Infectious Diseases – Steven A. Fischkoff, Joachim Kempeni, Roberta Weiss, AbbVie Biotechnology Ltd

Abstract for “Methods for administering anti-TNF?” antibodies”

“Methods for treating disorders that TNF is involved in” Subcutaneous administration of human antibodies to treat TNF-related disorders is possible. (hTNF?) are disclosed. You can administer the antibody with or without methotrexate. These antibodies are highly sensitive to hTNF. These antibodies have a high affinity for hTNF (e.g. Kd=10.8 M or lower), and a slower off rate for hTNF Koff=103 sec?1 or lower) to dissociate hTNF. Activity in vitro as well as in vivo. An antibody according to the invention may be either a full-length or antigen-binding component. The invention also includes kits containing a pharmaceutical composition, instructions for dosing, as well as preloaded syringes that contain pharmaceutical compositions.

Background for “Methods for administering anti-TNF?” antibodies”

“Tumor necrosis Factor?” (TNF?) This cytokine is produced by many cell types, including macrophages and monocytes. It was first identified because it induces necrosis in certain mouse tumors (see Old, L. (1985). Science 230:630-632). A factor called cachectin was later identified as the same molecule that TNF?. TNF? TNF has been implicated as a mediator of shock (see, e.g. Beutler, B., and Cerami A. (1988) Annu. Rev. Biochem. 57:505-518. Beutler, B., and Cerami. (1989) Annu. Rev. Immunol. 7:625-655). Furthermore, TNF? TNF has been implicated as a pathophysiology for a number of human diseases and disorders including infections, autoimmune disease, transplant rejection, and graft-versus host disease (see, e.g. Vasilli, P. (1992). Annu. Rev. Immunol. 10:411-452 Tracey, K. J., and Cerami A. (1994) Annu. Rev. Med. 45:491-503).”

“Murine anti-hTNF was created to address the issues associated with fully-murine human antibodies. Genetically engineered antibodies can be made to look more human-like. Chimeric antibodies have been made, where the variable regions of the antigen chains are murine-derived while the constant regions are human-derived (Knight, D. M. et al. (1993) Mol. Immunol. 30:1443-1453, PCT Publication Number. WO 92/16553 by Daddona, P. E., et al.). Humanized antibodies have been also prepared. The hypervariable domains in the antibody variable regions of the antibody variable are murine-derived, but the rest of the variable regions as well as the antibody constant regions, are human-derived. WO 92/11383 by Adair, J. R., et al.). These chimeric and humanized antibodies still contain some murine sequences. They may still elicit an undesirable immune reaction, the human-anti-chimeric antibody reaction (HACA), especially when given for long periods of time, as in the case of chronic indications such as rheumatoid. (see Elliott, M.J., et. al.). (1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet 344:1105-1110).”

“A preferred hTNF?” An entirely human anti-hTNF would be an inhibitory agent for murine mAbs and derivatives thereof (e.g. chimeric or humanized antibody). HAMA reaction should not be elicited by such an agent, even if it is used for long periods. Monoclonal human autoantibodies against the hTNF These were prepared using human hybridoma methods (Boyle P. et al. (1993) Cell. Immunol. 152:556-568; Boyle, P., et al. (1993) Cell. Immunol. 152:569-581; European Patent Application Publication No. 614 984 A2 by Boyle, et al.). These monoclonal, hybridoma-derived monoclonal antibodies had an affinity for hTNF. This was not possible to calculate using conventional methods. They were also unable to bind soluble HTNF. They were not able to neutralize hTNF-induced cytotoxicity (see Boyle et al. ; supra). The success of the human hybridoma procedure depends on the presence of human peripheral blood lymphocytes that produce autoantibodies against hTNF?. Certain studies have shown that serum antibodies against hTNF were detected in certain individuals. in human subjects (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog. Leukocyte Biol. 10B:447-452), while others have not (Leusch H-G. (1991) J. Immunol. Methods 139,145-147)

“An alternative to the naturally occurring human anti-hTNF?” A recombinant human anti-hTNF would be an antibody. antibody. Recombinant antibodies from human that bind to hTNF Griffiths, A. D. et al. have described antibodies that bind hTNF with a low affinity (Kd?107M) and a rapid off rate (Koff?102 sec?1). (1993) EMBO J. 12:725-734). These antibodies are not suitable for therapeutic purposes due to their fast dissociation kinetics. A recombinant anti-hTNF human antibody? It has been shown that it does not neutralize the hTNF. activation, but instead enhances binding of the hTNF enhances hTNF internalization and binds to cells’ surfaces (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45; Publication No. WO 92/03145 (Aston, R. and al.)

“Recombinant human antibodies which bind soluble HTNF With high affinity and slow dissociation rates and the ability to neutralize hTNF Activity, including hTNF-induced cytotoxicity in vitro and intracellularly, and hTNF-induced cell activation have been also described (see U.S. Pat. No. 6,090,382). The usual protocol for administering antibodies intravenously is performed on a weekly basis. The frequency of administering anti-/or drug therapy can make it more expensive and cumbersome. Intranasal administration has its limitations, as it is typically administered by someone who has medical training.

“The present invention allows for biweekly dosing regimens to treat TNF. Subcutaneous route is preferred for these disorders. There are many benefits to biweekly dosing, including a reduced number of injections, less injection site reactions (e.g. local pain and swelling), patient compliance (i.e. due to fewer injections), and lower costs for the patient and the provider. Subcutaneous dosing can be advantageous as the patient may administer a therapeutic substance (e.g., human TNF) themselves. This is convenient for both the patient as well as the health care provider.

“This invention offers methods to treat disorders that TNF is involved. Activity is harmful. These methods include subcutaneous injections of antibodies that are administered biweekly to a subject. Preferably, the antibodies are recombinant human antibodies which specifically bind to human thyroid hormone. This invention also provides methods to treat disorders that TNF? TNF is a dangerous agent. Combination therapy is where human antibodies are administered to a subject along with another therapeutic agent. This could include one or more antibodies that bind to other targets (e.g. antibodies that bind to cell surface molecules or cytokines), or one or more cytokines. receptor (see, e.g. PCT Publication Number. WO 94/06476), and/or one or several chemical agents that inhibit the hTNF. Production or activity (such cyclohexaneylidene derivatives, as described in PCT Publication No. WO 93/19751), preferably methotrexate. Preferably, the antibodies are recombinant human antibodies which specifically bind to human TNF’. The invention’s antibodies are known to be able to bind to hTNF? With high affinity and slow dissociation rates and neutralizing hTNF activity, including in vitro and in-vivo hTNF-induced cytotoxicity and cellular activation. You can have full-length antibodies (e.g. an IgG1 and IgG4 antibodies) or a portion that is antigen-binding (e.g. a F(ab?) or Fab). )2, scFv fraction or a single domain. D2E7 is the most popular recombinant antigen of the invention. It has a light-chain CDR3 domain that contains the amino sequence of SEQID NO: 3. A heavy-chain CDR3 domain comprises the amino sequence of SEQID NO: 4. (Apendix B). The D2E7 antibody should have a light-chain variable region (LCVR), which contains the amino acids sequence of SEQID NO: 1, and a heavy-chain variable region (HCVR), which includes the amino sequence of SEQU ID NO. 2. These antibodies are described by U.S. Pat. No. No. 6,090,382, incorporated herein in its entirety by reference.”

“In one embodiment, this invention provides methods for treating disorders that TNF is involved. activity is harmful. Inhibiting human TNF is one of these methods. Subcutaneous, biweekly administrations of anti-TNF can be used to inhibit human TNF? The disorder can be treated with an antibody. It could be sepsis, an immune disorder (e.g., rheumatoid, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis, and nephrotic disorder), or an infectious disease.

“Another embodiment of the invention offers methods for treating disorders that TNF is involved. activity is harmful. These methods include blocking human TNF? Subcutaneous administration of anti-TNF can be used to inhibit human TNF? The disorder can be treated with methotrexate and antibody. One aspect of methotrexate administration is that it is combined with anti-TNF. antibody. Methotrexate can be administered before anti-TNF is administered. antibody. Another aspect is that methotrexate can be administered after anti-TNF administration. antibody.”

“In another embodiment, the invention offers methods for treating disorders in which TNF is involved. Activity is reduced by subcutaneous, biweekly administration of a human antibody or an antigen-binding component to the subject. Preferably, the antibody or antigen binding portion of it has the following characteristics:

“b”) has a light-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 3. It can be modified from SEQID NO: 3 by one alanine substitution at position 1, 4, 5 or 8, or by one to five conservative amino acids substitutions at positions 1, 3, 4, 6, 7 and/or 9.

“c” has a heavy-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 4. Modified from SEQ ID No: 4 by one alanine substitution at position 2, 4, 5, 6, 8, 9 or 11, or by one to five conservative amino acids substitutions at positions 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

“In another embodiment, the invention provides methods for treating disorders that TNF is involved. Activity is harmful. The methods include subcutaneous administration of a human antibody or its antigen-binding component to the subject every two weeks. An LCVR with a CDR3 Domain containing the amino acids sequence of SEQID NO: 3, modified from SEQID NO: 3 by a single alanine substitut at position 1, 4, 5, 7, 8 or 8. A HCVR with a CDR3 Domain comprising the amino sequence of SEQ ID NO 4 or modified at position SEQID NO: 4 by a single alanine substitution at position 2, 3, 4, 6, 8, 9, 10, or 11. The LCVR has a CDR2 Domain that contains the amino sequence of SEQID NO: 5, while the HCVR has a CDR2 Domain that includes the amino sequence of SEQUID NO: 6. Even more preferable, the LCVR also has a CDR1domain that contains the amino sequence of SEQ ID NO. 7 and the HCVR has a CDR1domain that comprises the amino sequence of SEQ ID NO. 8.

“In another embodiment, the invention offers methods for treating disorders in which TNF is involved. Subcutaneous administration of an antibody or antigen-binding portion to the subject biweekly can be used to reduce activity. Preferably, the antibody or antigen binding portion of the antibody contains an LCVR that includes the amino acids sequence of SEQID NO: 1 and an HCVR that includes the amino acids sequence of SEQUID NO: 2. In some embodiments, the antibody contains an IgG1 heavy chain constant area or an IgG4 high chain constant region. Another embodiment of the antibody includes a Fab fragment or an F(ab) fragment. A single chain Fv fragment, or a Fab fragment can be used.

“In other embodiments, the invention offers methods for treating disorders that require the administration of anti-TNF. Subcutaneously administering one or more antiTNF to the subject biweekly is a good way to obtain an antibody. Antibodies, or antigen binding portions of them. Preferably, the antigen-binding or antibody portion of the LCVR has a CDR3 domain that contains an amino sequence chosen from the following groups: SEQID NO. 3, SEQID NO. 11, SEQID NO. 13, SEQID NO. 14, SEQID NO. 15, SEQID NO. 16, SEQID NO. 17, SEQID NO. 18, SEQID NO. 23, SEQID NO. 22, SEQID NO. 23, SEQID NO. 24, SEQID NO.

