti-TNFa anti-spasm. Even better, the treatment can be done by subcutaneous injection of human antibodies or antigen-binding sites thereof. The antibody 〆, the original 'Ό δ '^ position is preferably dissociated from human TNFa by Kd of 1 χ 1 Ο·8 Μ or less and Koff rate is 1 1 〇S or less (both by surface cells. Genomic resonance 敎) and in the L929 analysis standard towel neutralized human TNFot cytotoxicity with X10-7 or 1C50 under the armpit. More preferably, the isolated human antibody or antigen binding site is 5xl 〇 -4 s.i or less. "Even 1<S> or below K<jff is isolated from human TNFa. More preferably, the isolated human antibody or its antigen-binding site is in the range of 乂1 10 Μ or less in the in vitro l929 analytical standard, Even better 1><1〇-1〇 or less ICw neutralizes human TNFoc cytotoxicity. In another specific example, the present invention provides a method for treating a disorder in which TNFa activity is detrimental The human antibody or antigen-binding site thereof is administered. The antibody or antigen-binding site thereof preferably has the following characteristics: a) κ measured by surface cell plasmid genomic resonance. ^ is xl〇-3 si or the following dissociation from human TNFa; b) having an amino acid sequence comprising SEQ ID NO: 3 or substituted by a single alanine at position 1, 4, 5, 7 or 8 or by 1 to 5 conservative amino acids at positions 1, 3, 4, 0, 7, 8 and/or 9 and light chain modified from SEQ ID NO: 3 149555.doc 1353852 CDR3 region; c) having an amino acid sequence comprising SEQ ID NO: 4 or substituted by a single alanine at positions 2, 3, 4 , 5, 6, 8, 9, 10 or 11 or by 1 to 5 conservative amino acids in position 2 3, 4, 5, 6, 8, 9, 1 〇, η and/or 12 and the heavy chain CDR3 region modified from SEQ ID NO: 4. More preferably, the antibody or its antigen binding site is 5xl. Further, the antibody or its antigen-binding site is dissociated from human TNFoc by Kofl^1χ1 (Γ4 s·1 or less. In yet another specific example, the present invention provides A method of treating a disorder in which TNFa activity is detrimental. The method comprises subcutaneously administering to a subject a human antibody or an antigen binding site thereof. The antibody or antigen binding site thereof preferably comprises an amino acid sequence comprising SEQ ID NO: 3. Or replacing the LCVR of the CDR3 region modified from SEQ ID NO: 3 with position 1 , 4, 5, 7 or 8 by a single alanine and having the amino acid sequence comprising SEQ ID NO: 4 or substituted by a single alanine at position 2. 3, 4, 5, 6, 8, 9, 10 or 11 HCVR of the CDR3 region modified from SEQ ID NO: 4. More preferably, the LCVR has a CDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and HCVR CDR2 region having an amino acid sequence comprising SEQ ID NO: 6. Even better, the LCVR Further having a CDR1 region comprising the amino acid sequence of SEQ ID NO: 7 and HCVR having a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8. In yet another specific embodiment, the present invention provides a method for treating a disorder in which TNFa activity is detrimental The human antibody or antigen-binding site thereof is administered subcutaneously to the individual for two weeks. The antibody or antigen-binding portion thereof preferably comprises an amino acid sequence comprising SEQ ID NO: 1 LCVR&; including SEQ ID NO: [149] 149555. HCVR of the amino acid sequence of doc 2. In a specific example, the antibody has a 1 g G1 heavy chain constant region or an IgG4 heavy chain constant region. In yet another embodiment, the antibody is a Fab fragment, a F(ab,) 2 fragment or a single-chain Fv fragment. In still another embodiment, the invention provides a method of treating a disorder in which an individual is administered an anti-TNFcx antibody by subcutaneous administration of one or more anti-TNFot antibodies or antigen-binding sites thereof. Preferably, the antibody or antigen-binding portion thereof comprises a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 'SEQ ID NO: 16, SEQ ID NO: 17. 18. LC VR of the CDR3 region of the amino acid sequence of the ensemble number 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 An amine group selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 3, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 HCVR of the CDR3 region of the acid sequence. Another object of the invention is a kit comprising a formulation comprising a pharmaceutical composition. This kit includes an anti-TNFa antibody and a pharmaceutically acceptable carrier. The kit contains a copy indicating that the pharmaceutical composition is administered subcutaneously for two weeks to treat a disorder in which the anti-TNFa antibody is administered. In another aspect, the invention relates to a kit comprising a formulation comprising a pharmaceutical composition and further comprising an anti-inflammatory antibody, an alum, and a pharmaceutically acceptable carrier. The kit contains a copy indicating that the pharmaceutical composition is administered subcutaneously for two weeks to treat a disorder in which the anti-TNFot antibody is administered. 149,555.doc This July is also intended to provide a preloaded syringe containing a pharmaceutical composition comprising an anti-antibody and a pharmaceutically acceptable carrier. Still another object, the present invention provides a preloaded syringe of a pharmaceutical composition comprising an anti-TNFa antibody, an amidine and a pharmaceutical sensible carrier. [Embodiment] This is a method for treating a disorder in which anti-TNFa is beneficial in the present month, which comprises administering a single human antibody or a high affinity binding to human TNFa, a low dissociation rate, and a neutralizing ability. Its antigen binding site thus treats the disorder. Various objects of the invention relate to the treatment of such antibodies and antibody fragments and pharmaceutical compositions thereof. In order to better understand the present invention, certain nouns are first defined. As used herein, "administering" refers to administration of a substance (e.g., an anti-TNFa antibody) to achieve a therapeutic target (e.g., treatment of a TNFcc-related disorder). As used herein, the term 'biweek administration regimen', 'biweekly administration, and 'two-week administration," means that a substance (such as an anti-TNFa antibody) is administered to an individual to achieve a therapeutic target (eg, TNFa-related disorders) The time-lapse process. Bi-weekly medications do not want to include weekly medications. Better, every 9-19 days, preferably every 11_17 days, even better every 13-15 days and preferably every 14 days. As used herein, "combination therapy" refers to the administration of two or more therapeutic substances, such as an anti-TNFa antibody and a drug methotrexate. The methotrexate can be administered simultaneously with an anti-TNF 〇t antibody, in an anti-TNFct antibody. Pre- or post-administration. "Human TNFa" (abbreviated herein as hTNFa or reduced to hTNF) is intended to represent a 17 kD secretory state and a 26 kD membrane-associated state (a non-covalently bound 1 7 kD molecule terpolymer) The biologically active state of the composition is human [Cloud I49555.doc 12-cytokine. The structure of TNFa is described, for example, in Pennica, D., et al. (1984) Nature 312: 724-729; Davis, JM, et al. 1987)

Biochemistry 2β_ '· 1322-1326 ; and Jones, Ε.Υ., et al. (1989) Nature 338.: 225-228. The human TNFoc is intended to comprise recombinant human TNFoc, which can be prepared by standard recombinant expression methods or commercially available (R&amp;D Systems, Catalog No. 210-TA, Minneapolis, MN). As used herein, "antibody" is intended to mean an immunoglobulin molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains cross-linked by disulfide bonds). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three regions: CHI, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one region CL. The VH and VL regions can be subdivided into highly variable regions (referred to as complementarity determining regions (CDRs)) that are inter-configured with regions of more conservative network regions (FRs). Each of the VH and VL lines is composed of three CDRs and four FRs, and is arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An "antigen binding site" (or simply "antibody moiety") of an antibody as used herein refers to one or more fragments (e.g., hTNFot) of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be carried out by a fragment of a full length antibody. Examples of binding fragments contained in an "antigen-binding site" of an antibody include (i) a Fab fragment, a monovalent fragment composed of VL, VH, CL, and CH1 regions; (ii) a F(ab')2 fragment, which is composed of a disulfide The bridge links two 149555.doc -13-1353852 Fab fragments in the hinge region; (Hi) the Fd fragment composed of the vH and CH1 regions; (iv) the vl and VH of the antibody one arm The Fv fragment consisting of the region; (v) the dAb fragment (Ward et al. (1989) Nature 34L: 544-546) 'which consists of the VH region; and (vi) the independent complementarity determining region (CDR). Furthermore, although the two regions V1 and VH of the FV fragment are encoded by individual genes', they can be recombined to form a synthetic bond in which the VL and VH regions are paired to form a single protein chain of a monovalent molecule. (referred to as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 423-426; and Huston #A (1988) Proc. Natl. Acad. Sci. USA 5879-5883). This single chain antibody is also intended to be included in the term "antigen binding site" of the antibody. Other types of single chain antibodies such as diabodies are also included. A diabodies are one-valent dispecific antibodies that express VH and VL £ domains on a single polypeptide chain' but use too short a 4 linkage to allow pairing between the two regions on the same strand, thus driving the region to complement other chains. Regional pairing and production of two antigen binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ" et al. (1994) Structure 1121-1123 The guanidine antibody or antigen binding site thereof may be part of a larger immunoadhesive molecule formed by covalent or non-covalent association of an antibody or antibody site with one or more other proteins or peptides. Examples of such immunoadhesive molecules comprise Production of tetrameric scFv molecules using the streptavidin core region (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas color: 93-101) and use of cysteine residues, marker peptides and C-terminal polygroups Amino acid tag to produce a double [si 149555.doc -14-1353852 4 shell and biotinylated scFv molecule (Kipriyanov, SM, et al. (1994) Mol. Immunol Liao: 1047_1058) β antibody site such as Fat ^ F ( Ab,) 2 fragments can use conventional techniques From whole antibody preparation, such as whole antibody digestion with papain or pepsin, respectively, antibodies, antibody sites and immunoadhesive molecules can be obtained using the standard recombinant DNA technology described herein. "Human antibodies" as used herein, to be included An antibody having variable and constant regions and derived from a human germline immunoglobulin sequence. The human antibody of the present invention may comprise an amino acid residue but is not encoded by a human germline immunoglobulin sequence (such as a random or position-specific genetic mutation in vitro or a mutation introduced by in vivo somatic genetic variation). , for example, in Cdrs and in a specific CDR3. However, as used herein, "human antibodies, a female does not wish to include antibodies in which CDR sequences derived from other mammalian species, such as mouse germlines, have been grafted to human framework sequences. As used herein, "recombinant human antibodies," All human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described in detail later in the paragraph), human antibody gene pools isolated from recombinant combinations Antibodies (described in detail later in the paragraph), from isolated antibodies to human immunoglobulin genes that have gene transfer effects (eg, mice) (see, for example, Tayi〇r, lD, et al. (1992) Nucl· Acids Res. (6287-6295) or an antibody produced, expressed, produced or isolated by any other means involving the cleavage of a human immunoglobulin gene sequence into another DNA sequence having a human germline immunity Variable and constant regions of the globin sequence. However, in a specific example, the recombinant human antibody undergoes in vitro gene mutation (or when the in vitro somatic cell 149555.doc •15-1353852 gene mutation is used in the human ig sequence gene transfer animal) and thus the recombinant antibody VH and VL The amino acid sequence of the region, although derived from and associated with human germline VH and VL sequences, is not naturally occurring in the sequence of the human antibody germline library in vivo. As used herein, &quot;isolated antibody&quot; is intended to mean an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hTNFot is substantially free of antibodies that specifically bind to an antigen other than hTNFa). However, isolated antibodies that specifically bind hTNFa may have cross-reactivity to other antigens, such as hTNFa molecules from other species (discussed below). Furthermore, the isolated antibodies are substantially free of other cellular materials and/or chemicals. As used herein, &quot;neutralizing antibody&quot; (or &quot;antibody that neutralizes hTNFoc activity&quot;) is intended to represent an antibody that binds to hTNFoc and will result in inhibition of hTNFa biological activity. The inhibition of the biological activity of hTNFa can be assessed by measuring one or more indicators of hTNFot bioactivity, such as hTNFa-induced cytotoxicity (in vitro or in vivo), hTNFa-induced cell activation, and hTNFa binding to the hTNFa receptor. Such indicators of hTNFa biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see Example 4). Preferably, the ability of the antibody to neutralize hTNFa activity is assayed by inhibition of hTNFa-induced cytotoxicity of L929 cells. As for other or additional parameters of hTNFa, the ability of the antibody to inhibit the hTNFoc-induced expression of ELAM-1 on HUVEC (an indicator of hTNFa-induced cell activation) can be analyzed. As used herein, "surface plasmid cell genomic resonance" means that the protein concentration in the biosensor matrix can be detected by, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Switzerland and Pickup, NJ) [s] 149555.doc - 16. Analyze the optical properties of biospecific interactions in real time. For further description see Example 1 and Jonsson, U., et al. (1993) Ann. Biol. Clin, ϋ: 19-26; Jonsson, U., et al. (1991) Biotechniques Liao: 620-627; Johnsson, B., Et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem· 198: 268-277 » The term “Koff” is used herein to refer to an antibody. The rate constant for the dissociation of the antibody/antigen complex. The term "Kd" as used herein is intended to mean the dissociation constant of a particular antibody-antigen interaction. As used herein, a "nucleic acid molecule, which is intended to comprise a DNA molecule and an RNA molecule. The nucleic acid molecule may be single-stranded or double-stranded, but preferably double-stranded DNA. As used herein, an antibody or antibody protein (eg, VH, encoding an antibody that binds to hTNFa). A "isomeric nucleic acid molecule" as used in the nucleic acid of VL, CDR3) is intended to mean a nucleic acid in which the nucleic acid sequence encoding the antibody or antibody site does not contain other nucleotide sequences encoding the antibody or antibody site that binds to an antigen other than hTNFa. Molecules, the other sequence properties are flanked by nucleic acids in human genomic DNA. Thus, for example, the isolated nucleic acid encoding the 2VH region of the anti-hTNFa antibody of the present invention does not contain other sequences encoding other vh regions that bind to antigens other than hTNFa. "Vector, a nuclear knives that are intended to represent other nucleic acids that carry their linkages. One type of vector is a plastid, which represents a circular double-stranded DNA loop in which additional DNA fragments can be ligated. Another type of vector is a viral vector in which additional difficult fragments can be ligated into the viral genome. .doc -17- 1353852 The introduced host cells allow spontaneous replication (eg bacterial vectors with replicated bacterial regions and free gene sniffer vectors) other vectors (eg non-free gene mammalian vectors) can be integrated into host cells for integration into The genome of the host cell, thus replicating with the host genome. Further, certain vectors can direct the expression of the gene to its operably linked. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). In general, the utility of the expression vector in recombinant DNA technology is often in a qualitative state. In the present specification, "plastid, and "vector" can be used interchangeably 'because plastid is the most commonly used type of vector. However, the present invention Contains other types of expression vectors such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and gland-associated viruses) Equivalent function. ^ "Recombinant host cell," (or simply "host cell,") as used herein, is intended to mean a cell into which a recombinant expression vector has been introduced. It is to be understood that the term is not intended to represent a particular individual cell and represents the cell. Offspring. Since some modifications may occur in the subculture due to mutation or environmental influences, this descendant is in fact different from the mother cell, but is still included in the scope of the "host cell" used herein. The various purposes of the present invention will Further described in the following paragraphs: I. Human TNFA in combination with human TNFa The present invention provides a method of treating a disorder in which anti-TNFN is administered, which comprises bi-week subcutaneous administration with high affinity binding. The human antibody or antigen binding site thereof to human TNFa, low dissociation rate and high neutralizing ability. Preferably, the human antibody of the present invention is a recombinant neutralizing human anti-hTNFoc antibody. The optimal recombinant neutralizing antibody of the present invention is referred to herein. The amino acid sequence of D2E7 (D2E7 VL region is shown in SEQ ID NO: D2E7 VHg I; s 149555.doc • J 8 - 1353852 domain amino acid sequence is shown in Column No. 2). The nature of D2E7 is described in 881, 61, &lt;1, et al., <RTIgt; </RTI> <RTIgt; </RTI> <RTIgt; </RTI> <RTIgt; </RTI> <RTIgt; </RTI> 090, 382, which is incorporated herein by reference. A method of dysregulation, which comprises subcutaneous administration of a D2E7 antibody and an antibody to a biweekly subside, a D2E7-related antibody and an antibody site, and other human antibodies and antibody sites that are equivalent to D2E7 equivalent, such as high affinity binding to hTNFa, Low dissociation kinetics and sputum neutralization ability. In one specific example, the present invention provides φ to treat a human antibody or an antigen binding site thereof, which can be lxl〇-8 Μ or The following Kd&amp;lxi〇·3 s-丨 or below. Dissociation from human TNFa (both determined by surface plasmid cell genomic resonance) and neutralizing human TNFoc cytotoxicity with lxl 〇-7 M or below in the in vitro L929 assay standard. More preferably, the isolated human antibody or antigen binding site thereof can be detached from human TNFa at a price of 5xl0-4 s-i or less, or even better 1x10 s 1 or less. Preferably, the isolated human antibody or antigen-binding portion thereof is in the in vitro L929 analytical standard with 丨χ 〇 〇 8 m φ or less 1 C50, or even better 1 χ 1 〇 -9 以下 or less IC 5 〇 and preferably 1 x 10 1G Μ or less (10) neutralizes human TNFa cytotoxicity. In a preferred embodiment, the antibody is an isolated human recombinant antibody or an antigen binding site thereof. It is well known to those skilled in the art that the heavy and light chain cdr3 regions of the antibody play an important role in the binding specificity/affinity of the antibody to the antigen. Accordingly, in another aspect, the present invention relates to a method of treating a disorder in which an anti-TNFa antibody is administered, which is administered subcutaneously with a light structure which is associated with TNFa and which has a structurally identical or related structure to D2E7. And human antibodies in the heavy CDR3 region. Position 9 of the D2E7 VL CDR3 can be occupied by Ala 149555.doc 1353852 or Thr without substantially affecting the Kw. Accordingly, the conserved motif of the D2E7 VL CDR3 includes the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3). In addition, the position 12 of the D2E7 VH CDR3 can be occupied by Tyr or Asn without substantially affecting the Koff. Accordingly, the conserved motif of the D2E7 VH CDR3 includes the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 4). Furthermore, as demonstrated in Example 2, the CDR3 region of the D2E7 heavy and light chain was modified to be replaced with a single alanine residue (position 1, 4, 5, 7 or 8 within the VL CDR3 or position 2 within the VH CDR3) , 3, 4, 5, 6, 8, 9, 10 or 11) without affecting the K〇ff. Still better known to those skilled in the art, it will be apparent that assuming that the D2E7 VL and VH CDR3 regions are modified by alanine substitution, substitution of other amino acids in the CDR3 region is possible and still maintains a low dissociation rate constant of the antibody, especially with a conservative amino acid. Replace. As used herein, "conservative amino acid substitution" is one in which one of the amino acid residues is substituted with another amino acid residue having a similar side chain. Amino acid residues with similar side chains are defined in the art and include basic side chains (eg, aminic acid, fine acid, histidine), acidic side chains (eg aspartic acid). , valine acid), uncharged polar side chains (such as glycine, aspartame, amidoxime, serine, threonine, tyrosine, cysteine), non-polar side chains ( Such as alanine, lysine, leucine, isoleucine, valine, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine, Isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine) preferably no more than 1 to 5 conserved amines in the D2E7 VL and/or VH CDR3 region Substituted by acid. More preferably, no more than one to three conservative amino acid substitutions are made in the D2E7 VL and/or VH CDR3 regions. In addition, conservative amino acid substitutions are not made at positions of amino acids that are important for binding to f 149555.doc •20·1353852 to hTNFot. Positions 2 and 5 of D2E7 VL CDR3 and positions 1 and 7 of D2E7 VH CDR3 appear to be important for interaction with hTNFct. Therefore, conservative amino acid substitutions are better not performed at this position (but as described later, 'D2E7 VL CDR3 position 5 The alanine substitution is acceptable) (see USP 6,090,382). Accordingly, in another embodiment, the present invention relates to a method for treating an imbalance in which an anti-TNF a antibody is administered, which is administered subcutaneously to an isolated human antibody or antigen-binding site thereof. Preferably, the antibody or antigen binding site thereof has the following characteristics: a) £ determined by surface cell plasmid genomic resonance. Ting is lxl〇-3, or the following is dissociated from human TNFot; b) having an amino acid sequence comprising SEQ ID NO: 3 or substituted by a single alanine at position 1, 4, 5, 7 or 8 or 1 to 5 conservative Amino acid substitution of the light chain CDR3 region modified from SEQ ID NO: 3 at positions 1, 3, 4, 6, 7, 8, and/or 9; C) having an amino acid sequence comprising SEQ ID NO: 4 or Single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by 1 to 5 conservative amino acids at positions 2, 3, 4, 5, 6, 8, 9, Heavy CDR3 region modified from SEQ ID NO: 4 by 1 〇, U and/or 12. Better § hai antibody or its antigen binding site to 〖. Dissociated from human TNFoc by 5 X丨〇-4 s-i or below. Still more preferably, the antibody or antigen-binding site thereof is cleaved from human TNFa with Koff of lxio·4 S.1 or less. In still another embodiment, the invention provides a method of treating a disorder in which an anti-TNFa antibody is administered. These methods include biweekly subcutaneous 149555.doc • 21-1353852 administration of isolated human antibodies or antigen binding sites thereof. Preferably, the antibody or antigen-binding portion thereof comprises a light-weight CDR3 region having the amino acid sequence comprising SEQ ID NO: 3 or substituted with a single alanine at position 1, 4, 5, 7 or 8 and modified from SEQ ID NO: Chain variable region (LcvR) and having an amino acid sequence comprising SEQ ID NO: 4 or substituted by a single alanine at position 2, 3, 4, 5, 6, 8, 9, 1 or 11 and modified from sequence number 4 The heavy chain variable region (HCVR) of the CDR3 region. More preferably, the LCVR further has a CDR2 region comprising the amino acid sequence of SEQ ID NO: 5 (i.e., D2E7 VL CDR2) and HCVR further having a CDR2 region comprising the amino acid sequence of SEQ ID NO: 6 (i.e., D2E7 VH CDR2). Even more preferably, the LCVR further has a CDR1 region comprising the amino acid sequence of SEQ ID NO: 7 (i.e., D2E7 VL CDR1) and HCVR having a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8 (i.e., D2E7 VH CDR1). The framework region of VL is preferably obtained from a VKI human germline family, preferably from the human germline Vk gene and preferably the D2E7 VL framework sequence shown in Figures 1A and 1B of USP 6,090,382. The framework region of VH is preferably derived from the VH3 human germline family, preferably from the DP-3 1 human germline VH gene and preferably from the D2E7 VL framework sequences shown in Figures 2A and 2B of USP 6,090,382. In still another embodiment, the invention provides a method of treating a disorder in which administration of an anti-TNFa antibody is beneficial, wherein the individual is administered subcutaneously to the isolated human antibody or antigen binding site thereof. Preferably, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (LCVR) having an amino acid sequence comprising SEQ ID NO: 1 (i.e., D2E7 VL) and a heavy chain comprising the amino acid sequence of SEQ ID NO: Variable Area (HCVR) (also known as D2E7 VH). In a specific [s] 149555.doc -22-example, the antibody comprises a heavy chain region, such as an IgGl, IgG2, igG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy bond region is an IgGl heavy chain constant region or an IgG4 heavy chain constant region. Further, the antibody may comprise a light chain constant region, which is a kappa light chain constant region or a lambda light chain. Constant area. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody can be a Fab fragment or a single stranded ρν fragment. In still another embodiment, the present invention provides a method of treating a disorder in which an individual is administered an anti-TNFa antibody, which is administered subcutaneously to a human antibody or antigen-binding site thereof. Preferably, the antibody or antigen binding site thereof comprises a D2E7-related VL and VH CDR3 region, such as an antibody or antigen binding site thereof, comprising a sequence selected from SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 1, SEQ ID NO: 13, Sequence No. 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, and sequence number 26 The LCVR of the CDR3 region of the amino acid sequence of the group includes or is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 3, SEQ ID NO: 3 HCVR of the CDR3 region of the amino acid sequence of the ensemble number 3 3, SEQ ID NO: 3 4 and SEQ ID NO: 35. The antibody or antibody portion thereof of the present invention may be derivatized or linked to another functional molecule (eg, Another peptide or protein). Accordingly, the antibody or antibody portion of the invention is intended to comprise a derivatized and other modified human anti-hTNFa antibody herein, comprising an immunoadhesive molecule. For example, an antibody or antibody site of the invention 149555.doc -23- 1353852 can be functionally linked (by chemical coupling, gene fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody (eg, double a specific antibody or a diabodies), a detectable agent, a cytotoxic agent, a medicinal agent, and/or a protein or peptide (eg, a streptavidin core region or a polyhistidine tag) that modulates an antibody or antibody site with another molecule. . One type of derivatized anti-system is made by cross-linking two or more antibodies (of the same type or of different types, such as the production of bispecific antibodies). Suitable crosslinkers comprise heterofunctional groups having two different reactive groups separated by a spacer's spacer (eg, meta-maleimide benzoyl amide) A homobifunctional group (such as adiamyl succinate). This linkage was obtained from Pierce Chemicals Inc. Rockford, IL. Useful detectable agents which can be used to derivatize an antibody or antibody site of the invention comprise a fluorescent compound. Exemplary fluorescent detectable agents include luciferin, luciferin isothiocyanate, alkaline ruthenium red; 5-dimethylamine naphthalenesulfonium chloride, phycobilidin, and the like. Antibodies can also be used to detect enzyme derivatization, such as alkaline phosphate, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it uses an additional enzyme to produce an additional reagent that detects the reaction product. For example, when a detectable agent horseradish peroxidase is present, the addition of cyanogen peroxide and diaminobenzidine results in a colored reaction product which can be (4). Antibodies can also be derivatized by biotin and detected by indirect measurement of the binding of either inulin or streptavidin.抗体. Antibody expression Recombinant expression in the main cell For the expression of the antibody, the antibody or antibody site of the present invention can be prepared by the light and heavy chain genes of the phage globulin. 149555.doc -24- 1353852 Host cells are transfected with one or more recombinant expression vectors with DNA fragments that can be catalogued as immunoglobulins of light and heavy chains, allowing light and heavy chains to be expressed in host cells. Preferably, it is secreted into a medium in which the host cells are cultured, and the antibody is recovered from the medium. Standard recombinant DNA methods are used to obtain anti-alked heavy and light chain genes 'incorporating these genes into recombinant expression vectors and introducing vectors into host cells, such as Sambrook, Fritsch and Maniatis (eds.) 'Molecular selection; laboratory Handbook, 2nd edition, New York, Cold Spring Harbor Press (1989), Ausubel, FM, et al. (ed.) Existing Strategies in Molecular Biology, Greene Publishing Association (1989) and USP 4,816,397 to Boss et al. To express a D2E7 or D2E7-related antibody, a DNA fragment encoding a variable region of a light and heavy chain is first obtained. Such DNAs can be obtained by polymerase chain reaction (PCR) amplification and modification of germline light and heavy chain variable sequences. The germline DNA sequences of human heavy and light chain variable region genes are known in the art (see, for example, the "Vbase" human germline sequence database; see also Kabat, EA et al. (1991) Immunologically interesting protein sequences, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM., et al. (1992) ''Human germline % sequence collection reveals Group 5 of Vh fragments with different height-variable loops , j. M〇1 Bi〇1 776-798 and Cox, JPL, et al. (1994) “Management of human germline V78 fragments revealed a strong basis for its treatment” Eur j Immun〇丨亘: 827-836, Its inner valley is for reference in this article). To obtain a DNA fragment encoding a heavy chain variable region of a D2E7 or D2E7-related antibody, a VH3 family member of the human germline VH gene was amplified by standard PCR. Preferably, the Dp_149555.doc -25- 1353852 31 VH germline sequence is amplified. To obtain a DNA fragment encoding a light chain variable region of a D2E7 or D2E7-related antibody, a VKI family member of the human germline VL gene was amplified by standard PCR. Preferably, the A20 VL germline sequence is amplified. PCR primers suitable for amplifying the DP-31 germline VH and A20 germline VL sequences can be designed using standard methods disclosed in the references cited in the above references. When a germline VH and VL fragment is obtained, the sequences can be mutated to encode a D2E7 or D2E7-related amino acid sequence as disclosed herein. The amino acid sequence encoded by the VH and VL DNA sequences is first compared to the D2E7 or D2E7-related VH and VL amino acid sequences to identify amino acid residues other than the D2E7 or D2E7-related sequences of the germline. Next, the appropriate nucleotide mutation of the germline DNA sequence allows the mutated germline sequence to encode a D2E7 or D2E7-related amino acid sequence using a genetic code to determine the nuclear station acid change to be made. The gene mutation of the germline sequence is carried out by standard methods such as PCR-regulated gene mutation (in which the mutated nucleotide is incorporated into the PCR primer such that the PCR product contains the mutation) or gene mutation at the leader position. When a DNA fragment encoding a D2E7 or D2E7-related VH and VL fragment is obtained (by the above-described germline VH and VL gene amplification and gene mutation), the DNA fragments can be manipulated by standard recombinant DNA techniques, for example, The region gene is transformed into a full-length antibody chain gene and transformed into a Fab fragment gene or scFv gene. In such operations, the VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, "operably linked" means that the combination of two DNA fragments allows the amino acid sequence encoded by the two DNA fragments to remain in frame 149555.doc -26-1353852. The single DN A encoding the VH region can be converted into a full length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule (CHI, CH2 and CH3) encoding a constant region of the heavy chain. Human heavy chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Immunologically interesting protein sequences, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242) And DNA fragments comprising such regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgGl or IgG4 constant region. In the case of a Fav fragment heavy chain gene, the VH-encoding DNA is operably linked to another DNA molecule encoding only the constant region of the heavy chain CH1. The isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule CL encoding a constant region of the light chain. Human light chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Immunologically interesting protein sequences, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242) And DNA fragments comprising such regions can be obtained by standard PCR amplification. The light chain constant region may be a κ or λ constant region, but is preferably a κ constant region. To generate the scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible link (eg, encoding an amino acid sequence (Gly4-Ser) 3) such that the VH and VL sequences It can be expressed as a continuous single-stranded protein, while the VL and VH regions are linked by a flexible linker (see, for example, Bird et al. 149555.doc -27- 1353852 (1988) Science 242: 423-426; Huston et al. (1988) Proc Natl. Acad. Sci. USA ϋ: 5879-5883; McCafferty et al, Nature (1990) Ml: 552-554). To express an antibody or antibody site of the invention, the coding portion or the full length light and heavy chain DNA fragments obtained above are inserted into an expression vector such that the gene is operably linked to the transcriptional and translational control sequences. In the present specification, "operatively linked" means that the antibody is conjugated to the vector such that the transcriptional and translational control sequences within the vector have the desired function of regulating the transcription and translation of the antibody gene. The expression vector and the expression control sequence are selected to be compatible with the performance host cells used. The antibody light chain gene and the antibody heavy chain gene can be inserted into another vector or more typically, and the two genes are inserted into the same expression vector. The antibody gene can be expressed by a standard method (such as binding of an antibody gene fragment and a complementary restriction site of the vector, or a blunt end junction if there is no restriction position) ^ before inserting a D2E7 or D2E7-related light or heavy chain sequence, the expression vector can be The antibody constant region sequence has been taken. For example, one of the methods for converting a D2E7 or D2E7-related VH and VL· sequence into a full-length antibody gene is to insert it into a expression vector that encodes a heavy chain constant region and a light chain constant region, respectively, and thus the VH fragment is operably The CH fragment and the ?^^ fragment linked to the vector are operatively linked to the CL fragment in the vector. In addition or in addition, the &apos;recombinant expression vector can encode a mono-peptide which promotes secretion of the antibody chain from the host cell. The antibody chain gene is selected in a human vector such that the mono-peptide is linked in the framework to the amine terminus of the antibody bond gene. The mono-peptide may be an immunoglobulin mono-peptide or a hetero-single peptide (i.e., a mono-peptide derived from a non-immunoglobulin protein). In addition to the antibody chain gene, the recombinant expression vector of the present invention carries a regulatory sequence of the expression of the t antibody chain gene of the host cell of I49555.doc • 28-!353852. A "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. This regulatory sequence is described, for example, in Goeddel; Gene Expression Technology: Enzyme Methods 185, College Press, San Diego, CA (1990). It is well known in the art that the design of the performance vector includes the selection of regulatory sequences depending on several factors, the choice of host cell to be transformed, the degree of protein expression desired, and the like. Preferred regulatory sequences for mammalian host cell expression include viral elements that are introduced into high-protein expression in mammalian cells, such as promoters/enhancers derived from cytomegalovirus (CMV) (eg CMV promoter/enhancer), Simon (Simian) virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus (such as the adenovirus major delayed promoter (AdMLP)), and polyomavirus. Further description of the viral regulatory elements and their sequences can be found in, for example, USP 5,168,062 to Stinski, USP 4,510,245 to Bell et al., and US Patent 4,968,615 to Schaffner et al. In addition to the antibody bond genes and regulatory sequences, the recombinant expression vectors of the present invention may carry additional sequences, such as sequences (e.g., replication sources) and selectable marker genes that modulate vector replication in a host cell. The selection of the standard is due to the selection of host cells into which the vector has been introduced (see, for example, Axd et al., USP 4,399,216; 4,634,665 and 5,179,017). For example, a selectable marker gene confers resistance to a drug such as G418, hygr〇mycin or aminoguanidine to a host cell into which the vector has been introduced. A preferred alternative marker gene comprises: the genus 土3_虱 folate reductase (DHFR) gene (for immortalized host cells with methotrexate selectivity/amplification) and the neo gene (for G418 selection) effect). 149555.doc •29· 1353852 For the performance of light and heavy chains, the expression vector encoding this heavy and light chain is transfected into host cells by standard techniques. Various types of "transfection" are intended to encompass a wide variety of techniques commonly used to introduce foreign DN A into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibody of the present invention in a prokaryotic or eukaryotic host cell, since eukaryotic cells, and particularly mammalian cells, appear to be easier to assemble and secrete appropriately folded and immunologically active antibodies than prokaryotic cells, they are preferably eukaryotic. Antibodies are expressed in cells, especially in mammalian cells. Prokaryotic expression of antibody genes has been reported to be ineffective in the production of high-yield active antibodies (Boss, Μ.A. and Wood, CR (1 985) Immunology Today barrier: 12-13) ° Preferred mammals for the expression of recombinant antibodies of the invention The host cell contains Chinese hamster nest (CHO cells) (containing dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, with DHFR selectable marker genes for use , as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to express the antibody in the host cell, or better for secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from culture media using standard protein purification methods. Host cells can also be used to produce intact antibody sites, such as Fab fragments or scFv molecules. It is to be understood that variations of the above described procedures are within the scope of the invention. For example, it may be desirable to transfect a host cell with DNA encoding one (but not both) of the light or heavy chain of an antibody of the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light or heavy chains, which is not necessary for binding to hTNFa, 149555.doc -30- 353852. Molecules from which the truncated dna molecule is expressed are also included in the antibody of the present invention. In addition, the antibody of the present invention can be conjugated to a second antibody by a standard chemical crosslinking method to produce a bifunctional antibody. One heavy chain and one light chain are the antibodies of the present invention and other heavy and light chains are other than hTNFa. The antigen is specific. A preferred system for recombinant expression of an antibody or antigen-binding portion thereof of the present invention, • a recombinant expression vector encoding both the heavy chain of the antibody and the light chain of the antibody, is transfected into dhfr-CHO cells by calcium phosphate. In recombinant expression vectors, antibody heavy and light chain genes are each operably linked to a CMV enhancer/AdMLp promoter regulatory element to confer a high degree of gene transfection. The recombinant expression vector also carries the DHFR gene, which can select CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformed host cell cultures allow for antibody heavy and light chain expression and recovery of intact antibodies from the culture medium. Standard biomolecular techniques are used to prepare recombinant expression vectors, transfected host cells, selected transformants, cultured host cells, and recovered from culture media. IH.t group human antibody release In addition to the D2E7 or its antigen binding site or D2E7_related antibody disclosed herein, the recombinant human antibody of the present invention can be isolated by screening the recombinant antibody database, preferably using self-derived The human VL and VH cDNA prepared by the human lymphocyte mRNA was displayed as a scFv phage display library. Methods of preparing and screening such databases are known in the art. In addition to commercially available kits that produce phage display databases (eg, pharmaceutical weight 149555.doc • 31· 1353852)