“Another aspect of the invention is kits that contain a formulation containing a pharmaceutical composition. Kits contain an anti-TNF. The kits contain an anti-TNF? antibody and a pharmaceutically acceptable carrier. Instructions for subcutaneous administration of the pharmaceutical composition to treat a disorder requiring anti-TNF injection are included in the kits. It is advantageous to use antibody. Another aspect of the invention is kits that contain a pharmaceutical composition and an anti-TNF. anti-TNF?, methotrexate and a pharmaceutically acceptable carrier. Instructions for subcutaneous administration of the pharmaceutical composition are included in the kits. It is very beneficial to have an antibody.

“Another aspect of the invention is a preloaded needle containing a pharmaceutical compound containing an anti-TNF. anti-TNF and a pharmaceutically acceptable carrier. Another aspect of the invention is a preloaded needle containing a pharmaceutical composition containing anti-TNF. “Antibody, methotrexate, as well as a pharmaceutically acceptable carrier.”

“This invention relates to treatments for disorders that require the administration of anti-TNF. It is possible to administer isolated human antibodies or antigen-binding components of antibody that binds to human TNF. This is called an antibody. The treatment of the disorder can be achieved by using antibodies that have high affinity, low off rates and high neutralizing capacities. The invention relates to the treatment of antibodies and fragments of antibodies, as well as pharmaceutical compositions.

“For the invention to be easier to understand, certain terms must first be defined.”

“Dosing” is the act of administering a substance (e.g. an anti-TNF). “Anti-TNF? treatment) in order to reach a therapeutic goal.

“Biweekly dosing schedule?,??biweekly dose? and?biweekly administering? refer to the time it takes to administer a substance (e.g. an anti-TNF). antibodies) to a subject in order to achieve a therapeutic goal (e.g. the treatment of a TNF-associated disorder). A weekly dosing schedule is not recommended for the biweekly regimen. The substance should be administered approximately every 9-19 days. It is more preferable to administer the substance every 11-17 days. Even more preferably every 13-15 days. Most preferably every 14 days.

“Combination therapy” is the administration of two or more therapeutic drugs, e.g. an anti-TNF. combination therapy includes the administration of anti-TNF and methotrexate. You may administer methotrexate concurrently with, before, or after an anti-TNF injection. antibody.”

“The term “human TNF?” “Human TNF” is an acronym for a human cytokine. It is also known as hTNF or simply hTNF. The biologically active form is made up of a trimer consisting of 17 kD molecules that are noncovalently bound. What is the structure of TNF? Pennica, D., and others. provide more information. (1984) Nature 312:724-729; Davis, J. M., et al. (1987) Biochemistry 26.1322-1326. Jones, E. Y. et al. (1989) Nature 338, 225-228. What is the meaning of human TNF? Recombinant human TNF is also included in the term human TNF? (rhTNF? ), which can either be made using standard recombinant expression techniques or bought commercially (R & D Systems Catalog No. 210-TA, Minneapolis, Minn.).”

“Antibody” is a term that refers to immunoglobulin molecules made up of four polypeptide chains: two heavy (H), and two light (L), chains interconnected by disulfide bond. Each heavy chain is composed of a heavy-chain variable region (abbreviated as HCVR, VH or VH) along with a heavy-chain constant region. The three domains that make up the heavy chain constant area are CH1, CH2 or CH3. Each light chain is composed of a light-chain variable region (abbreviated as LCVR, VL or VL), and a light-chain constant region. One domain, CL, is the light chain constant region. You can further subdivide the VH and VL regions into regions of hypervariability (CDR), as well as regions that are conserved (FR). Each VH/VL is made up of four FRs and three CDRs. They are arranged in the following order from amino-terminus through carboxy-terminus: FR1, CDR1, CDR2, CDR2, CDR3, CDR3, and FR4.

“The term ‘antigen-binding part? An antibody (or simply the?antibody section?) is a portion of an antibody. As used herein, “antibody portion” refers to one or more fragments that can bind to an antigen. A fragment of a full length antibody can perform the antigen-binding function. The term “antigen-binding section” includes examples of binding fragments. A Fab fragment is a monovalent fragment that includes the VL and VH domains. It also contains the CL, CH1 and CL domains. A bivalent fragment that consists of two Fab fragments linked together by a disulfide link at the hinge region, and (iii), a F(ab) fragment. Although VL, and VH are distinct domains of Fv fragments, they can be linked using recombinant techniques to create a single protein chain in the form of monovalent molecules. This is known as single chain Fv (scFv); see Bird et. al. (1988) Science 242(4):423-426. Huston et. al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. These single-chain antibodies are also included in the term “antigen binding portion?” An antibody. Diabodies and other single-chain antibodies are also included. Bivalent, bispecific antibodies called dibodies contain VH and L domains. However, the linker is too short to permit pairing between the two domains. This creates two antigen binding site and forces them to pair with each other. (see Holliger, P. and al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2121-1123

“But, further, an antigen-binding or antibody portion of an antibody may form part of larger immunoadhesion molecule, which is formed by covalent or notcovalent association of the antibody/antibody portion with one or several other proteins or peptides. To make a tetrameric, scFv-molecule, one example of such immunoadhesion molecular is the use of the streptavidin central region (Kipriyanov S. M., and others). (1995) Human Antibodies and Hybridomas 6 :93-101. The use of a cysteine, marker peptide and C-terminal histidine tag to create bivalent and biotinylated ScFv molecules (Kipriyanov S. M. et al. (1994) Mol. Immunol. 31:1047-1058). F(ab) and Fab are antibody fragments. You can prepare Fab and F(ab)2 fragments from whole antibodies by using traditional techniques such as papain or pipsin digestion of whole antibodies. Standard recombinant DNA techniques can also be used to obtain antibodies, immunoadhesion molecules, and portions thereof, as discussed herein.

“Human antibody” is a term that refers to antibodies with variable and constant regions derived primarily from human germline immunoglobulin sequencings. The invention could include amino acid residues that are not encoded in human germline immunoglobulin sequencings. This includes mutations introduced by random, site-specific or somatic mutations in vitro or in vivo. The term “human antibody” as used in this invention does not include antibodies in which CDRs sequences derived from another mammalian species (e.g., a mouse) have been grafted onto human frames sequences.

“Recombinant human antibodies” is a term that refers to all human antibodies that have been prepared, expressed, created, or isolated using recombinant methods. This includes antibodies generated from a recombinant combination of human antibodies (described in Section H), antibodies obtained from a recombinant library of human antibodies (described in Section III below), and antibodies taken from animals (e.g. a mouse) that has been transgenic for human immunoglobulin gene (see Taylor, L. D. (1992) Nucl. Acids Res. 20:6287-6295). or antibodies that have been prepared, expressed, created, or isolated using any other method that involves the splicing human immunoglobulin gene sequencing to other DNA sequences. These recombinant antibodies are derived from the human germline immunoglobulin gene sequences and have constant and variable regions. However, certain embodiments of recombinant human antibodies are subject to in vitro mutation (or when an animal transgenic to Ig sequences for humans is used, in-vivo somatic mutation). The VH and L regions of the recombinant antibody sequences are sequences that are derived from and related with human germline VH sequences.

An “isolated antibody” is an antibody that is substantially devoid of antibodies with different antigenic specificities. It is significantly free from antibodies that specifically bind to antigens other hTNF? Is there an isolated antibody that binds specifically to hTNF However, cross-reactivity with other antigens such as hTNF may exist. molecules of other species (discussed below). An isolated antibody might be completely free from other cellular material or chemicals.

“A?neutralizing antibody? as described herein (or an antibody that neutralized the hTNF) activity? () is an acronym for an antibody that binds to hTNF This results in the inhibition of the biological activities of hTNF? What is the effect of this inhibition on the biological activity of hTNF You can measure one or more indicators for hTNF to assess the level of inhibition. biological activity such as hTNF-induced cytotoxicity in vitro and in vivo, hTNF-induced cell activation, and hTNF-binding to hTNF in vivo. receptors. These are indicators of hTNF. One or more of the many standard in vitro and in vivo tests that are available in the art can assess biological activity (see Example 4). The ability of an antibody neutralize hTNF is preferred. Activity is measured by the inhibition of hTNF-induced cytotoxicity in L929 cells. Another parameter that can be used to measure hTNF activity is the ability of an antibody to inhibit hTNF-induced expression of ELAM-1 on HUVEC. Activity can be measured by assessing the ability of an anti-hTNF antibody to inhibit hTNF-induced expression of ELAM-1 in HUVEC. This is a measure of hTNF-induced cellular activation.

“Koff” is the off rate constant that allows an antibody to be dissociated from its antibody/antigen complex.

“Kd” is the dissociation constant for a specific antibody-antigen interaction.

“Nucleic Acid Molecule” is a term that refers to both DNA molecules and RNA molecules. One-stranded or two-stranded nucleic acids molecules are possible, but it is preferred to have double-stranded DNA.

“Isolated nucleic Acid molecule” is a term that refers to nucleic acids that encode antibodies or portions of antibodies (e.g. VH, L, CDR3) that bind to hTNF. It can also be used to describe a nucleic molecule in which nucleotide sequencings encoding antibodies are not interrupted by any other sequences. These other sequences could naturally flank the nucleic molecule in human genomic DNA. For example, an isolated nucleic acids of the invention that encodes a VH region for anti-hTNF could be used. Antibody does not contain any other sequences that encode VH regions binding antigens other then hTNF?

The term “vector” is used herein to describe a nucleic acids molecule that can transport another nucleic acids to which it has been attached. A?plasmid? is one type of vector. This refers to a circular, double-stranded DNA loop in which additional DNA segments can be ligated. A viral vector is another type of vector that allows for additional DNA segments to be inserted into the virus genome. Some vectors can be self-replicating in the host cell they are introduced into (e.g., episomal mammalian and bacterial vectors with a bacterial origin. Other vectors, such as non-episomal mammals vectors, can be integrated into a host’s genome and reproduced with the host genome. Some vectors can also be used to control the expression of genes they are linked. These vectors are called?recombinant Expression Vectors? (or simply, ?expression vectors?). Generic expression vectors of utility for recombinant DNA techniques often take the form of plasmids. The present specification uses the terms?plasmid? The terms?plasmid? und?vector are interchangeable in the present specification. The terms?vector? and plasmid may be interchangeable as the vector most commonly used is the plasmid. The invention does not include viral vectors. These vectors serve similar functions.

“The term “recombinant host cells” is used. (or simply ‘host cell) As used in this document, “host cell” is a cell into whom a recombinant gene vector has been introduced. These terms do not refer to the specific subject cell, but also to its progeny. Certain modifications can occur in subsequent generations due to mutations or environmental influences. These progeny are not necessarily identical to the parent cells, but they are still included in the definition of ‘host cell. as used herein.”

“Various aspects of the invention will be described in detail in the subsections below.”

“I. “I.