Group phage antibody system, catalog number 27-9400-01; and Stratagene SurfZAPTM phage display kit, catalog number 240612), examples of methods and reagents specifically adapted for generating and screening antibody display libraries can be found, for example, in Ladner et al USP 5,223,409 Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Announcement No. WO 93/01288; McCaffery et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9. · 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas: 81-85; Hues et al. (1989) Science 246: 1275-1281;

McCafferty et al, Nature (1990) 348. · 552-554; Griffiths et al. (1993) EMBO J ϋ: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89.: 3576-3580; Garrard et al. (1991) Bio/Technology 9_: 1373-1377;

Hoogenboom et al. (1991) Nuc Acid Res 19_ '4133-4137; and Barbas et al. (1991) PNAS M: 7978-7982. In a preferred embodiment, in order to isolate a human antibody having high affinity and low dissociation constant for hTNFoc, a mouse anti-hTNFoc antibody having high affinity to hTNFtx and a low dissociation constant (such as MAK 195, having the accession number ECACC) is first used. The fusion tumor of 87 050801) selects human heavy and light chain sequences having similar hTNFot binding activity by the method of using the antigen-determining gene described in Hoogenboom et al. PCT Publication No. WO 93/0621. This method [S 1 149555.doc • 32- used antibody database is preferably McCafferty et al. PCT Publication No. WO 92/01047, McCafferty et al, Nature (1990) 348_: 552-554; and Griffiths et al. (1993). EMBO J ϋ: The scFv database prepared and screened as described in 725-734. The scFv antibody library is preferably screened using recombinant human TNFa as an antigen. Once the original human VL and VH fragments were selected, a &quot;mixing and pairing&quot; experiment was performed in which the initially selected VL and VH fragments of the hTNFa binding were screened differently to select the preferred VL/VH pairwise combination. In addition, in order to further improve the affinity and/or reduce the dissociation constant of hTNFa binding, the VL/VH paired VL and VH fragments may be similar to those in the body wall mutation process in which the antibody affinity mutation is responsible for the natural immune reaction. Random mutations, preferably in the CDR3 region of VH and/or VL. This in vitro affinity mutation can be performed by amplifying the VH and VL regions using PCR primers complementary to the VH CDR3 or VL CDR3, respectively, which have been "spiked" in a random mixture of 4 nucleotide bases at certain positions. The resulting PCR product can encode VH and VL fragments in which the random mutation has been introduced into the VH and/or VL CDR3 regions. The VH and VL fragments of these random mutations are rescreened for binding to hTNFa and optionally exhibit high binding to hTNFa. Affinity and sequence of low dissociation rate. After screening and recombining the anti-hTNF a antibody of the present invention from the recombinant immunoglobulin display database, the selection antibody can be encoded from the display package (eg, from the bacterium genome) The nucleic acid and the standard recombinant DNA technology are sub-selected into other expression vectors. If desired, the nucleic acid can be manipulated to produce other antibody types of the invention (e.g., linked to nucleic acids encoding other immunoglobulin regions, such as other constant regions). In order to demonstrate the 149555.doc •33· 1353852 recombinant human antibody, the dna encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell as further described in detail in the above paragraph η. IV. Pharmaceutical Compositions and Pharmaceutical Authors The antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to an individual by the methods described herein (e.g., biweekly subcutaneous administration). Typically, the pharmaceutical composition comprises an antibody (or antibody site) of the invention and/or an amidine and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delays that are physiologically compatible and suitable for administration to an individual by the methods herein. An agent, etc. Examples of the pharmaceutically acceptable carrier include water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., or a combination thereof, and many combinations thereof, preferably containing an isotonic agent in the composition. Such as sugars, polyols such as mannitol, sorbitol or sodium chloride. Pharmaceutically acceptable carriers can also include trace amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers d, which can enhance the preservation of antibodies or antibody sites. The composition of the present invention may be in various forms, including, for example, liquid, semi-solid, and solid dosage forms such as liquid solutions (e.g., injection of xin (e.g., primary and perfusion solutions), dispersions or suspensions, bonds Agents, pills, powders, fines and suppositories. The preferred form depends on the mode of administration and the therapeutic use. 疋 Typical preferred compositions are injectable or solutions, such as humans. Other antibodies used for passive immunization: composition. The preferred mode of administration is parenteral (eg intravenous, subcutaneous, 1U specific example, the antibody is administered by intravenous infusion 4 injections. Another preferred specific 翟 / main or. Intraspecific antibody straw muscle injection injection. In the specific case, the antibody Saguxia, ± ^ 杈 杈 Le. Subcutaneous injection (such as biweekly subcutaneous injection ' [S ] H9555.doc -34- 1353852 treatment combination The material is generally sterile and (4) stable under the conditions of cutting and cutting. The compound can be formulated as a solution 'microemulsion, dispersion, liposome or other grade structure suitable for high-grade soil. Sterile injectable solution can be borrowed The desired amount of the compound (i.e., the antibody or antibody site) is prepared if necessary in combination with the above ingredients: or the composition is incorporated into a suitable solvent, followed by filtration sterilization. Usually, the dispersion can be incorporated by containing the active compound. The sterile carrier of the dispersion medium and other components required for the preparation. In the case of preparing a sterile injectable solution, the preferred method of preparation is vacuum drying and cold-drying, which can produce active ingredient powder plus on Any additional desired ingredient in any of the foregoing sterilizing solutions may be maintained, for example, by the use of a coating agent such as (iv) vinegar, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants to maintain proper flow of the bath. The extended absorption of the injectable composition can be achieved by the use of a reagent which delays absorption, such as monostearate and gelatin, in the composition. The antibody and antibody site of the present invention can be administered by various methods known in the art, but In many cases, the preferred mode of administration is subcutaneous injection. As the skilled artisan knows, the route and/or mode of administration will vary depending on the desired result. In some specific examples, the active compound may protect the compound from Preparation of carriers released in Shaanxi, such as controlled release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable biocompatible polymers such as ethylene glycol can be used. Ethylene vinegar, polyethylene glycol (PEG), polyacid needle, polyglycolic acid, prion, polyglycol and polylactic acid. Many of the methods for preparing such formulations are patented and generally known to those skilled in the art. See, for example, Continuous and Controlled Release Drug Delivery Systems, JRI-Editor, Marcel Dekker, New York, 1978. 149555.doc -3$. 1353852 In certain embodiments, the benzene inventive antibody or antibody site can be administered orally, for example, as an inert diluent or a digestible carrier. The compound (and other ingredients if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into a keying agent or incorporated directly into the individual's diet. For oral therapeutic administration, the compound can be incorporated with an excipient and used in digestible tablets, buccal tablets, troches, capsules, sweets, suspensions, syrups, wafers and the like. In order to administer a compound of the invention excreted parenterally, it is necessary to avoid coating the deactivated material or co-administering with the compound. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, an antibody or antibody site of the invention is co-administered and/or co-administered with one or more other therapeutic agents. For example, the anti-hTNF〇c antibody or antibody site of the present invention may be combined with methotrexate, one or more other antibodies that bind to other targets (such as antibodies that bind to other cytokines or bind cell surface molecules), one or more cytokines, and soluble. TNFa receptor (see, for example, PCT Publication No. WO 94/06476) and/or one or more chemical agents that inhibit the production or activity of hTNFa (such as the cyclohexylene derivative described in PCT Publication No. WO 93/19751) Co-allocation or co-administration. Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. This combination therapy may facilitate the administration of a lower dose of the therapeutic agent, thus avoiding possible toxicity or complications associated with various monotherapies. The use of an antibody or antibody site of the invention in combination with other therapeutic agents is further detailed in Section IV. Non-limiting examples of therapeutic agents for rheumatoid arthritis that can be used in combination with an antibody or antibody site of the invention include the following: non-sterol anti-inflammatory agents (NSAIDs); cytokine inhibitory anti-inflammatory agents (CSAIDs); CDP-571/BAY-10 -3356 (person 149555.doc -36 - 1353852

Anti-TNFoc antibody; Celltech/Bayer; cA2 (chemotaxis anti-TNFa antibody Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see eg Arthritis &amp; Rheumatism (1994) Volume 21, S295; J. Invest. Med. (1996), 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non- Censored primate anti-CD4 antibody; IDEC/SmithKline; for example see Arthritis &amp; Rheumatism (1995) Vol. 11, S185); DAB 486-IL-2 and/or DBA 389-IL-2 (IL-2 Fusion protein; Seragen; see eg Arthritis &amp; Rheumatism (1993), pull, 1223); anti-Tac (humanized anti-IL-2Ra; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokines; DNAX/ Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonist (eg agonist antibody); IL -1RA (IL-1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF-binding protein, see, for example, Arthritis &amp; Rheumatism (1996), Vol. 9 (Supplement) S284Amer. J. Physiol.-Heart and Circulatory Physiology (1 995), vol. 268, pp. 37-42); R973401 (acid diesterase type IV inhibitor; see, for example, Arthritis &amp; Rheumatism (1996) vol. Phase 9 (Supplementary), S282); MK-966 (COX-2 inhibitor; see, for example, Arthritis &amp; Rheumatism (1996) Vol. 9 (Supplement), S81); Iloprost (see, for example, Arthritis &amp; Rheumatism ( 1996) Volume 9 (Supplementary), S82); Amine; Amidoxime 83⁄4 (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 2, No. 9 (Added 149555.doc -37- 1353852) Supplementary) 'S282) and from the chloramines _ related drugs (such as ceigen); leflunomide (anti-inflammatory and cytokine inhibitors, see for example Arthritis &amp; Rheumatism (1996) , No. 9 (Supplementary), S131; Inflammation Research (1996), Vol. 45, pp. 103-107); tranexamic acid (inhibitor of plasma-priming; see, for example, Arthritis &amp; Rheumatism (1996), vol.9 Period (supplementary version), S284" τ_614 (cytokine inhibitor; see for example Arthriti s &amp; Rheumatism (1996) Vol. 9 (Supplementary), S282); prostaglandin e 1 (see, for example,