“This invention provides treatments for disorders that require anti-TNF administration. It is advantageous to use antibody. This includes the subcutaneous, biweekly administration of human antibodies or antigen binding portions thereof that bind to human TNF. High affinity, low off rate, and high neutralizing power. The invention prefers that the human antibodies are recombinant and neutralize human anti-hTNF. antibodies. D2E7 is the preferred recombinant neutralizing antibody according to the invention. The SEQ ID No: 1 shows the amino acid sequence for the D2E7 L region; SEQ ID no: 2 shows the sequence for the D2E7 H region. Salfeld et al. U.S. Pat. No. No. 6,090,382, which can be incorporated by reference in this document.”

“It is well-known that the binding specificity/affinity for antigens by antibodies can be affected by the CDR3 domains of heavy and light chains. In another aspect of the invention, methods for treating disorders that require administration of anti-TNF are described. Subcutaneous administration of anti-TNF antibodies by human beings is beneficial. These antibodies have CDR3 domains with light and heavy chains that are structurally identical or similar to D2E7. Ala or Th can occupy position 9 of D2E7’s VL CDR3 without affecting the Koff. Accordingly, a consensus motif for the D2E7 VL CDR3 comprises the amino acid sequence: Q-R?Y?N?R-A-P?Y-(T/A) (SEQ ID NO: 3). Tyr or Asn can also occupy position 12 of D2E7’s VH CDR3, without affecting the Koff. Accordingly, a consensus motif for the D2E7 VH CDR3 comprises the amino acid sequence: V?S?Y-L-S-T-A-S?S-L-D-(Y/N) (SEQ ID NO: 4). As shown in Example 2 of U.S. Pat. No. No. Further, the skilled artisan will be able to appreciate that substitutions of other amino acids within CDR3 domains, such as those with conservative amino acids, may be possible due to the D2E7 VL or VH CDR3 domains being able to accept substitutions by alanine. As used herein, a “conservative amino acids substitution” is when one amino acid residue has been replaced by another with a similar sidechain. The art identifies several families of amino acid residues with similar side chains. These include basic side chains like lysine and arginine or glutamine as well as acidic side chain (e.g. aspartic acid, glutamic acids), uncharged polar sidechains (e.g. glycine asparagine or glutamine), nonpolar sidechains (e.g. alanine and valine), isoleucine, proline), phenylalanine) and aromatic sidechains such as tryptophan, tyrosine toptophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, tidine, ttophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, trytophan, tyrosine It is preferable that no more than five conservative amino acids are substituted within the D2E7 and/or VH CDR3 domains. It is preferred that no more than three conservative amino acids substitutions are made within D2E7 VL or VH CDR3 domains. You should also avoid making conservative amino acid substitutions at positions that are critical for binding to the hTNF?. The interaction of hTNF with D2E7 VL CD3 positions 2 and 5 and D2E7 H CDR3 positions 1 and 7 seem to be crucial. Therefore, it is important to avoid making conservative amino acid substitutions at these positions. However, an alanine substitution at position 5, of the D2E7 L CDR3, would be acceptable (see U.S. Pat. No. 6,090,382).”

“Accordingly, another embodiment of the invention provides methods for treating disorders that require the administration of anti-TNF. Subcutaneous, biweekly administration of an antibody or antigen binding portion from a human is a good way to obtain antibody. Preferably, the antibody or antigen binding portion of it contains the following characteristics:

“b”) has a light-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 3. It can be modified from SEQID NO: 3 by one alanine substitution at position 1, 4, 5 or 8, or by one to five conservative amino acids substitutions at positions 1, 3, 4, 6, 7 and/or 9.

“c” has a heavy-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 4. Modified from SEQ ID No: 4 by one alanine substitution at position 2, 4, 5, 6, 8, 9 or 11, or by one to five conservative amino acids substitutions at positions 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

“Another embodiment of the invention offers methods for treating disorders that require anti-TNF administration. Subcutaneous, biweekly administration of an antibody or an antigen binding portion is beneficial. A light chain variable area (LCVR), which contains the amino acids sequence of SEQID NO: 3, modified from SEQID NO: 3 by one alanine substitution, and a heavy chain variable (HCVR) with a CDR3domain comprising the amino sequence of the SEQID NO: 4, modified from the SEQID NO: 4 by one alanine substitution, at positions 2, 3, 4, 6, 8, 9, 10, or 11. The LCVR has a CDR2domain that contains the amino sequence of the SEQ ID No: 5, (i.e. the D2E7VL CDR2), and the HCVR has a CDR2domain that comprises the amino sequence of the SEQ ID ID NO: 6(i.e. the D2E7VH CDR2). The LCVR also has a CDR1domain that contains the amino sequence of the SEQ ID No: 7 (i.e. the D2E7 CDR1), and the HCVR has a CDR1domain that contains the amino sequence of the SEQ ID no: 8 (i.e. the D2E7 H CDR1). VL’s framework regions preferably come from the V?I human germline, but more preferably from A20 human germline Vk genes and most preferably from D2E7 VL frame sequences as shown in FIGS. 1A and 1B in U.S. Patent. No. 6,090,382. Preferably, the VH framework regions are from VH3 Human Germline Family. More preferably, they are from DP-31 Human germline VH gene. Most preferably, the D2E7 VH frame sequences shown in FIGS. 2A and 2B U.S. Patent No. 6,090,382.”

“Another embodiment of the invention offers methods for treating disorders that require anti-TNF administration. Subcutaneous, biweekly administration of an antibody or an antigen-binding portion of the isolated human antibody can be beneficial. Preferably, the antibody or antigen binding portion of the antibody contains a light-chain variable region (LCVR), which includes the amino acids sequence of the SEQ ID No: 1 (i.e. the D2E7 LL) and a heavier chain variable area (HCVR), which comprises the amino acids sequence of the SEQ ID no: 2 (i.e. the D2E7 H). In some embodiments, the antibody includes a heavy-chain constant region such as an IgG1, IgG2, IgG3, IgG4, IgG5, IgG4, IgG3, IgG3, IgG4, IgG5, IgE, IgM, or IgD constant area. The IgG1 or IgG4 heavy chains constant regions are preferred. The antibody may also contain a light-chain constant region. This could be either a kappa or lambda light-chain constant region. The antibody should consist of a kappa-light chain constant region. Alternately, the antibody can be a Fab fragment, or a single-chain Fv fragment.

“In other embodiments, the invention offers methods for treating disorders that require the administration of anti-TNF. Subcutaneous administration of an antibody or antigen-binding portion can be done biweekly. Antibody or the antigen-binding part thereof should contain D2E7-related VL or VH CDR3domains.

“An antibody, or a portion thereof, of the invention may be derivatized and linked to another functional molecular (e.g. another peptide or proteins). The invention’s antibodies and antibody parts can be derivatized or linked to other functional molecules (e.g., another peptide or protein). antibodies described herein, including immunoadhesion molecules. An antibody or part of an antibody described herein can be functionally linked to other molecular entities (e.g., chemical coupling, genetic fuse, noncovalent association or any other way), such as another antibody, a cytotoxic or pharmaceutical agent and/or a protein peptide that can act as a mediator between the antibody or portion of the invention with another molecule (e.g., streptavidin core area or a polyhistidine label).

Crosslinking multiple antibodies of the same or different type to make a derivatized type of antibody can create a bispecific type. Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). These linkers can be purchased from Pierce Chemical Company in Rockford, Ill.

Fluorescent compounds are useful detectable agents that can be used to derivatize an antibody or a portion thereof. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. A detectable enzyme can be used to derivatize an antibody, including horseradish peroxidase or glucose oxidase. An antibody can be derivatized using a detectable enzyme by adding additional reagents to the enzyme’s reaction product. If horseradish peroxidase has been detected, the addition of hydrogenperoxide and diaminobenzidine will result in a colorless reaction product that can be detected. A biotin derivatization may be used to detect an antibody. This can be done by indirect measurement of streptavidin binding or avidin.

“II. Expression of Antibodies

Recombinant expression in a host cell of immunoglobulin heavy and light chain genes can create an antibody or a portion thereof. A host cell is transfected using one or more recombinant vectors that contain DNA fragments encoding the antibody’s immunoglobulin heavy and light chains. The host cell then expresses the antibody recombinantly. Standard recombinant DNA methods are used to obtain heavy and light chains of antibody genes. These genes are then incorporated into recombinant expression Vectors and introduced into host cells. (eds.) (eds.) Current Protocols in Molecular Biology. Greene Publishing Associates. (1989). Also in U.S. Pat. No. 4,816,397 by Boss et al.”

To express D2E7, or a D2E7 related antibody, you must first obtain DNA fragments that encode the light and heavy chains variable regions. These DNAs are obtained using the polymerase chains reaction (PCR) to amplify and modify germline light- and heavy-chain variable sequences. The art has a number of germline DNA sequences that are suitable for human heavy and light-chain variable genes (see, e.g. the?Vbase?). Human germline sequence database. See also Kabat, E. A. et al. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. 91-3242; Tomlinson, I. M., et al. (1992)?The Repertoire Human Germline VH Sequences Reveals About Fifty Groups Of VH Segments With Different Hypervariable Loops J. Mol. Biol. 227:776-798. Cox, J. P. L. and others. (1994)?A Directory of Human Genem-line V78 Segments Reveals Strong Bias in Their Use? Eur. J. Immunol. 24:827-836. The contents of each are included herein by reference. Standard PCR is used to amplify a DNA fragment that encodes the D2E7 heavy chain variable region or an antibody related to D2E7. Amplification is most preferred for the DP-31 VH gene sequence. Standard PCR is used to amplify a DNA fragment that encodes the D2E7 light chain variable region or an antibody related to D2E7. Amplification of the A20 VL germline sequence would be preferred. Using standard methods, you can create PCR primers that are suitable for amplifying the DP31 germline VH or A20 germline L sequences.

These sequences can be mutated once the germline VH or VL fragments have been obtained to encode the D2E7 and D2E7-related sequences of amino acids disclosed herein. First, the VH and VL sequences of the amino acids are compared with the D2E7 and D2E7-related VH or VL amino sequences to identify any amino acid residues that are different from the germline D2E7 and D2E7-related sequence. Next, the appropriate nucleotides from the germline DNA sequences must be mutated so that the D2E7/D2E7-related sequence of amino acids is encoded. The genetic code will determine the nucleotide modifications to be made. The standard methods for mutagenesis of germline sequences include PCR-mediated mutagenesis (where the mutated nucleotides in the PCR primers are incorporated so that the PCR product contains mutations), and site-directed mutagenesis.

“Once DNA fragments encoding D2E7 and D2E7-related DH and D2E7 VL segments have been obtained (by amplification of germline VH or VL genes, described above), these DNA pieces can be further modified by standard recombinant techniques, such as to convert variable region genes into full-length antibody chains genes, Fab fragment genes, or to a SCFv gene. A VL- or VH encoding DNA fragment can be operatively linked with another DNA fragment, such as an antibody constant area or a flexible connecter, in these manipulations. In this context, the term “operatively linked” is used to refer to the joining of two DNA fragments so that their amino acid sequences are preserved in-frame.