Arthritis &amp; Rheumatism (1996), vol. 9, vol. 9 (S282); Tenidap (non-sterol anti-inflammatory drugs; see, for example, Arthritis &amp; Rheumatism (1996) 巧巧_, Issue 9 (Supplementary), S280); Naproxen (non-sterol anti-inflammatory drugs; see for example

Neuro Report (1996) Vol. 2_, pp. 1209-1213); Mel〇xicam (non-sterol anti-inflammatory drugs); Ibupr〇feil (non-sterol anti-inflammatory drugs); Piroxicam (non-sterol anti-inflammatory drugs); Dicl〇fenac (non-sterol anti-inflammatory drugs); Indomethacin (non-sterol anti-inflammatory drugs); Sulfasalazine (for example) See Arthritis &amp; Rheumatism (1996), vol. 9 (Supplementary), S281); Azathioprine (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 9 (Supplement)' S281); ICE inhibitor (enzyme interleukin-1β converting enzyme); zap_7〇 and/or rick inhibitor (inhibitor of tyrosine kinase zap-7〇 or lck); VEgf inhibitor and/or VEGF_R inhibitor (vascular endothelial growth factor or vascular endothelial growth factor receptor; inhibitor of angiogenesis); corticosteroid anti-inflammatory drugs (eg 149555.doc •38· SB203580); TNF-converting enzyme inhibitor; anti-IL -12 antibody; interleukin-11 (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 9 (Supplement) S296); Interleukin-13 (see, for example, Arthritis &amp; Rheumatism (1996), Vol. 9 (Supplement), S308); Interleukin-17 inhibitor (see, for example, Arthritis &amp; Rheumatism (1996) Vol. 39 '9th (additional version), S120); gold; penicillamine; chlorinated porphyrin; hydroxychloroporphyrin; chlorflurane; cyclophosphamide; cyclosporine; total lymphoid radiation; Globulin; anti-CD4 antibody; CD5-toxin; oral administration of skin and collagen, lobenzarit disodium; cytokine modulators (CRAs) HP228 and HP466 (Houghten Pharmaceuticals); ICAM-1 Isothiophosphate oligodeoxynucleotide: y: acid (ISIS 2302; Isis Pharmaceuticals); soluble complementary receptor 1 (ΤΡΙΟ; T Cell Science); prednisone; orgotein; glucose Aminoglycan polysulfate; diamine tetracycline; anti-IL2R antibody; mouse and plant lipids (fish and plant seed fatty acids; see for example DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 2J_: 759-777 ); auranofin; phenylbutyrate; meclofenac; flufenic acid; Internal immunoglobulin; zileuton; mold age acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); Amiprilose (therafectin); cladribine (2-gas deoxygenation); and azoribine. Non-limiting examples of therapeutic agents for inflammatory bowel disease for use in combination with an antibody or antibody site of the invention include the following: budenoside; epidermal growth factor; corticosteroid; cyclosporine; sfaphine; amine Salicylic acid 149555.doc -39- 1353852 ester; 6-hydrosulfanyl hydrazine; azathiopurine; metronidazole; lipoxygenase inhibitor; mesalamine; olsalazine; Balsalazide; antioxidant; thromboxane inhibitor; IL-1 receptor antagonist; anti-IL-Ιβ monoclonal antibody; anti-IL-6 monoclonal antibody; growth factor; elastase inhibitor; pyridyl Imidazole compound; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer; cA2 (chemotaxis anti-TNFot antibody Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein) Immunex; see for example Arthritis &amp; Rheumatism Π 994), Vol. 37, S295; J. Invest. Med. (1996) Vol. 4i_, 235 A); 55 kdTNFR-IgG (55 kD TNF Receptor-IgG fusion protein; Hoffmann-LaRoche ); Interleukin-10 (SCH 52000; Schering Plough); IL-4; IL-10 and/or IL-4 Agent (such as agonist antibody); interleukin-11; pannisone, dexamethasone or Bhutan taste of glucosamine- or dextran-conjugated prodrug; ICAM-1 Isothiophosphate oligodeoxynucleotides (ISIS 23 02; Isis Pharmaceuticals); Soluble Complementary Receptor 1 (ΤΡΙΟ; T Cell Science); low release of manhole; ammonia; platelet activating factor PAF) antagonist; ciprofloxacin; and lidocaine (lignocaine) °

Non-limiting examples of therapeutic agents for multiple sclerosis that can be combined with an antibody or antibody site of the invention include the following: corticosteroids; pannisone; sulfhydryl decandenone; azathiazepine; cyclophosphamide; cyclosporine Amino acid; 4-amino-based p-bite; Tizanidine; interferon-pla (AvonexTM; Biogen); interferon-βΐϊ) (BetaseronTM; Chiron/Berlex); copolymerization [SI 149555.doc •40- 1 (Cop-1; C0paxoneTM; Teva Pharmaceuticals) High specific gravity oxygen; intravenous immunoglobulin; crabiribine; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer); cA2 (hostile anti-TNFa antibody; Centocor) 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; immunoex; see for example Arthritis &amp;

Rheumatism (1994) Vol. 12, S295; J. Invest. Med. (1996) ϋ '235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IL-10; IL- 4; and IL-10 and / or IL-4 agonist (such as agonist antibodies). Non-limiting examples of therapeutic agents for sepsis that can be combined with an antibody or antibody site of the invention include the following: an isotonic saline solution; an antibiotic; an intravenous gamma globulin; a continuous sputum sputum; a carbaenes (such as a merlot) Ning (meropenem); cytokine antagonists such as TNFa, IL-Ιβ, IL-6 and/or IL-8; CDP-571/BAY-10-3356 (humanized anti-TNFtx antibody; Celltech/Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see for example Arthritis &amp; Rheumatism (1994) Vol. 12, S295; J. Invest. Med. 1996) mad, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); cytokine modulators (CRAs) HP228 and HP466 (Houghten Pharmaceuticals); SK&amp;F 107647 (low Molecular Peptide; SmithKline Beecham); Tetravalent Indole-based CNI-1493 (Picower Association); Tissue Factor Pathway Inhibitor (TFPI; Chiron); PHP (Chemically Modified Erythropoietin; APEX Bioscience); Iron Chelating Agent and Chelate , 149555.doc • 41- 1353852 contains di-ethyltriamine Acid-iron (III) complex (DTPA iron (III); Molichem medicine); lyophilized lisofylline (synthesis small molecule methylxanthine; cell therapy company); PGG-glucan (aqueous soluble) Β-1,3-glucan; Alpha-Beta Technology Inc.; apolipoprotein Α-1 with lipid reconstitution; palmitic hydroxy-anthronic acid (synthetic antibacterial agent can inhibit lipid A biosynthesis); anti-endotoxin antibody E5531 (synthetic lipid A antagonist; Eisai, USA); rBPI21 (recombinant N-terminal fragment of human bactericidal/increasing osmotic protein); and synthetic anti-endotoxin peptide (SAEP; BioYnth Research Laboratories). Non-limiting examples of therapeutic agents for adult respiratory distress syndromes that can be combined with an antibody or antibody site of the invention include the following: anti-IL-8 antibody; surfactant replacement therapy; CDP-571/BAY-10-3356 (humanized antibody -TNFoc antibody; Celltech/Bayer); cA2 (anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see eg Arthritis &amp; Rheumatism Π 994) Volume 3 7, S295; J. Invest. Med. (1996) Μ, 235Α); and 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche) ° The composition of the invention may comprise "therapeutically effective amount "Or a "prophylactically effective amount" of an antibody or antibody site of the invention. "Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result during administration and during the desired period. The therapeutically effective amount of an antibody or antibody site can depend on several factors, such as the individual's disease state, age, sex, and weight, and the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount can also be one in which the therapeutic benefit of the antibody or antibody site is greater than any toxic or deleterious effect. The "preventive effective amount" represents the amount of the preventative result that can be effectively achieved during the period of administration and [S i 149555.doc -42 - 1353852. Typically, since the preventive agent I is applied to the individual at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount. The administration regimen can be adjusted to provide the optimal desired response (eg, a therapeutic or prophylactic response). For example, a single pellet can be administered, and a divided dose can be administered for a certain period of time or the dose can be proportionally decreased or increased. As indicated by the urgency of the therapeutic position. Formulating a parenteral composition in a unit dosage form is particularly advantageous in terms of ease of administration and uniformity in the agent. The unit dosage form as used herein refers to a unit that is physically separated by unit dosages for the individual to be treated; each unit contains a predetermined amount of active compound and a desired carrier for the preparation of the desired therapeutic effect. The unit dosage form of the invention is subject to and directly related to: (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the field of mixing the active compound for the treatment of the individual's inductivity. . A therapeutically or prophylactically effective amount of an antibody or antibody site of the invention is exemplified by a non-limiting range of 10 to 100 mg, more preferably 20 to 80 mg, and most preferably 4 mg. Need/Important I can be used as a temperament;