“The VH region’s DNA can be isolated and converted into a full-length, heavy-chain gene by linking it to another DNA molecule that encodes heavy chain constant regions (CH1,CH2 or CH3). The sequences of human genes encoding the heavy chain constant regions are well-known (see E. A. Kabat et al. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. Standard PCR amplification can yield DNA fragments encompassing these areas. You can choose to have the heavy chain constant area as an IgG1, IgG2, IgG3, IgG4, IgA or IgE constant region. However, it is preferred that you have an IgG1 constant region. The VH-encoding DNA of a Fab fragment can be operatively linked with another DNA molecule encoding the heavy chain CH1 constant area.

“The VL region-encoding DNA can be transformed to a full-length light-chain gene (as a well as a Fab light chain gene) by linking the VL-encoding DNA with another DNA molecule that encodes the light-chain constant region, CL. The sequences of human light-chain constant region genes are well known (see E. A. Kabat et al. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. Standard PCR amplification can yield DNA fragments encompassing these areas. You can choose to have the light chain constant region as either a lambda or kappa region, but it is more preferred to be a region that is kappa.

“To create a SCFv gene, VH- and VL encoding DNA fragments must be operatively linked with another fragment encoding an amino acid sequence (Gly4Ser)3. This allows the VH and VL sequences to be expressed as a single-chain contiguous protein, with the VL, and VH regions joining by the flexible linker (see Bird et. al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).”

“To express antibodies, or the invention’s antibody portions, DNAs encoding partial- or full-length light or heavy chains are inserted into expression vectors so that they are operatively linked with transcriptional and translational control sequencings. The term “operatively linked” is used in this context. The term?operatively linked? refers to the fusion of an antibody gene into a vector in order that the transcriptional and translational control sequences contained within the vector fulfill their intended functions of controlling the transcription and expression of the antibody gene. The expression vector and control sequences must be compatible with the host cell being used. Either the antibody light chain gene or the antibody heavy chain genes can be placed in a separate vector, or both genes can be placed into the same vector. Standard methods are used to insert the antibody genes into the expression vector. These include ligation of complementary restriction sites on the fragment or vector of the antibody gene, or blunt end-ligation if there are no restriction sites. The expression vector may contain existing antibody constant region sequences before the D2E7 and D2E7-related light chain or heavy chain sequences are inserted. One way to convert the D2E7 and D2E7-related VH or VL sequences into full-length antibodies genes is to insert them in expression vectors that already encode heavy chain constant and/or light chain constant regions. The VH segment should be operatively linked with the CH segment(s) within vector, and the VL segment to the CL segment within vector. The signal peptide can be encoded in the recombinant expression vector to facilitate the secretion from a host cells. The vector can be cloned with the antibody chain gene so that the signal protein is linked in-frame to its amino terminus. You can choose to clone the signal peptide as an immunoglobulin signal protein or as a heterologous signalpeptide (i.e. a signal from a non-immunoglobulin peptide).

“In addition to the antibody chains genes, the regulatory sequences in the invention recombinant expression channels contain regulatory sequences that regulate the expression of antibody chain genes within a host cell. “Regulatory sequence” is a term that refers to the following: The term “regulatory sequence” refers to promoters, enhancers, and other expression control elements (e.g. polyadenylation signal) that regulate the translation or transcription of the antibody chain gene genes. These regulatory sequences can be found in Goeddel’s Gene Expression Technology: Methods in Enzymology, 185, Academic Press. San Diego, Calif. (1990). The art of designing expression vectors, including selecting regulatory sequences, is dependent on factors such as the type of host cell being transformed and the desired level of protein expression. The preferred regulatory sequences for mammalian hosts include viral elements that can direct high levels in protein expression in mammalian cell. These include promoters and/or enhancements derived from cytomegalovirus CMV (such the CMV promoter/enhancer), Simian Virus40 (SV40), adenovirus (e.g. the adenovirus major later promoter (AdMLP), and polyoma. U.S. Pat. provides more information about viral regulatory elements and their sequences. No. No. No. 4,510,245 as reported by Bell et.al. U.S. Pat. No. 4,968,615 by Schaffner et al.”

“In addition to regulatory sequences and antibody chain genes, recombinant expression viruses of the invention can contain additional sequences such as sequences that regulate the replication of the vector within host cells (e.g. origins of replication) or selectable marker genes. The selectionable marker gene allows for the selection of host cells in which the vector can be introduced (see, e.g. U.S. Pat. Nos. Nos. The selectable marker gene is usually able to confer resistance to drugs such as G418, methotrexate, hygromycin, or G418, on the host cell in which it has been introduced. The preferred selectable marker genes are the dihydrofolate reducetase gene (DHFR) (for use with dhfr). host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).”

“For expression of light and heavy chains, standard methods are used to transfect the expression vector(s), encoding heavy and light chains into host cells. There are many ways to define the term “transfection”. The various forms of the term “transfection” are meant to cover a variety of methods used for the introduction exogenous DNA into prokaryotic and eukaryotic host cells. Though it is theoretically possible for the invention to be expressed in prokaryotic and eukaryotic host cell, the preferred method of expression is in eukaryotic, most preferably mammalian, host cells. This is because eukaryotic, in particular mammalian, cells are more likely to produce and secrete an immunologically active, properly folded antibody. According to Wood, C.R. (1985), Immunology Today 6-13, prokaryotic expressions of antibodies genes have been shown to not be effective in producing high yields active antibodies.

“Chinese Hamster Ovary (CHO) cells are the preferred mammalian host cell for the expression of the recombinant antibody of the invention (including dhfr CHO cells, as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77.4216-4220, used in conjunction with a DHFR selectable markers, e.g. as described by R. J. Kaufman, and P. A. Sharp (1982). Mol. Biol. Biol. Recombinant expression vectors that encode antibody genes are introduced to mammalian host cell cells. The antibodies are made by cultivating the host cells for a sufficient time to allow the expression or secretion of the antibody within the culture medium. Standard protein purification methods can be used to recover antibodies from culture medium.

“Host cells may also be used to make intact antibodies. You will understand that there are many variations to the above process. It may be possible to transfect host cells with DNA that encodes either the light or heavy chains (but not both) for an antibody of the invention. Recombinant DNA technology can also be used to eliminate some or all the DNA that encodes either one or both of the heavy and light chains. This is not required for binding to the hTNF?. The antibodies of the invention also include molecules expressed from these truncated DNA molecules. Bifunctional antibodies can also be made in which one of the heavy and one of the light chains are an antibody according to the invention, and the other is specific for another antigen than hTNF. Crosslinking an invention antibody to another antibody using standard chemical crosslinking techniques.

“In a preferred method for recombinantly expression of an antigen, or antigen binding portion thereof, a recombinant vector encoding both of the antibody heavy chains and the antibody light chains is introduced into dhfrCHO cells via calcium phosphate-mediated transcription. The recombinant vector contains both the heavy and light chains genes. This allows for high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. Transformed host cells from selected recipients are grown to express the antibody heavy and lightweight chains. The culture medium is then used to recover the intact antibody. Standard molecular biology methods are used to create the recombinant expression virus, transfect host cells, select transformants, culture host cells, and then recover the antibody from culture medium.

“III. “III. Selection of Recombinant Human Antibodies

“Recombinant human antibody of the invention, in addition to D2E7, an antigen binding section thereof, or D2E7 related antibodies, can be isolated by screening a recombinant combinatorial library, preferably one scFv, made using VL and VH DNAs prepared from mRNA obtained from human lymphocytes. These libraries can be prepared and screened using known methods. There are also commercially available kits that can be used to generate phage display libraries, such as the Pharmacia Recombinant Phage Antibody System (catalog no. 27-9400-01; Stratagene SurfZAP phage display kit, catalog no. 240612), are examples of methods and reagents that can be used in generating or screening antibody display libraries. See, for instance, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. Publication no. WO 92/18619; Dower et al. PCT Publication Number. WO 91/17271; Winter et al. No. WO 92/20791; Markland et al. PCT Publication Number. WO 92/15679; Breitling et al. PCT Publication Number. WO 93/01288; McCafferty et al. No. WO 92/01047; Garrard et al. PCT Publication Number. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; 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; Barbas et.al. (1991) PNAS:7978-7982

“In a preferred embodiment to isolate human antibodies with high affinity? and a low rate constant for hTNF?, we use a murine anti hTNF? antibody with high affinity and low off rate constants for hTNF (e.g. MAK 195 hybridoma, deposit number ECACC 87 050801), is used first to select human heavy- and light-chain sequences with similar binding activity towards hTNF? using the epitope printing methods described in Hoogenboom et. al., PCT Publication Number. WO 93/06213. Preferably, the antibody libraries used in this procedure are scFv libraries that have been prepared and screened according to McCafferty et., PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990) 348:552-554; and Griffiths et al., (1993) EMBO J. 12:725-734. The scFv antibodies libraries should be screened with recombinant human TN? as the antigen.”

“Once the initial human VL segment and VH segments have been selected, mix and match? Different pairs of VL and H segments from the initial selection are tested for hTNF in experiments. To select the most desirable VLNH pairing combinations, binding is performed. To further increase the affinity and/or lower down the off rate constant of hTNF, The VL and/or VH segments of the preferred pair(s of VLNH) can be randomly modified, preferably in the CDR3 region or VL. This process is analogous to in vivo somatic mutation processes that result in affinity maturation. In vitro affinity maturation can also be achieved by amplifying the VH and L regions with complementary PCR primers to the VH CDR3 and VL CDR3, respectively. These primers have been “spiked?” With a random mix of the four nucleotide base bases at specific positions so that the PCR products encode VH or VL segments into the which random mutations were introduced into the VH or VL CDR3 areas. These VH and L segments randomly mutated can be screened for binding to the hTNF. sequences with high affinity and low off rates for hTNF? You can choose to bind.”

“Following screening, isolation of an anti-hTNF The invention can be obtained from a recombinant immuneglobulin display database. Nucleic acid encoding selected antibody can also be extracted from the display package (e.g. from the phage genome). Subcloning into other expression vectors can be done using standard recombinant DNA methods. The nucleic acids can be further modified to create additional antibody forms. The DNA encoding an antibody from a human is cloned into the recombinant expression vector. This vector can then be introduced into mammalian host cells as described in Section II.

“IV. “IV.

“The invention’s antibodies and portion of the invention may be included in pharmaceutical compositions that can be administered to a subject according to the methods described herein. The pharmaceutical composition typically contains an antibody (or portion thereof) from the invention, methotrexate, and a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” is defined herein. Any and all solvents and dispersion media, coatings and antibacterial and/or fungal agents, as well as isotonic or absorption delay agents and the like, that are physiologically compatible, are acceptable for administration to a subject in accordance with the methods described herein. One or more of the following pharmaceutically acceptable carriers are examples: water, saline and phosphate buffered saltsaline; dextrose; glycerol; ethanol, and combinations thereof. It is preferable to include isotonic substances, such as sugars, polyalcohols like mannitol or sorbitol in many cases. A small amount of pharmaceutically acceptable carriers can also contain auxiliary substances, such as preservatives or buffers, wetting agents, emulsifying or buffers that enhance the shelf-life or effectiveness of the antibody.