The professional judgment of the administration or administration of the composition is adjusted over time, and the dosage ranges described herein are merely exemplary and not intended to limit the scope or operation of the invention. V. The use of the present invention can be carried out by using a conventional immunoassay such as an enzyme bond and the ability to bind to hTNFa, and the immunosorbent assay of the anti-hTNFcx antibody or enzyme linkage of the present invention is 149,555. Doc -43- 1353852 Method (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry to detect hTNFot (as in biological samples such as serum or plasma). The present invention provides a method for detecting hTNFot in a biological sample, comprising contacting a biological sample with an antibody of the present invention or an antibody site thereof, and detecting an antibody (or antibody site) or an unbound antibody (or antibody site) that binds to hTNFot. . The antibody is labeled, directly or indirectly, with a detectable substance to accelerate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent materials and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, phosphatase, beta-galactosidase or acetaminocholine; examples of suitable prosthetic complexes include chain enzyme peptide/biotin and avidin/biotin Examples of suitable fluorescent materials include carbaflorin, luciferin, luciferin isothiocyanate, alkaline ruthenium, dichlorotriamine fluorescein, 5-diguanamine-1- Sulfonium or phycoerythrin; examples of luminescent materials comprising luminol; and examples of suitable radioactive materials comprising 1251, 1311, 35S or 3H. In addition to labeling the antibody, hTNFot can be analyzed in a biological fluid by competitive immunoassay using a detectable substance-labeled rhTNFoc substance and an unlabeled anti-hTNFa antibody. In this assay, the biological sample, the labeled rhTNFa standard, and the anti-hTNFot antibody can be mixed and assayed for the amount of labeled rhTNFa standard bound to the unlabeled antibody. The amount of hTNFa in the biological sample is inversely proportional to the amount of labeled hTNFa standard bound to the anti-hTNFcx antibody. The D2E7 antibody of the present invention can also be used for detecting TNFocs derived from septicemia other than humans, especially from primates (such as chimpanzees, baboons, baboons, baboons and rhesus monkeys), pigs and mice, because D2E7 It can be bound to each of these TNFocs. 149555.doc -44- 1353852 The antibodies and antibody sites of the invention can neutralize hTNFa activity in vitro and in vivo (see USP 6,090,382). Furthermore, at least some antibodies of the invention such as D2E7 can neutralize hTNFoc activity from other species. Accordingly, the antibodies and antibody sites of the invention can be used, for example, in cell cultures containing hTNF〇c, human subjects or with Inhibition of hTNFa activity is inhibited in other mammalian individuals (such as chimpanzees, baboons, baboons, baboons, rhesus monkeys, pigs, and mice) in which the antibody is reacted with TNFas. In one embodiment, the invention provides a method of inhibiting TNFa activity comprising contacting 7^11?〇1 with an antibody or antibody site of the invention thereby inhibiting TNFoc activity. Preferably, the TNFa is human TNFcc. For example, in a cell culture containing or suspending TNFa, the antibody or antibody site of the present invention can be added to the culture medium to inhibit hTNFa activity in the culture. In a preferred embodiment, the invention provides a method of treating a disorder in which administration of an anti-TNFa antibody is beneficial&apos; comprising subcutaneous administration of an antibody or antibody site of the invention to an individual for biweekly treatment of the disorder. In a particularly preferred embodiment, the anti-system is administered subcutaneously in a biweekly manner. In another particularly preferred embodiment, the anti-system is administered subcutaneously before, during or after the administration of ammonia. Preferably, the individual is a human individual. Alternatively, the individual can be a mammal having TNFa that is reactive with the antibody of the invention. Still other individuals may be mammals in which hTNFa has been introduced (e.g., by administration of hTNFa or by hTNFa gene transfer). The antibodies of the invention can be administered to a human subject for therapeutic purposes (as discussed above). Further, the antibody of the present invention can be administered to a non-human mammal (e.g., primate, pig or mouse) having TNFa reactive with the antibody of the present invention for veterinary purposes or as an animal model of human disease. Regarding the latter, this animal model can be used to evaluate the therapeutic effect of the antibody of the present invention (e.g., test dose and administration time) by evaluating 149555.doc -45·1353852. As used herein, "the disorder in which an anti-TNFot antibody is administered is beneficial, and is intended to include a disease and other disorders in which an individual having the disorder has a pathology or a disorder in which the TNFot has been shown or is expected to be responsible for the disorder. Factors or have shown that other anti-TNFa antibodies or biologically active sites thereof have been successfully used to treat the disease. Accordingly, a disorder in which TNFa activity is detrimental is a condition in which inhibition of TNFa is expected to soothe the symptoms and/or progression of the disorder. It can be demonstrated by an increase in the concentration of TNFa in a biological fluid, such as an individual suffering from the disorder (e.g., serum, plasma, synovial fluid, etc. of an individual), which can be detected using, for example, an anti-TNFa antibody as described above. Examples of the use of the antibodies and antibody sites of the invention in the treatment of specific diseases are further discussed as follows: A · sepsis tumor necrosis factor has established a role in the pathology of sepsis, and biological effects include hypotension, myocardial depression, vascular infiltration Leakage syndrome, organ necrosis, stimulation of release of toxic minor regulators and activation of clot cascade ^ ^ W-ko ^ ^Tracey, K.J.^Cerami, A. (1994) Annu. Rev. 45. 491-503, Russell, Thompson, R.C. (1993)

Cun·. 〇pin. Biotech•生: 714 72i). Accordingly, the human antibodies and antibody sites of the invention can be used to treat any clinically defined disorder, including asymptomatic shock, endotoxic shock, Gram-negative sepsis, and toxic shock syndrome. [S1 again, 'A treatment of sepsis, the anti-(4)~antibody or anti-caries of the present invention may be combined with one or more other therapeutic agents for restoring sepsis. I49555.doc-46 - Administration, such as interleukin-1 Inhibitors (as described in PCT Publication Nos. WO 92/16221 and WO 92/17583), cytokine interleukin-6 (see, for example, PCT Publication No. WO 93/1 1793) or platelet activating factor antagonists (see, for example, Europe) Patent Application Bulletin EP 374 510). Further, in a preferred embodiment, the anti-TNFot or antibody site of the present invention is administered to a human subject, and the subgroup of the end septicemia group has a serum or plasma concentration of IL-6 higher than 500 pg/ml at the time of treatment. And more preferably 1000 pg/ml (see Daum, L et al. PCT Bulletin No. WO 95/20978). B. Autoimmune diseases Tumor necrosis factors are associated with roles in the pathology of various autoimmune diseases. For example, TNFot is associated with inflammation of activated tissues and joint destruction that causes rheumatoid arthritis (see, for example, Tracey and Cerami, supra; Arend, WP and Dayer, JM. (1995) Arth. Rheum. 38. '151-160; Fava , RA, et al. (1993) Clin. Exp. Immunol. 94: 261-266). TNFot is associated with promoting island cell death and modulating diabetic insulin resistance (see, for example, Tracey and Cerami, supra; PCT Publication No. WO 94/08609). TNFoc is also associated with modulation of cytotoxicity in oligodendrocytes and induction of inflammatory plaques in multiple sclerosis (see, for example, Tracey and Cerami, supra). Intended and humanized mouse anti-hTNFa antibodies have undergone clinical testing for the treatment of rheumatoid arthritis (see, for example, Elliott, MJ, et al. (1994) Lancet 344: 1 125-1127; Elliot, MJ, et al. (1994) Lancet 344: 1105-1110; Rankin, EC, et al. (1995) Br. J. Rheumatol. Μ: 334-342). The human antibody and antibody site of the present invention can be used to treat autoimmune diseases 149555.doc -47-1353852 Diseases, especially those associated with inflammation, including rheumatoid arthritis, rheumatic spondylitis, osteoarthritis and gout arthritis, allergies, Multiple sclerosis, autoimmune diabetes, autoimmune uveitis, and nephrotic syndrome. Typically, the antibody or antibody site is administered systemically, but for certain disorders, it may be beneficial to localize the antibody or antibody site at the site of inflammation (eg, topical administration of rheumatoid arthritis or ulceration of diabetes) Topical administration of cancer can be administered alone or in combination with a cyclocyclohexylene derivative as described in PCT Publication No. WO 93/19751). C. Infectious diseases Tumor necrosis factors are associated with the physiological effects seen in the regulation of various infectious diseases. For example, TNFa is associated with brain inflammation and capillary embolism and infarction that regulate dysentery (see, for example, Tracey and Cerami, supra). TNFa is also associated with the regulation of brain inflammation, including meningitis, cerebral dysfunction, restriction of septic shock syndrome, and activation of venous infarction (see, for example, Tracey and Cerami, supra). TNFa is also associated with stimulation of viral proliferation and regulation of central nervous system damage in septic disease, acquired immunodeficiency syndrome (AIDS) (see, for example, Tracey and Cerami, supra).

Accordingly, the antibodies and antibody sites of the invention can be used to treat infectious diseases, including bacterial meningitis (see, for example, European Patent Publication No. Ep 585 7〇5), cerebral dysentery, AIDS, and AIDS-related complications (ARC) (see, for example, European Patent Publication No. EP 230 574) and secondary cytomegalovirus infections for transplantation (see, for example, FietZe, E., et al. (1994) Transplant 58: 675-680). The antibody and the antibody site of the invention can also be used to alleviate the syndrome associated with infectious diseases, including fever caused by infection (such as influenza) and myalgia and the recurrence of infection [W I49555.doc -48- quality disease (such as Continued AIDS or ARC). D. Transplantation of tumor necrosis factor is associated with graft rejection and a major regulator of graft-to-host disease (GVHD) and when rat antibody 0KT3 associated with anti-T cell receptor CD3 complex is used to inhibit renal transplant rejection The regulation of adverse reactions seen is related (see, for example, Tracey and Cerami, supra; Eason, JD, et al. (1995) Transplantation 300-305; Suthanthiran, Μ. and Strom, Τ·Β., (1994) New Engl. J Med. 331 : 365-3 75). Accordingly, the antibody and antibody sites of the present invention can be used to inhibit transplant rejection, including allograft and allogeneic transplantation, and inhibition of GVHD. Although the antibody or antibody site can be used alone, it is preferably used in combination with one or more other drugs which inhibit the immune response against allografts or inhibit GVHD. For example, in one embodiment, an antibody or antibody site of the invention can be used in combination with OKT3 to inhibit OKT3-induced responses. In another embodiment, an antibody or antibody site of the invention is used in combination with one or more antibodies directed to other targets that modulate an immune response, such as the cell surface molecule CD25 (interleukin-2 receptor-a), CDlla (LFA- 1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment, the antibody or antibody site of the invention is used in combination with one or more general immunosuppressive agents such as cyclosporin A or FK506. E. Malignant Diseases Tumor necrosis factor is associated with malignant disease-induced malignant disease, stimulation of tumor growth, enhancement of migration potential, and regulation of cytotoxicity (see, for example, Tracey and Cerami, supra). Accordingly, the antibody and antibody sites of the present invention can be used in the treatment of malignant diseases to inhibit tumor growth or migration and/or to alleviate the malignant disease of malignant disease. The antibody or antibody site can be administered systemically or locally to the tumor site. F. Lung disorders Tumor necrosis factor is associated with the pathology of adult respiratory distress syndrome, including stimulation of white jk ball-endothelial cell activation, introduction of cytotoxicity into lung cells, and induction of vascular leakage syndrome (see, for example, Tracey and Cerami, supra) ). Accordingly, 'the antibody and antibody sites of the invention can be used to treat various lung disorders' including adult respiratory distress syndrome (see, for example, PCT Bulletin No. 〇91/04054), pulmonary shock, chronic pulmonary inflammatory disease, pulmonary sarcoma, pulmonary fibrosis And bauxite deposits. The antibody or antibody site can be administered systemically or locally to the surface of the lung, e.g., as an aerosol. G. Intestinal disorders Tumor necrosis factor is associated with the pathology of inflammatory bowel disease (see, for example, Tracey, KJ, et al. (1986) Science 214: 470-474; Sun Χ·Μ., et al. (1988) J. Clin. Invest. 81 : 1328-1331 *

MacDonald, T.T., et al. (1990) Clin. Exp. Immunol. Red: 301-305). The anti-hTNFa antibody has been clinically tested for Cologne's disease (van Dullemen, HM, et al. (1995) Gastroenterology: 129_13 5). The human antibodies and antibody sites of the present invention can also be used to treat intestinal disorders such as Idiopathic inflammatory bowel disease 'It contains two syndromes: Cologne's disease and colon ulcer. H. Heart Disorders The antibodies and antibody sites of the invention can also be used to treat various heart disorders, scoops

[S 149555.doc -50· Contains cardiac anemia (see, for example, European Patent Publication EP 453 898) and cardiac insufficiency (cardiac muscle weakness) (see, for example, PCT Bulletin No. WO 94/20139) » I. Other inventions Antibodies and antibody sites can also be used to treat a variety of other disorders in which TNFa activity is detrimental. Examples of other diseases and disorders in which TNFa activity is associated with pathology and which can be treated using the antibodies or antibody sites of the invention include inflammatory bone disorders and bone resorption diseases (see, for example, Bertolini, DR, et al. (1986) Nature 3j. 9: 5 16-5 18 ; Konig, A., et al. (1988)]·

Bone Miner. Res. 1: 621-627; Lerner, UH and Ohlin, A. (1993) J. Bone Miner· Res. 147-155; and Shankar, G. and Stern, PH (1993) Bone ϋ : 871- 876) Hepatitis comprises alcoholic hepatitis (see, for example, McClain, CJ and Cohen, DA (1989) Hepatology 349-351; Felver, ME, et al. (1990) Alcohol. Clin. Exp.