“The compositions described in this invention can be made in many forms. These can include liquid, semi-solid, and solid dosage forms such as dispersions, suspensions, dispersions, tablets, pills and powders. The intended use and therapeutic purpose of the preferred form will determine which one is chosen. The preferred compositions are either injectable or infusible, similar to compositions used for passive immunization. Parenteral administration is preferred (e.g. intravenous subcutaneous, intraperitoneal and intramuscular). Preferably, the antibody can be administered intravenous injection or infusion. Another preferred embodiment is intramuscular injection. A preferred embodiment of the antibody is administered subcutaneously (e.g., biweekly subcutaneous injection).

“Therapeutic compositions must be stable and sterile under all conditions. You can formulate the composition as a microemulsion or dispersion, liposome or other orderly structure that is suitable for high drug concentrations. You can prepare sterile injectable solutions by combining the active compound (i.e. the antibody or portion thereof) with the appropriate solvent. Then, filter sterilization is performed. Dispersions can be prepared by adding the active compound to a sterile medium that includes the basic dispersion medium as well as the other required ingredients. For the preparation of sterile injection solutions, vacuum drying and freeze drying are preferred methods. This produces a powder of active ingredient as well as any desired ingredients from a previously sterilized solution. A coating, such as lecithin or surfactants, can help maintain the fluidity of a solution. You can prevent prolonged absorption of injectable formulations by including an agent that delays absorption (e.g. monostearate salts or gelatin).

Although there are many methods for administering the antibodies and their portions, subcutaneous injection is the preferred method of administration. The route and/or method of administration will depend on the desired results. The active compound can be prepared in certain embodiments with a carrier to prevent rapid release. This could include implants, microencapsulated delivery systems, or transdermal patches. You can use biodegradable and biocompatible polymers such as ethylene vinyl, polyethylene glycol, (PEG), polyanhydrides or polyglycolic acids, collagen, polyorthoesters and polylactic acid. Many of the methods used to prepare such formulations have been patented. See, e.g., Sustained Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

“In some embodiments, the antibody or portion of an invention can be administered orally, for example with an inert or edible carrier. You can also include the compound and other ingredients in a capsule or tablet, or compress them into tablets. The compounds can be combined with excipients to be used for oral therapeutic administration. They may be taken as tablets, buccal tablets or capsules. It may be necessary to co-administer a compound of invention with a material to inhibit its inactivation to administer the compound other than via parenteral administration.

Supplementary active compounds may also be included in the compositions. An antibody or a portion thereof may be coformulated with or coadministered by one or more therapeutic agents in certain embodiments. An example of this is an anti-hTNF. An anti-hTNF antibody may be combined with methotrexate and/or administered in combination with one or more other antibodies (e.g. antibodies that bind to other cytokines or cell surface molecules), one cytokines, or soluble TNF? See e.g. PCT Publication Number. WO 94/06476), and/or one or several chemical agents that inhibit the hTNF. Production or activity (e.g. cyclohexaneylidene derivatives, as described in PCT Publication No. WO 93/19751. One or more of these therapeutic agents may also be combined with one or more of the antibodies described in the invention. Combination therapies can be advantageous because they use lower doses of therapeutic agents. This avoids potential toxicities and complications that may arise from monotherapies. In subsection IV, we discuss the use of the antibody, or portions thereof, in combination with other therapeutic drugs.

Non-limiting examples of therapeutic agents to treat rheumatoidarthritis with which an antibody or a portion of an invention can be combined are: non-steroidal antiinflammatory drug(s), cytokine suppressive drug(s), CSAIDs; CDP-571/BAY-10-33556 (humanized antiTNF). Celltech/Bayer; cA2 [chimeric anti-TNF antibody; Centocor] 75 kdTNFRIgG (75 kD IgG fusion protein, Immunex; Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. (1996) Vol. 38, S 185; DAB 486 -IL-2 and/or DAB389 -IL-2 (IL-2 fusion protein; Seragen); see e.g. Arthritis & Rheumatism (1993). Vol. 36, 1223); Anti-Tac (humanized anti-IL-2R? ; Protein Design Labs/Roche; IL-4 (antiinflammatory cytokine, DNAX/Schering); IL-10 SCH 52000 (recombinant IL-10; DNAX/Schering); IL-1RA; IL-1 receptor antagonist, Synergen/Amgen; TNF-bp/sTNFR (soluble TNF binding proteins; see e.g. Arthritis & Rheumatism, Vol. 39, No. 9 (supplement), 5284; Amer. J. Physiol. Physiol. 268, pp. 268 pp. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic (inhibitor plasminogen activation; e.g. Arthritis & Rheumatism (1996) Vol. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 7, pp. 12, pp. 39, No. 39, No. 39, No. 39, No. converting enzyme; zap70 and/or lck inhibit (inhibitor the tyrosine kinase zap70 or lck); VEGF inhibition and/or VEGFR inhibitor (inhibitors or receptors for vascular endothelial cells growth factor; inhibitors of angiogenesis); corticosteroid antiinflammatory drugs (e.g. SB203580); anti-IL-12 antibody; TNF-convertase inhibitors; TNF-convertase; IL-12; interleukin-11; Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S120); gold; penicillamine; chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; orally-administered peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.”

Non-limiting examples of therapeutic agents to treat inflammatory bowel diseases with which an antibody or a portion of the invention could be combined include: budenoside, epidermal growth factors; corticosteroids, cyclosporins, sulfasalazine, aminosalicylates, 6-mercaptopurine, metronidazole, lipoxygenase inhibitions, mesalamine, olsalazine, balsalazine, IL-1 receptor antagonists, anti-IL-1 monoclonal antibody; anti-IL-6 monoclonal antibody; growth factors; elastase inhibitors; pyridinyl?imidazole compounds CDP-571/BAY-10-333356 (humanized antiTNF). antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kdTNFRIgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 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 agonists (e.g., agonist antibodies); interleukin-11; glucuronide- or dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); slow-release mesalazine; methotrexate; antagonists of Platelet Activating Factor (PAF); ciprofloxacin; and lignocaine.”

“Non-limiting examples of therapeutic agents to multiple sclerosis with the which an antibody or a portion of an invention can be combined include: prednisolone, methylprednisolone, azathioprine, cyclophosphamide, methotrexate, cyclophosphamide, cyclosporine, cyclophosphamide, cyclosporine, methotrexate; 4-aminopyridine, tizanidine, interferon-?1a (Avonex?) ; Biogen); interferon-?1b (Betaseron? ; Chiron/Berlex; Copolymer 1; Copaxone; Copolymer 2? Teva Pharmaceutical Industries Inc.; hyperbaric oxygen; intravenous immuneglobulin; Clabribine; CDP-571/BAY-10-33556 (humanized antiTNF). antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kDTNFR-IgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. Vol.

“Non-limiting examples of therapeutic agents to treat sepsis that an antibody or a portion thereof can be combined with include: antibiotics; hypertonic saline solution; intravenous gamma globulin (IgG); continuous hemofiltration; carbapenems, e.g., meropenem); antagonists cytokines like TNF?, IL-113, IL-6, IL-8; CDP-571/BAY-10-33556 (humanized anti-TNF) antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kdTNFRIgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular peptide; SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (Picower Institute); Tissue Factor Pathway Inhibitor (TFPI; Chiron); PHP (chemically modified hemoglobin; APEX Bioscience); iron chelators and chelates, including diethylenetriamine pentaacetic acid-iron (III) complex (DTPA iron (III); Molichem Medicines); lisofylline (synthetic small molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan (aqeuous soluble ?1,3glucan; Alpha-Beta Technology); apolipoprotein A-1 reconstituted with lipids; chiral hydroxamic acids (synthetic antibacterials that inhibit lipid A biosynthesis); anti-endotoxin antibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.); rBPI21 (recombinant N-terminal fragment of human Bactericidal/Permeability-Increasing Protein); and Synthetic Anti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories);”

Nonlimiting examples of therapeutic agents to treat adult respiratory distress syndrome (ARDS), with which an antibody or a portion of the invention could be combined are: anti-IL-8 antibody; surfactant therapy; CDP-571/BAY-10-33556 (humanized antiTNF). antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kdTNFRIgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. Vol.

“The compositions of the pharmaceutical invention may contain a?therapeutically efficacious amount? Or a?prophylactically efficacious amount? An antibody or a portion of the invention. Therapeutically effective amounts? A therapeutically effective amount is one that is effective at the prescribed dosages and over the required time to achieve the desired therapeutic effect. The therapeutically effective amount or portion of an antibody may differ depending on factors like the person’s age, gender, and health. It also depends on the individual’s ability to produce the desired response. Therapeutically effective amounts are those that have no toxic or harmful effects. They also include therapeutically beneficial effects. What is a ‘prophylactically efficient amount? A prophylactically effective amount is one that can be administered in the dosages and over the time required to produce the desired prophylactic effect. The prophylactically-effective amount is typically lower than the therapeutically-effective amount, as it is usually administered to subjects at an earlier stage of the disease.

The dosage regimens can be altered to achieve the desired result (e.g., therapeutic or prophylactic). One bolus can be given, or several doses may be divided over time. The dose may also be adjusted to meet the needs of the therapeutic situation. For ease of administration and uniform dosage, it is particularly advantageous to create parenteral compositions using dosage unit form. As used herein, dosage unit form refers to physically distinct units that can be used as unitary doses for mammalian subjects. Each unit contains a predetermined amount of active compound to produce the desired therapeutic effect when combined with the appropriate pharmaceutical carrier. Specification for dosage units forms according to the invention is determined by and directly dependent upon (a) the unique characteristics and prophylactic effects of the active compound, and (b), the limitations inherent in the art and science of compounding such active compounds for treatment of sensitive individuals.

“An example, non-limiting range of therapeutically or prophylactically efficacious amount of an antibody/antibody portion of the invention is 10-100 mg, more preferably 20-40 mg, and most preferably around 40 mg. Dosage values can vary depending on the severity and type of the condition being treated. It should also be noted that specific dosage regimens for any subject should be adjusted over time depending on the individual need and professional judgment of those administering or supervising the administration. The dosage ranges herein are only an example and do not limit the practice or scope of the claimed composition.

“V. Uses for the Antibodies to the Invention”

“Given their binding ability to hTNF, the anti-hTNF is? The invention allows for the detection of hTNF using antibodies or portions thereof. (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method of detecting hTNF. In a biological specimen, the method involves contacting the biological sample with an antibody or antibody portion of the invention, and detecting the antibody (or portion) bound to the hTNF? Unbound antibody (or a portion thereof) to detect hTNF. The biological sample. To facilitate detection of bound or unbound antibodies, the antibody can be directly or indirectly labeled using a detectable substance. There are many suitable detectable substances, including prosthetic groups, enzymes, fluorescent materials, luminescent material and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ?-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125I, 131I, 35S or 3H.”

“Alternative to the labeling of the antibody, hTNF?” Can be tested in biological fluids using a competition immunoassay that uses rhTNF Standards labelled with detectable substances and unlabeled anti hTNF? antibody. The biological sample is the one that has been labeled with rhTNF. The standards and the anti -hTNF are combined. The amount of labeled anti-hTNF is determined by combining the two. The standard bound to unlabeled antibodies is determined. How much hTNF is in the biological sample? The amount of hTNF in biological samples is proportional to their labeled levels. What is the standard bound to anti-hTNF antibody.”

“A D2E7 antibody can be used to detect TNF’s from other species, such as primates (e.g., monkey, baboon and marmoset), pig, and mouse. D2E7 is able to bind to each TNF?”

“The invention’s antibodies and anti-tumor components are capable of neutralizing the hTNF?” activity in vitro as well as in vivo (see U.S. Pat. No. 6,090,382). D2E7 and at least some other antibodies in the invention can neutralize hTNF. activity from other species. The invention’s antibody and antibody parts can therefore be used to inhibit the hTNF. activity in cell cultures containing hTNF, or in mammalian subjects with TNF?s with whom an antibody of this invention crosses-reacts (e.g. chimpanzees, marmosets, cynomolgus, rhesus, pig, and mouse. The invention is a method of inhibiting TNF in one embodiment. activation that involves contacting TNF? With an antibody or a portion of the invention so that TNF? activation is blocked. The TNF is preferred. Is human TNF?. To inhibit hTNF, you can add an antibody or portion of the invention to a cell culture that contains, or is suspected to contain, TNF?. “Activity in the culture.”

“In a preferred embodiment of the invention, methods are provided for treating disorders that require anti-TNF administration. It is possible to subcutaneously give the subject an antibody or a portion of the invention biweekly. This will make the disorder easier to treat. The preferred embodiment of the antibody is given subcutaneously on an every other week basis. Another preferred embodiment is to administer the antibody subcutaneously prior, during, or after administrations of methotrexate. The subject should be a human. You can also use a mammal that expresses TNF. An antibody of the invention can cross-react with it. Further, the subject could be a mammal in which hTNF has been introduced? (e.g. by administration of hTNF.c or expression of an hTNF. transgene). The invention allows for the administration of an antibody to human subjects for therapeutic purposes. An antibody of the invention may also be administered to non-human mammals that express TNF. The antibody can cross-react with any animal (e.g., a primate or pig, or mouse) to test for human disease or to veterinary purposes. These animal models can be used to evaluate the therapeutic efficacy and administration times of antibodies.

“A disorder in which an anti-TNF agent is administered?, as used herein. Is antibody beneficial? TNF is present in diseases and other conditions. If a person suffering from the disorder is shown or suspected to be responsible for the disease’s pathophysiology or contributing to its worsening, or where another anti-TNF antibody has been found to be effective, The disease has been successfully treated with an antibody or a biologically active component. TNF-inhibiting disorders are those in which TNF is not present. Activity is considered to be harmful if inhibition of TNF is involved. Activity is expected to reduce the severity and/or progression of the condition. TNF may increase in these cases. In a biological fluid of a person with the disorder (e.g. an increase in TNF concentration). in serum, plasma, synovial fluid, etc. Anti-TNF can detect these antibodies. As described above, TNF can be used to treat many disorders. TNF? activity can be harmful. Below is more information about the use of antibodies and the antibody portions of the invention for the treatment of certain disorders:

“A. Sepsis”

Summary for “Methods for administering anti-TNF?” antibodies”

“Tumor necrosis Factor?” (TNF?) This cytokine is produced by many cell types, including macrophages and monocytes. It was first identified because it induces necrosis in certain mouse tumors (see Old, L. (1985). Science 230:630-632). A factor called cachectin was later identified as the same molecule that TNF?. TNF? TNF has been implicated as a mediator of shock (see, e.g. Beutler, B., and Cerami A. (1988) Annu. Rev. Biochem. 57:505-518. Beutler, B., and Cerami. (1989) Annu. Rev. Immunol. 7:625-655). Furthermore, TNF? TNF has been implicated as a pathophysiology for a number of human diseases and disorders including infections, autoimmune disease, transplant rejection, and graft-versus host disease (see, e.g. Vasilli, P. (1992). Annu. Rev. Immunol. 10:411-452 Tracey, K. J., and Cerami A. (1994) Annu. Rev. Med. 45:491-503).”

“Murine anti-hTNF was created to address the issues associated with fully-murine human antibodies. Genetically engineered antibodies can be made to look more human-like. Chimeric antibodies have been made, where the variable regions of the antigen chains are murine-derived while the constant regions are human-derived (Knight, D. M. et al. (1993) Mol. Immunol. 30:1443-1453, PCT Publication Number. WO 92/16553 by Daddona, P. E., et al.). Humanized antibodies have been also prepared. The hypervariable domains in the antibody variable regions of the antibody variable are murine-derived, but the rest of the variable regions as well as the antibody constant regions, are human-derived. WO 92/11383 by Adair, J. R., et al.). These chimeric and humanized antibodies still contain some murine sequences. They may still elicit an undesirable immune reaction, the human-anti-chimeric antibody reaction (HACA), especially when given for long periods of time, as in the case of chronic indications such as rheumatoid. (see Elliott, M.J., et. al.). (1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet 344:1105-1110).”

“A preferred hTNF?” An entirely human anti-hTNF would be an inhibitory agent for murine mAbs and derivatives thereof (e.g. chimeric or humanized antibody). HAMA reaction should not be elicited by such an agent, even if it is used for long periods. Monoclonal human autoantibodies against the hTNF These were prepared using human hybridoma methods (Boyle P. et al. (1993) Cell. Immunol. 152:556-568; Boyle, P., et al. (1993) Cell. Immunol. 152:569-581; European Patent Application Publication No. 614 984 A2 by Boyle, et al.). These monoclonal, hybridoma-derived monoclonal antibodies had an affinity for hTNF. This was not possible to calculate using conventional methods. They were also unable to bind soluble HTNF. They were not able to neutralize hTNF-induced cytotoxicity (see Boyle et al. ; supra). The success of the human hybridoma procedure depends on the presence of human peripheral blood lymphocytes that produce autoantibodies against hTNF?. Certain studies have shown that serum antibodies against hTNF were detected in certain individuals. in human subjects (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog. Leukocyte Biol. 10B:447-452), while others have not (Leusch H-G. (1991) J. Immunol. Methods 139,145-147)

“An alternative to the naturally occurring human anti-hTNF?” A recombinant human anti-hTNF would be an antibody. antibody. Recombinant antibodies from human that bind to hTNF Griffiths, A. D. et al. have described antibodies that bind hTNF with a low affinity (Kd?107M) and a rapid off rate (Koff?102 sec?1). (1993) EMBO J. 12:725-734). These antibodies are not suitable for therapeutic purposes due to their fast dissociation kinetics. A recombinant anti-hTNF human antibody? It has been shown that it does not neutralize the hTNF. activation, but instead enhances binding of the hTNF enhances hTNF internalization and binds to cells’ surfaces (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45; Publication No. WO 92/03145 (Aston, R. and al.)

“Recombinant human antibodies which bind soluble HTNF With high affinity and slow dissociation rates and the ability to neutralize hTNF Activity, including hTNF-induced cytotoxicity in vitro and intracellularly, and hTNF-induced cell activation have been also described (see U.S. Pat. No. 6,090,382). The usual protocol for administering antibodies intravenously is performed on a weekly basis. The frequency of administering anti-/or drug therapy can make it more expensive and cumbersome. Intranasal administration has its limitations, as it is typically administered by someone who has medical training.

“The present invention allows for biweekly dosing regimens to treat TNF. Subcutaneous route is preferred for these disorders. There are many benefits to biweekly dosing, including a reduced number of injections, less injection site reactions (e.g. local pain and swelling), patient compliance (i.e. due to fewer injections), and lower costs for the patient and the provider. Subcutaneous dosing can be advantageous as the patient may administer a therapeutic substance (e.g., human TNF) themselves. This is convenient for both the patient as well as the health care provider.

“This invention offers methods to treat disorders that TNF is involved. Activity is harmful. These methods include subcutaneous injections of antibodies that are administered biweekly to a subject. Preferably, the antibodies are recombinant human antibodies which specifically bind to human thyroid hormone. This invention also provides methods to treat disorders that TNF? TNF is a dangerous agent. Combination therapy is where human antibodies are administered to a subject along with another therapeutic agent. This could include one or more antibodies that bind to other targets (e.g. antibodies that bind to cell surface molecules or cytokines), or one or more cytokines. receptor (see, e.g. PCT Publication Number. WO 94/06476), and/or one or several chemical agents that inhibit the hTNF. Production or activity (such cyclohexaneylidene derivatives, as described in PCT Publication No. WO 93/19751), preferably methotrexate. Preferably, the antibodies are recombinant human antibodies which specifically bind to human TNF’. The invention’s antibodies are known to be able to bind to hTNF? With high affinity and slow dissociation rates and neutralizing hTNF activity, including in vitro and in-vivo hTNF-induced cytotoxicity and cellular activation. You can have full-length antibodies (e.g. an IgG1 and IgG4 antibodies) or a portion that is antigen-binding (e.g. a F(ab?) or Fab). )2, scFv fraction or a single domain. D2E7 is the most popular recombinant antigen of the invention. It has a light-chain CDR3 domain that contains the amino sequence of SEQID NO: 3. A heavy-chain CDR3 domain comprises the amino sequence of SEQID NO: 4. (Apendix B). The D2E7 antibody should have a light-chain variable region (LCVR), which contains the amino acids sequence of SEQID NO: 1, and a heavy-chain variable region (HCVR), which includes the amino sequence of SEQU ID NO. 2. These antibodies are described by U.S. Pat. No. No. 6,090,382, incorporated herein in its entirety by reference.”

“In one embodiment, this invention provides methods for treating disorders that TNF is involved. activity is harmful. Inhibiting human TNF is one of these methods. Subcutaneous, biweekly administrations of anti-TNF can be used to inhibit human TNF? The disorder can be treated with an antibody. It could be sepsis, an immune disorder (e.g., rheumatoid, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis, and nephrotic disorder), or an infectious disease.

“Another embodiment of the invention offers methods for treating disorders that TNF is involved. activity is harmful. These methods include blocking human TNF? Subcutaneous administration of anti-TNF can be used to inhibit human TNF? The disorder can be treated with methotrexate and antibody. One aspect of methotrexate administration is that it is combined with anti-TNF. antibody. Methotrexate can be administered before anti-TNF is administered. antibody. Another aspect is that methotrexate can be administered after anti-TNF administration. antibody.”

“In another embodiment, the invention offers methods for treating disorders in which TNF is involved. Activity is reduced by subcutaneous, biweekly administration of a human antibody or an antigen-binding component to the subject. Preferably, the antibody or antigen binding portion of it has the following characteristics:

“b”) has a light-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 3. It can be modified from SEQID NO: 3 by one alanine substitution at position 1, 4, 5 or 8, or by one to five conservative amino acids substitutions at positions 1, 3, 4, 6, 7 and/or 9.