Res. ϋ: 255-259; and Hansen, J., et al. (1994) Hepatology Welcome: 46 474) and Viral Hepatitis (Sheron, N., et al. (1991) Journal of Hepatology 12: 241-245 And Hussain, MJ, et al. (1994), (: 1111· Pathol·1112-1115), condensation jk interference (see, for example, van der Poll, T., et al. (1990) N. Engl. J. Med_ 32 ^ : 1622-1627 ; and van der Poll. T. et al. (1991) Prog. Clin. Biol. Res. 367. : 55-60), burns (see, for example, Giroir, BP, et al. (1994) Am. J. Physiol. Ml: H118-124; and Liu, XS, et al. (1994) Burns. 40-44), Reperfusion injury (see, for example, scaies, wE, et al. (1994) Am. J. Physiol. 267: G1122-1127; Serrick, C., et al. (1994) Transplantation Society: 1158-1162; and Yao, YM., et al. (1995) 149555.doc -51- 1353852

Resucitation 29_: 157-168), neoplasia (see, for example, McCauley R.L. (1992) J. Clin. Immunol.: 300-308), formation of flash marks, and fever. The invention is further illustrated by the following examples, which should not be construed as limiting. All references, patents and published patent applications cited in this specification are hereby incorporated by reference. Example 1: Efficiency of D2E7' Subcutaneous Administration with Anti-TNFa Antibody In this study, 24 patients with RA received a dose of 0.5 mg/kg D2E7 (n=18) or placebo (n=6) per week. Treated by sc injection for 3 months. The study of patients enrolled in this study had an average age of 10.1 years and a disease index (D a §) of 4.87 and an average of 3.4 DMARDs (disease-modifying anti-rheumatic drugs); again reflecting comparable disease activity. The sensor continued to receive D2E7 open-label treatment, while patients who did not respond to the 0.5 mg/kg dose or who lost the DAS response to the 05 mg/kg dose were given a booster dose at week 12 of the study to s c. injected j mg/kg. The first group of patients was enrolled for up to 60 injections and the study drug was therefore received for 6 weeks. Up to 78% of patients achieved DAS and ACR2〇 responses during the first week of treatment. Subcutaneously administered at a dose of 0.5 mg/kg/week £2) can be reduced by 54 in 12 weeks. /. Joint expansion (SWJ), softened joint number (TJC) 61%, and cRp were 39% (compared to baseline), while all parameters in the placebo group increased. After completion of the placebo control during the study, the patient continued to be treated with sustained efficiency (4) solid month. These results therefore show that subcutaneous administration of D2E7 at a dose of 55 mg/kg/week can safely self-administer the drug with good local tolerance. 149555.doc -52- D2E7 and Aminoguanidine Administration In this study, in addition to its initial treatment with (aminopterin) MTX, a dose of 1 mg / kg sc or iv administered placebo or D2E7. 54 diseases Patients enrolled in the study and 18 patients received iv received D2E7 and sc received placebo, 18 patients received placebo and sc received D2E7 and 18 iv and sc received placebo. The patient received the second dose only after losing her blind response, no earlier than 4 weeks after the first dose. Subsequently, all patients received an open label for biweekly s.c. injection of D2E7. The demographic characteristics of the study included an average RA period of 11 · 1 years, an average of 3.6 DMARDs before exposure (non-MTX), and an average DAS of 4.81 when included in the study. On day 29, 72% of i.v. D2E7 treated patients and 44% of s.c. D2E7 treated patients had reached the DAS standard response compared to only 28% of placebo-treated patients (described in Figure 5). Of the participants in the study, 28% of placebo-treated patients maintained ACR20 response on day 29 compared to 72% of iv-treated D2E7 patients and 67% of sc-treated D2E7 patients, 1 and 3 Maintain its response during the month. Example 2: Total human dose of subcutaneous administration of anti-TNFoc antibody D2E7 was administered subcutaneously weekly. This study included 284 patients with RA and was designed to determine the optimal total human dose for subcutaneous administration of D2E7. Patients were randomized to receive 20, 40 or 80 mg of D2E7 or placebo for 12 weeks per week, and then placebo-treated patients were blindly switched to 40 mg D2E7/week. About 49% of patients had ACR20 at 20 mg, 55% of patients had ACR20 at 40 mg and 54% of patients had ACR20 at 80 mg, and only 10% received placebo 149555.doc -53-1353852. Figure 1A). Approximately 23% of patients had ACR50 at 20 mg, 27% had ACR50 at 40 mg, and 20% had ACR50 at 80 mg, and only 2% received placebo for ACR50. These data indicate that subcutaneous administration of D2E7 (especially at a dose of 40 mg/week) produces a good response. Example 3: Biweekly subcutaneous administration of anti-TNFot antibody to subcutaneously administered D2E7 in two doses at several doses per week subcutaneously (sc) placebo or D2E7 for 24 weeks and continued MTX treatment to explore clinical efficacy, safety, immunogen And tolerance to RA patients who are potentially responsive to MTX. The study was designed for placebo-controlled, double-blind, randomized, rehospital studies of RA patients with inadequate or tolerable MTX. During the trial, the patient's sustained MTX dose and dose range were included in the specific inclusion criteria below. The study consisted of two parts: 1) a "washout period" 4 weeks prior to the first dose of the drug, during which DMARDs were excluded (except for MTX); and 2) a "placebo-controlled period" during which the patient was randomly assigned to 67 One-fourth of patients received 20, 40 or 80 mg of D2E7 (total human dose) administered every other week for 24 weeks. The doses of each study drug were administered by two s.c. injections of 1.6 ml each. The first dose of the patient is administered by a medical practitioner as part of the patient training. Subsequent doses were self-administered by the participating patients under the direct surveillance of the first 4 weeks. Subsequently, the dose is administered by the patient, the patient designated by the patient, or by a medical professional outside the study site. Dosing for 4 or 5 weeks was assigned after each clinical assessment. On the 1st, 2nd, 3rd, 4th, 6th, 8th, 12th, 16th, 149555.doc -54- 1353852% of the study, 2 weeks and 24 weeks of continuous examination of the patient' while the joint examination was performed by the unidentified assessor The doctor has nothing to do. This study included 271 RA patients. The study population is representative of the moderate to severe RA population in North America: approximately 70% of women and most of them over 4 years of age. The population is selected using predetermined inclusion and exclusion criteria (as known to those skilled in the art), such as patients requiring RA diagnosis, such as the 1987-Revised American College Rheumatology (ACR) standard (described in Appendix A). Results Figures IB and 2-4 show that biweekly administration of D2E7 with an amine subcutaneously was significantly superior to placebo in reducing RA signals and signs after 24 weeks. All three D2E7 dose effects were statistically significantly better than placebo administered weekly. Furthermore, 4 mg and 80 mg D2E7 have superior effects than the 20 mg dose. Equivalents It will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; This equivalent is intended to be included in the scope of the present invention. 149555.doc 55· 1353852 RA ACR Definitions Classification tree criteria and functional criteria for rheumatoid arthritis (RA) in 1987 Definition 1.3 or more joint areas of arthritis Observed by a physician at least 3 joints with soft tissue expansion or Fluid (not just bone overgrowth). 14 possible joint areas are right or left PIP, MDP, wrist, knee, ankle and MTP joints 2. Hand joint arthritis Wrist joint MCP MCP or wrist joint MCP and wrist joints are observed by physicians with soft tissue expansion or fluid (not just bone overgrowth). There are 2 specific areas that need to be included at the same time. 3. Symmetrical swelling (arthritis), including the same joint area on both sides of human Zhao (including bilateral PIPs, MCP'^MTPs g and not absolutely symmetrical) (eg 丨中定4. Serum rheumatism index number^ Constant 4, the result 5. Rheumatoid arthritis X-ray changes j wet joint χ _ light map typical change ΐ ί ϋ ί in or most adjacent to the involved arthritis, or non-suspicious bone road separation classification (Only bones and joints • If the patient is included in the $·ρ Δ Λ listed in Table 7, &lt; SRA subgroup and especially the clinical diagnosis of RA, the patient is suffering from ra. ^Huai, ^铋准1 , 2 and 3 need to last for at least 6 weeks.

Arthritis &amp; Rheumatism' Volume 31, ^ 3 (March 1988) [Simple description of the diagram] Figure 1A and Figure 1B show that suffering from wind-thirsty Guan Ming* m / ·, f-sheng is inflammation (RA) The patient was administered subcutaneously with antibody D2E7 for 12 weeks (1 Α, total ..β, parental administration with antibody DZE7 and cyanmethoxime for 24 weeks) (After the lm order, the American University of Rheumatology 2 〇 ACR20) and ACR50 reaction. The number of wall flips _ a temple number media is not every week to be administered as | weekly drug-effective. Figure 2 shows patients with RA subcutaneously administered antibody bile and 氦149555.doc • 56 · 1353852 ACR20, ACR50, and ACR70 responses 24 weeks after sputum. Figures 3A and 3B show weak joint counts 24 weeks after subcutaneous administration of D2E7 and amoxime in patients with RA ( 3 A) and the expansion joint count (3B) over the course of 24 weeks. Figure 4 shows the short-term health examination results of patients with RA after subcutaneous administration of D2E7 and methotrexate every other week (SF-36) RP, physiological role; PF, physiological function; BP, physical pain; GH, general health; V, vitality; SF, social function; RE, Emotional role; and ME, emotional health.

Figure 5 shows the percentage of ACR responders after single intravenous injection of antibody D2E7 and ammonia in patients with RA.

149555.doc 57-

Claims (1)

135385, 099124845 Patent Chinese patent application for seven, patent application --T^10^7 Riba/3 early from 1. Α ^ «The use of the body or its antigen-binding medicinal composition, 1 in the field ^ I # is used to prepare drugs for the treatment of intestinal diseases, wherein the drug is biweekly Hepatic and bacteriological to treat the disease, and wherein the pharmaceutical composition comprises a human anti-TNFot antibody or an antigen 8 binding site thereof, and the pharmaceutical composition is suitable a dosage form administered subcutaneously in a biweekly manner, and the human antibody or antigen-binding site thereof is κ^ΐχΐ〇 Μ or below and Κ. &quot;Rate constant ··乌〗 χ 1 Λ·3 -丨&gt; f heart dry * number of horses 1X10 s丨 or below from human Ding ^^ to dissociate (both by surface slinky Qiu Zhenzhong, 曰 you W plasma 搌 搌), and in vitro L929 analysis standard lxl 〇 7M or below Φ> ί i 4 to the ICso neutralized human TNFa cytotoxicity. 2. The use of the scope of the patent application, wherein the human antibody or its antigen binding site is K. &quot; Rate constant is 5x 1〇-4 s-i or below dissociated from human TNFa. 3. The use of the first aspect of the patent application, wherein the human antibody or its antigen binding site is ruthenium. The rate constant is 1 X 1 〇·4 s-丨 or below dissociated from human TNFot. 4. The use of claim 1, wherein the human antibody or antigen binding site thereof is neutralized by human TNFa cytotoxicity in an in vitro L929 assay standard with an IC50 of 1 χΐ〇 8 M or less. 5. The use of claim 1, wherein the human antibody or antigen binding site thereof is neutralized by human TNFa cytotoxicity at an IC50 of 1x1 9·9 μ or less in the in vitro L929 assay standard. 6. The use of the first aspect of the patent application 'where the human antibody or its anti-149555-1000715.doc 1353852 original binding site is in the in vitro L929 assay standard and neutralizes human TNFct with an IC50 of lx1〇-i〇M or below. Cytotoxicity. 7. The use of the first aspect of the patent application. The human antibody or its anti-pro-binding site is a recombinant antibody or a recombinant antigen binding site thereof. 8* Use of a pharmaceutical composition for the preparation of a medicament for inhibiting human TNFa activity in a human subject having TNFa activity which is a harmful intestinal disease, wherein the drug is administered subcutaneously to a human subject in a biweekly course of treatment, Wherein the pharmaceutical composition comprises 20 to 80 mg of a human anti-TNFa antibody or antigen-binding site thereof, and wherein the human antibody or antigen-binding site thereof has the following characteristics: a) by surface electropolymerization resonance measurement. (^ is 1 X1 〇·3 s.1 or below dissociated from human TNFot; b) has a light chain CDR3 region comprising the following amino acid sequence: amino acid sequence of SEQ ID NO: 3 or substituted by a single alanine at position 1, 4, 5, 7 or 8 or by 1 to 5 conservative amino acids substituted amino acid sequence modified from position number 3 at positions 1, 3, 4, 6, 7, 8 and/or 9; And c) having a heavy chain CDR3 region comprising the following amino acid sequence: the amino acid sequence of the sequence number 4 or substituted by a single alanine at positions 2, 3, 4, 5, 6, 8, 9, 10 or 11 or an amino acid sequence modified from SEQ ID NO: 4 at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12 by 1 to 5 conservative amino acids. 9. The use according to claim 8, wherein the human anti-TNFa antibody or antigen-binding site thereof is dissociated from human TNFa with a K()ff rate constant of 5xl〇4 or less. 149555-1000715.doc 1353852 I 10. Use of a pharmaceutical composition for the preparation of a medicament for inhibiting human TNFa activity in a human subject having intestinal diseases harmful to TNFa activity, wherein the medicament is administered in a biweekly manner Subcutaneously administering a pharmaceutical composition comprising 20 to 80 mg of a human anti-TNFa antibody or antigen-binding site thereof and wherein the human antibody or antigen-binding site thereof has an amino acid sequence comprising SEQ ID NO: 3 or a single propylamine Acid-substituting the light chain variable region (LCVR) of the CDR3 region modified from SEQ ID NO: 3 at position 1, 4, 5, 7 or 8 and having an amino acid sequence comprising SEQ ID NO: 4 or substituted with a single alanine Heavy chain variable region (HCVR) of the CDR3 region modified from SEQ ID NO: 4 at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. 11. The use of claim 1, wherein the LCVR of the human antibody or antigen binding site thereof further comprises a CDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the HC VR further comprises an amine comprising SEQ ID NO: 6. The CDR2 region of the base acid sequence. 12. The use according to claim U, wherein the LCVR of the human antibody or antigen binding site thereof further has a CDR1 region comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR further comprises an amino acid comprising SEQ ID NO: 8. The CDR1 region of the sequence. 13' Use of a pharmaceutical composition for the preparation of a medicament for inhibiting human TNFa activity in a human subject having an intestinal disease in which τΝΡα activity is harmful, wherein the drug is administered subcutaneously in a biweekly treatment, wherein the medicament The composition comprises 20 to 80 mg of a human anti-TNFa antibody or antigen binding site thereof and wherein the human antibody or antigen binding site thereof has a light chain variable region (Lcvr) comprising 149555-1000715.doc 1353852 and a package Heavy chain variable region (HCVR). Use of the human antibody having the amino acid sequence of SEQ ID NO: 1 comprising the amino acid sequence of SEQ ID NO: 2, as claimed in claim 13 of the IgGl heavy chain constant region. 15. If the use of the third paragraph of the patent scope is cold, the use of 冷, 疋 疋 疋 其中 该 该 该 该 该 该 该 该 该 该 该 该 该 该 该 该 该 该 人类 人类 人类 人类Wherein the human antibody is a Fab 16. The use of the article of claim 13 of the patent application, wherein the human antibody is a single-chain Fv fragment. 1 8. Use of a pharmaceutical composition for the preparation of a medicament for inhibiting human TNFa activity in a human subject having intestinal diseases harmful to τΝρα activity, wherein the medicament is administered subcutaneously in a biweekly treatment, wherein The pharmaceutical composition comprises 20 to 80 mg of a human anti-TNFa antibody or antigen binding site thereof, and wherein the human antibody or antigen binding site thereof comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 1, SEQ ID NO:1 3. Sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, and The light chain variable region (LCVR) of the CDR3 region of the amino acid sequence of the SEQ ID NO: 26, or having the sequence consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 3〇, SEQ ID NO: 3 1, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34 The heavy chain variable region of the CDR3 region of the amino acid sequence of the group 149555-1000715.doc 1353852 I (HCVR). 19. Use of a pharmaceutical composition comprising a D2E7 antibody or an antigen binding site thereof for the preparation of a medicament for inhibiting human TNFa activity in a human subject having intestinal diseases harmful to TNFa activity, wherein the drug is biweekly The treatment is administered subcutaneously, and wherein the pharmaceutical composition comprises from 2 to 8 mg of the D2E7 antibody. 20. The use of the intestinal disease is inflamed as in the scope of claims 1, 8, 1, 13, 18 or _ Sexual bowel disease. 21. The use of claim 20, wherein the inflammatory bowel disease is Cologne's disease or colon cancer A pharmaceutical composition for treating intestinal diseases, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a 20-80 mg dose of a single human anti-TNFa antibody or antigen-binding site thereof, and wherein the pharmaceutical composition is suitable A bi-weekly subcutaneous administration to a dosage form of a human individual and wherein the human antibody or antigen-binding site thereof has a Kd of 1 X 1 〇·8 Μ or less and a ruler. The rate constant is lx ΙΟ·%·1 or less dissociated from human TNFa (both measured by surface plasma resonance), and neutralized human TNFa cells with IC50 of lxl〇.7 M or less in the L929 assay standard in vitro. toxicity. A pharmaceutical composition according to claim 22, wherein the human antibody or antigen-binding site thereof is dissociated from human TNFa with a Kw rate constant of 5X 1 〇·4 s-i. 24. The pharmaceutical composition of claim 22, wherein the human antibody or antigen binding site thereof is dissociated from human TNFa with a Kw rate constant of ixl 〇 -4 s '丨 or below. 149555-1000715.doc 1353852 25. 26. 27. 28. 29. The pharmaceutical composition of claim 22, wherein the human antibody or antigen binding site thereof is in the in vitro L929 analytical standard at 1χ1〇·8 M Or the following ICS0 neutralizes human TNFa cytotoxicity. The pharmaceutical composition according to claim 22, wherein the human antibody or antigen-binding site thereof is neutralized by human TNFa cytotoxicity by ICso at 1 X 1 〇·9 M or less in the in vitro L929 assay standard. The pharmaceutical composition according to claim 22, wherein the human antibody or antigen-binding site thereof is neutralized by human TNFa cytotoxicity with lxi〇-iQ M or below in the L929 assay standard. The pharmaceutical composition according to claim 22, wherein the human antibody or antigen-binding site thereof is a recombinant antibody or a recombinant antigen binding site thereof. The pharmaceutical composition according to claim 22, wherein the human anti-TNFa antibody or antigen-binding site thereof has the following characteristics: a) dissociation from human TNFa by si or the following as determined by surface plasma resonance; b) having the following The light chain CDR3 region of the amino acid sequence: the amino acid sequence of SEQ ID NO: 3 or substituted by a single alanine at position 1, 4, 5, 7 or 8 or |fl to 5 conservative amino acid substitutions at position 1 , a 3, 4, 6, 7, 8 and/or 9 amino acid sequence modified from SEQ ID NO: 3; and c) a heavy chain CDR3 region comprising the following amino acid sequence: amino group of SEQ ID NO: Acid sequence or substitution by single alanine at position 2, 3, 4, 5, 6, 8, 9, 1 or 11 or by 1 to 5 conservative amino acids at positions 2, 3, 4, 5, 6 Amino acid sequence modified from SEQ ID NO: 4, 8, 9, 10, 11 and/or 12. The pharmaceutical composition of claim 29, wherein the human anti-TNFa antibody or antigen-binding site thereof has a Keff rate constant of 5xl〇-4 s-1 or less from human TNFa. Dissociation. 31. The pharmaceutical composition of claim 22, wherein the human anti-TNFa antibody or antigen binding site thereof has an amino acid sequence comprising SEQ ID NO: 3 or substituted with a single alanine at positions 1, 4, 5, Light chain variable region (LCVR) of the CDR3 region modified from SEQ ID NO: 3 and having an amino acid sequence comprising SEQ ID NO: 4 or substituted with a single alanine at positions 2, 3, 4, 5' 6, 8, 9' 1 or 11 and the heavy chain variable region (Hcvr) of the CDR3 region modified from SEQ ID NO: 4. 32. The pharmaceutical composition of claim 31, wherein the LCVR of the human antibody or antigen binding site thereof further has a CDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the HCVR further comprises SEQ ID NO: 6. The CDR2 region of the amino acid sequence. 33. The pharmaceutical composition of claim 32, wherein the LCVR of the human antibody or antigen binding site thereof further has a CDR1 region comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR further comprises an amine comprising SEQ ID NO: 8. The CDR1 region of the base acid sequence. 34. The pharmaceutical composition of claim 22, wherein the human anti-TNFa antibody or antigen binding site thereof has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and comprises a sequence number The heavy chain variable region (HCVR) of the amino acid sequence of 2. 35. The pharmaceutical composition of claim 34, wherein the human antibody has an IgGl heavy chain constant region. 149555-I000715.doc 1353852 36. The pharmaceutical composition of claim 34, wherein the human antibody has an IgG 4 heavy bond region. 37. The pharmaceutical composition of claim 34, wherein the human antibody is a Fab fragment. 38. The pharmaceutical composition of claim 34, wherein the human antibody is a single chain F v fragment. 39. The pharmaceutical composition of claim 22, wherein the human anti-TNFoc antibody or antigen binding site thereof comprises a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, Group No. 15, Sequence No. 16, Sequence Number 17, Sequence Number 18, Sequence Number 19, Sequence Number 20, Sequence Number 21, Sequence Number 22, Sequence Number 23, Sequence Number 24, Sequence Number 25, and Sequence Number 26 a light chain variable region (LCVR) of the CDR3 region of the amino acid sequence of the group, or having a sequence selected from SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28 SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 The heavy chain variable region (HCVR) of the CDR3 region of the amino acid sequence of the SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. 40. The pharmaceutical composition of claim 22, wherein the human anti-TNFa antibody or antigen binding site thereof is a D2E7 antibody or antigen binding site thereof. The pharmaceutical composition according to any one of claims 22 to 40, which comprises a 40 mg dose of a human anti-TNFa antibody or an antigen binding site thereof. 42. The pharmaceutical composition of any one of claims 22 to 4, wherein the intestinal disease is an inflammatory bowel disease. 149555-1000715.doc 1353852 The pharmaceutical composition of claim 4, wherein the inflammatory bowel disease is a Cologne disease or a colon ulcer, contains a formulation comprising: a The pharmaceutical composition according to any one of claims 22 to 43; and b) the pharmaceutical composition for subcutaneous administration of the pharmaceutical composition for biweekly treatment of intestinal diseases. 45_ The kit of claim No. 1, wherein the intestinal disease is an inflammatory bowel disease. 46. The kit of claim 45, wherein the inflammatory bowel disease is Cologne's disease or colon cancer. 47. A preloaded syringe&apos; is a composition comprising 20 to 80 mg of a human anti-TNFa antibody or antigen binding site thereof and a pharmaceutically acceptable carrier. ' 48, For the syringe of the 47th patent application, apply for any combination of the first to the 18th. Wherein the human antibody comprises the antibody or antigen thereof as described in 149555-1000715.doc