“c” has a heavy-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 4. Modified from SEQ ID No: 4 by one alanine substitution at position 2, 4, 5, 6, 8, 9 or 11, or by one to five conservative amino acids substitutions at positions 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

“In another embodiment, the invention provides methods for treating disorders that TNF is involved. Activity is harmful. The methods include subcutaneous administration of a human antibody or its antigen-binding component to the subject every two weeks. An LCVR with a CDR3 Domain containing the amino acids sequence of SEQID NO: 3, modified from SEQID NO: 3 by a single alanine substitut at position 1, 4, 5, 7, 8 or 8. A HCVR with a CDR3 Domain comprising the amino sequence of SEQ ID NO 4 or modified at position SEQID NO: 4 by a single alanine substitution at position 2, 3, 4, 6, 8, 9, 10, or 11. The LCVR has a CDR2 Domain that contains the amino sequence of SEQID NO: 5, while the HCVR has a CDR2 Domain that includes the amino sequence of SEQUID NO: 6. Even more preferable, the LCVR also has a CDR1domain that contains the amino sequence of SEQ ID NO. 7 and the HCVR has a CDR1domain that comprises the amino sequence of SEQ ID NO. 8.

“In another embodiment, the invention offers methods for treating disorders in which TNF is involved. Subcutaneous administration of an antibody or antigen-binding portion to the subject biweekly can be used to reduce activity. Preferably, the antibody or antigen binding portion of the antibody contains an LCVR that includes the amino acids sequence of SEQID NO: 1 and an HCVR that includes the amino acids sequence of SEQUID NO: 2. In some embodiments, the antibody contains an IgG1 heavy chain constant area or an IgG4 high chain constant region. Another embodiment of the antibody includes a Fab fragment or an F(ab) fragment. A single chain Fv fragment, or a Fab fragment can be used.

“In other embodiments, the invention offers methods for treating disorders that require the administration of anti-TNF. Subcutaneously administering one or more antiTNF to the subject biweekly is a good way to obtain an antibody. Antibodies, or antigen binding portions of them. Preferably, the antigen-binding or antibody portion of the LCVR has a CDR3 domain that contains an amino sequence chosen from the following groups: SEQID NO. 3, SEQID NO. 11, SEQID NO. 13, SEQID NO. 14, SEQID NO. 15, SEQID NO. 16, SEQID NO. 17, SEQID NO. 18, SEQID NO. 23, SEQID NO. 22, SEQID NO. 23, SEQID NO. 24, SEQID NO.

“Another aspect of the invention is kits that contain a formulation containing a pharmaceutical composition. Kits contain an anti-TNF. The kits contain an anti-TNF? antibody and a pharmaceutically acceptable carrier. Instructions for subcutaneous administration of the pharmaceutical composition to treat a disorder requiring anti-TNF injection are included in the kits. It is advantageous to use antibody. Another aspect of the invention is kits that contain a pharmaceutical composition and an anti-TNF. anti-TNF?, methotrexate and a pharmaceutically acceptable carrier. Instructions for subcutaneous administration of the pharmaceutical composition are included in the kits. It is very beneficial to have an antibody.

“Another aspect of the invention is a preloaded needle containing a pharmaceutical compound containing an anti-TNF. anti-TNF and a pharmaceutically acceptable carrier. Another aspect of the invention is a preloaded needle containing a pharmaceutical composition containing anti-TNF. “Antibody, methotrexate, as well as a pharmaceutically acceptable carrier.”

“This invention relates to treatments for disorders that require the administration of anti-TNF. It is possible to administer isolated human antibodies or antigen-binding components of antibody that binds to human TNF. This is called an antibody. The treatment of the disorder can be achieved by using antibodies that have high affinity, low off rates and high neutralizing capacities. The invention relates to the treatment of antibodies and fragments of antibodies, as well as pharmaceutical compositions.

“For the invention to be easier to understand, certain terms must first be defined.”

“Dosing” is the act of administering a substance (e.g. an anti-TNF). “Anti-TNF? treatment) in order to reach a therapeutic goal.

“Biweekly dosing schedule?,??biweekly dose? and?biweekly administering? refer to the time it takes to administer a substance (e.g. an anti-TNF). antibodies) to a subject in order to achieve a therapeutic goal (e.g. the treatment of a TNF-associated disorder). A weekly dosing schedule is not recommended for the biweekly regimen. The substance should be administered approximately every 9-19 days. It is more preferable to administer the substance every 11-17 days. Even more preferably every 13-15 days. Most preferably every 14 days.

“Combination therapy” is the administration of two or more therapeutic drugs, e.g. an anti-TNF. combination therapy includes the administration of anti-TNF and methotrexate. You may administer methotrexate concurrently with, before, or after an anti-TNF injection. antibody.”

“The term “human TNF?” “Human TNF” is an acronym for a human cytokine. It is also known as hTNF or simply hTNF. The biologically active form is made up of a trimer consisting of 17 kD molecules that are noncovalently bound. What is the structure of TNF? Pennica, D., and others. provide more information. (1984) Nature 312:724-729; Davis, J. M., et al. (1987) Biochemistry 26.1322-1326. Jones, E. Y. et al. (1989) Nature 338, 225-228. What is the meaning of human TNF? Recombinant human TNF is also included in the term human TNF? (rhTNF? ), which can either be made using standard recombinant expression techniques or bought commercially (R & D Systems Catalog No. 210-TA, Minneapolis, Minn.).”

“Antibody” is a term that refers to immunoglobulin molecules made up of four polypeptide chains: two heavy (H), and two light (L), chains interconnected by disulfide bond. Each heavy chain is composed of a heavy-chain variable region (abbreviated as HCVR, VH or VH) along with a heavy-chain constant region. The three domains that make up the heavy chain constant area are CH1, CH2 or CH3. Each light chain is composed of a light-chain variable region (abbreviated as LCVR, VL or VL), and a light-chain constant region. One domain, CL, is the light chain constant region. You can further subdivide the VH and VL regions into regions of hypervariability (CDR), as well as regions that are conserved (FR). Each VH/VL is made up of four FRs and three CDRs. They are arranged in the following order from amino-terminus through carboxy-terminus: FR1, CDR1, CDR2, CDR2, CDR3, CDR3, and FR4.

“The term ‘antigen-binding part? An antibody (or simply the?antibody section?) is a portion of an antibody. As used herein, “antibody portion” refers to one or more fragments that can bind to an antigen. A fragment of a full length antibody can perform the antigen-binding function. The term “antigen-binding section” includes examples of binding fragments. A Fab fragment is a monovalent fragment that includes the VL and VH domains. It also contains the CL, CH1 and CL domains. A bivalent fragment that consists of two Fab fragments linked together by a disulfide link at the hinge region, and (iii), a F(ab) fragment. Although VL, and VH are distinct domains of Fv fragments, they can be linked using recombinant techniques to create a single protein chain in the form of monovalent molecules. This is known as single chain Fv (scFv); see Bird et. al. (1988) Science 242(4):423-426. Huston et. al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. These single-chain antibodies are also included in the term “antigen binding portion?” An antibody. Diabodies and other single-chain antibodies are also included. Bivalent, bispecific antibodies called dibodies contain VH and L domains. However, the linker is too short to permit pairing between the two domains. This creates two antigen binding site and forces them to pair with each other. (see Holliger, P. and al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2121-1123

“But, further, an antigen-binding or antibody portion of an antibody may form part of larger immunoadhesion molecule, which is formed by covalent or notcovalent association of the antibody/antibody portion with one or several other proteins or peptides. To make a tetrameric, scFv-molecule, one example of such immunoadhesion molecular is the use of the streptavidin central region (Kipriyanov S. M., and others). (1995) Human Antibodies and Hybridomas 6 :93-101. The use of a cysteine, marker peptide and C-terminal histidine tag to create bivalent and biotinylated ScFv molecules (Kipriyanov S. M. et al. (1994) Mol. Immunol. 31:1047-1058). F(ab) and Fab are antibody fragments. You can prepare Fab and F(ab)2 fragments from whole antibodies by using traditional techniques such as papain or pipsin digestion of whole antibodies. Standard recombinant DNA techniques can also be used to obtain antibodies, immunoadhesion molecules, and portions thereof, as discussed herein.

“Human antibody” is a term that refers to antibodies with variable and constant regions derived primarily from human germline immunoglobulin sequencings. The invention could include amino acid residues that are not encoded in human germline immunoglobulin sequencings. This includes mutations introduced by random, site-specific or somatic mutations in vitro or in vivo. The term “human antibody” as used in this invention does not include antibodies in which CDRs sequences derived from another mammalian species (e.g., a mouse) have been grafted onto human frames sequences.

“Recombinant human antibodies” is a term that refers to all human antibodies that have been prepared, expressed, created, or isolated using recombinant methods. This includes antibodies generated from a recombinant combination of human antibodies (described in Section H), antibodies obtained from a recombinant library of human antibodies (described in Section III below), and antibodies taken from animals (e.g. a mouse) that has been transgenic for human immunoglobulin gene (see Taylor, L. D. (1992) Nucl. Acids Res. 20:6287-6295). or antibodies that have been prepared, expressed, created, or isolated using any other method that involves the splicing human immunoglobulin gene sequencing to other DNA sequences. These recombinant antibodies are derived from the human germline immunoglobulin gene sequences and have constant and variable regions. However, certain embodiments of recombinant human antibodies are subject to in vitro mutation (or when an animal transgenic to Ig sequences for humans is used, in-vivo somatic mutation). The VH and L regions of the recombinant antibody sequences are sequences that are derived from and related with human germline VH sequences.

An “isolated antibody” is an antibody that is substantially devoid of antibodies with different antigenic specificities. It is significantly free from antibodies that specifically bind to antigens other hTNF? Is there an isolated antibody that binds specifically to hTNF However, cross-reactivity with other antigens such as hTNF may exist. molecules of other species (discussed below). An isolated antibody might be completely free from other cellular material or chemicals.

“A?neutralizing antibody? as described herein (or an antibody that neutralized the hTNF) activity? () is an acronym for an antibody that binds to hTNF This results in the inhibition of the biological activities of hTNF? What is the effect of this inhibition on the biological activity of hTNF You can measure one or more indicators for hTNF to assess the level of inhibition. biological activity such as hTNF-induced cytotoxicity in vitro and in vivo, hTNF-induced cell activation, and hTNF-binding to hTNF in vivo. receptors. These are indicators of hTNF. One or more of the many standard in vitro and in vivo tests that are available in the art can assess biological activity (see Example 4). The ability of an antibody neutralize hTNF is preferred. Activity is measured by the inhibition of hTNF-induced cytotoxicity in L929 cells. Another parameter that can be used to measure hTNF activity is the ability of an antibody to inhibit hTNF-induced expression of ELAM-1 on HUVEC. Activity can be measured by assessing the ability of an anti-hTNF antibody to inhibit hTNF-induced expression of ELAM-1 in HUVEC. This is a measure of hTNF-induced cellular activation.

“Koff” is the off rate constant that allows an antibody to be dissociated from its antibody/antigen complex.

“Kd” is the dissociation constant for a specific antibody-antigen interaction.

“Nucleic Acid Molecule” is a term that refers to both DNA molecules and RNA molecules. One-stranded or two-stranded nucleic acids molecules are possible, but it is preferred to have double-stranded DNA.

“Isolated nucleic Acid molecule” is a term that refers to nucleic acids that encode antibodies or portions of antibodies (e.g. VH, L, CDR3) that bind to hTNF. It can also be used to describe a nucleic molecule in which nucleotide sequencings encoding antibodies are not interrupted by any other sequences. These other sequences could naturally flank the nucleic molecule in human genomic DNA. For example, an isolated nucleic acids of the invention that encodes a VH region for anti-hTNF could be used. Antibody does not contain any other sequences that encode VH regions binding antigens other then hTNF?

The term “vector” is used herein to describe a nucleic acids molecule that can transport another nucleic acids to which it has been attached. A?plasmid? is one type of vector. This refers to a circular, double-stranded DNA loop in which additional DNA segments can be ligated. A viral vector is another type of vector that allows for additional DNA segments to be inserted into the virus genome. Some vectors can be self-replicating in the host cell they are introduced into (e.g., episomal mammalian and bacterial vectors with a bacterial origin. Other vectors, such as non-episomal mammals vectors, can be integrated into a host’s genome and reproduced with the host genome. Some vectors can also be used to control the expression of genes they are linked. These vectors are called?recombinant Expression Vectors? (or simply, ?expression vectors?). Generic expression vectors of utility for recombinant DNA techniques often take the form of plasmids. The present specification uses the terms?plasmid? The terms?plasmid? und?vector are interchangeable in the present specification. The terms?vector? and plasmid may be interchangeable as the vector most commonly used is the plasmid. The invention does not include viral vectors. These vectors serve similar functions.

“The term “recombinant host cells” is used. (or simply ‘host cell) As used in this document, “host cell” is a cell into whom a recombinant gene vector has been introduced. These terms do not refer to the specific subject cell, but also to its progeny. Certain modifications can occur in subsequent generations due to mutations or environmental influences. These progeny are not necessarily identical to the parent cells, but they are still included in the definition of ‘host cell. as used herein.”

“Various aspects of the invention will be described in detail in the subsections below.”

“I. “I.

“This invention provides treatments for disorders that require anti-TNF administration. It is advantageous to use antibody. This includes the subcutaneous, biweekly administration of human antibodies or antigen binding portions thereof that bind to human TNF. High affinity, low off rate, and high neutralizing power. The invention prefers that the human antibodies are recombinant and neutralize human anti-hTNF. antibodies. D2E7 is the preferred recombinant neutralizing antibody according to the invention. The SEQ ID No: 1 shows the amino acid sequence for the D2E7 L region; SEQ ID no: 2 shows the sequence for the D2E7 H region. Salfeld et al. U.S. Pat. No. No. 6,090,382, which can be incorporated by reference in this document.”

“It is well-known that the binding specificity/affinity for antigens by antibodies can be affected by the CDR3 domains of heavy and light chains. In another aspect of the invention, methods for treating disorders that require administration of anti-TNF are described. Subcutaneous administration of anti-TNF antibodies by human beings is beneficial. These antibodies have CDR3 domains with light and heavy chains that are structurally identical or similar to D2E7. Ala or Th can occupy position 9 of D2E7’s VL CDR3 without affecting the Koff. Accordingly, a consensus motif for the D2E7 VL CDR3 comprises the amino acid sequence: Q-R?Y?N?R-A-P?Y-(T/A) (SEQ ID NO: 3). Tyr or Asn can also occupy position 12 of D2E7’s VH CDR3, without affecting the Koff. Accordingly, a consensus motif for the D2E7 VH CDR3 comprises the amino acid sequence: V?S?Y-L-S-T-A-S?S-L-D-(Y/N) (SEQ ID NO: 4). As shown in Example 2 of U.S. Pat. No. No. Further, the skilled artisan will be able to appreciate that substitutions of other amino acids within CDR3 domains, such as those with conservative amino acids, may be possible due to the D2E7 VL or VH CDR3 domains being able to accept substitutions by alanine. As used herein, a “conservative amino acids substitution” is when one amino acid residue has been replaced by another with a similar sidechain. The art identifies several families of amino acid residues with similar side chains. These include basic side chains like lysine and arginine or glutamine as well as acidic side chain (e.g. aspartic acid, glutamic acids), uncharged polar sidechains (e.g. glycine asparagine or glutamine), nonpolar sidechains (e.g. alanine and valine), isoleucine, proline), phenylalanine) and aromatic sidechains such as tryptophan, tyrosine toptophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, tidine, ttophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, trytophan, tyrosine It is preferable that no more than five conservative amino acids are substituted within the D2E7 and/or VH CDR3 domains. It is preferred that no more than three conservative amino acids substitutions are made within D2E7 VL or VH CDR3 domains. You should also avoid making conservative amino acid substitutions at positions that are critical for binding to the hTNF?. The interaction of hTNF with D2E7 VL CD3 positions 2 and 5 and D2E7 H CDR3 positions 1 and 7 seem to be crucial. Therefore, it is important to avoid making conservative amino acid substitutions at these positions. However, an alanine substitution at position 5, of the D2E7 L CDR3, would be acceptable (see U.S. Pat. No. 6,090,382).”

“Accordingly, another embodiment of the invention provides methods for treating disorders that require the administration of anti-TNF. Subcutaneous, biweekly administration of an antibody or antigen binding portion from a human is a good way to obtain antibody. Preferably, the antibody or antigen binding portion of it contains the following characteristics:

“b”) has a light-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 3. It can be modified from SEQID NO: 3 by one alanine substitution at position 1, 4, 5 or 8, or by one to five conservative amino acids substitutions at positions 1, 3, 4, 6, 7 and/or 9.

“c” has a heavy-chain CDR3 domain that contains the amino acid sequence SEQ ID No: 4. Modified from SEQ ID No: 4 by one alanine substitution at position 2, 4, 5, 6, 8, 9 or 11, or by one to five conservative amino acids substitutions at positions 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

“Another embodiment of the invention offers methods for treating disorders that require anti-TNF administration. Subcutaneous, biweekly administration of an antibody or an antigen binding portion is beneficial. A light chain variable area (LCVR), which contains the amino acids sequence of SEQID NO: 3, modified from SEQID NO: 3 by one alanine substitution, and a heavy chain variable (HCVR) with a CDR3domain comprising the amino sequence of the SEQID NO: 4, modified from the SEQID NO: 4 by one alanine substitution, at positions 2, 3, 4, 6, 8, 9, 10, or 11. The LCVR has a CDR2domain that contains the amino sequence of the SEQ ID No: 5, (i.e. the D2E7VL CDR2), and the HCVR has a CDR2domain that comprises the amino sequence of the SEQ ID ID NO: 6(i.e. the D2E7VH CDR2). The LCVR also has a CDR1domain that contains the amino sequence of the SEQ ID No: 7 (i.e. the D2E7 CDR1), and the HCVR has a CDR1domain that contains the amino sequence of the SEQ ID no: 8 (i.e. the D2E7 H CDR1). VL’s framework regions preferably come from the V?I human germline, but more preferably from A20 human germline Vk genes and most preferably from D2E7 VL frame sequences as shown in FIGS. 1A and 1B in U.S. Patent. No. 6,090,382. Preferably, the VH framework regions are from VH3 Human Germline Family. More preferably, they are from DP-31 Human germline VH gene. Most preferably, the D2E7 VH frame sequences shown in FIGS. 2A and 2B U.S. Patent No. 6,090,382.”

“Another embodiment of the invention offers methods for treating disorders that require anti-TNF administration. Subcutaneous, biweekly administration of an antibody or an antigen-binding portion of the isolated human antibody can be beneficial. Preferably, the antibody or antigen binding portion of the antibody contains a light-chain variable region (LCVR), which includes the amino acids sequence of the SEQ ID No: 1 (i.e. the D2E7 LL) and a heavier chain variable area (HCVR), which comprises the amino acids sequence of the SEQ ID no: 2 (i.e. the D2E7 H). In some embodiments, the antibody includes a heavy-chain constant region such as an IgG1, IgG2, IgG3, IgG4, IgG5, IgG4, IgG3, IgG3, IgG4, IgG5, IgE, IgM, or IgD constant area. The IgG1 or IgG4 heavy chains constant regions are preferred. The antibody may also contain a light-chain constant region. This could be either a kappa or lambda light-chain constant region. The antibody should consist of a kappa-light chain constant region. Alternately, the antibody can be a Fab fragment, or a single-chain Fv fragment.

“In other embodiments, the invention offers methods for treating disorders that require the administration of anti-TNF. Subcutaneous administration of an antibody or antigen-binding portion can be done biweekly. Antibody or the antigen-binding part thereof should contain D2E7-related VL or VH CDR3domains.

“An antibody, or a portion thereof, of the invention may be derivatized and linked to another functional molecular (e.g. another peptide or proteins). The invention’s antibodies and antibody parts can be derivatized or linked to other functional molecules (e.g., another peptide or protein). antibodies described herein, including immunoadhesion molecules. An antibody or part of an antibody described herein can be functionally linked to other molecular entities (e.g., chemical coupling, genetic fuse, noncovalent association or any other way), such as another antibody, a cytotoxic or pharmaceutical agent and/or a protein peptide that can act as a mediator between the antibody or portion of the invention with another molecule (e.g., streptavidin core area or a polyhistidine label).

Crosslinking multiple antibodies of the same or different type to make a derivatized type of antibody can create a bispecific type. Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). These linkers can be purchased from Pierce Chemical Company in Rockford, Ill.

Fluorescent compounds are useful detectable agents that can be used to derivatize an antibody or a portion thereof. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. A detectable enzyme can be used to derivatize an antibody, including horseradish peroxidase or glucose oxidase. An antibody can be derivatized using a detectable enzyme by adding additional reagents to the enzyme’s reaction product. If horseradish peroxidase has been detected, the addition of hydrogenperoxide and diaminobenzidine will result in a colorless reaction product that can be detected. A biotin derivatization may be used to detect an antibody. This can be done by indirect measurement of streptavidin binding or avidin.

“II. Expression of Antibodies

Recombinant expression in a host cell of immunoglobulin heavy and light chain genes can create an antibody or a portion thereof. A host cell is transfected using one or more recombinant vectors that contain DNA fragments encoding the antibody’s immunoglobulin heavy and light chains. The host cell then expresses the antibody recombinantly. Standard recombinant DNA methods are used to obtain heavy and light chains of antibody genes. These genes are then incorporated into recombinant expression Vectors and introduced into host cells. (eds.) (eds.) Current Protocols in Molecular Biology. Greene Publishing Associates. (1989). Also in U.S. Pat. No. 4,816,397 by Boss et al.”

To express D2E7, or a D2E7 related antibody, you must first obtain DNA fragments that encode the light and heavy chains variable regions. These DNAs are obtained using the polymerase chains reaction (PCR) to amplify and modify germline light- and heavy-chain variable sequences. The art has a number of germline DNA sequences that are suitable for human heavy and light-chain variable genes (see, e.g. the?Vbase?). Human germline sequence database. See also Kabat, E. A. et al. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. 91-3242; Tomlinson, I. M., et al. (1992)?The Repertoire Human Germline VH Sequences Reveals About Fifty Groups Of VH Segments With Different Hypervariable Loops J. Mol. Biol. 227:776-798. Cox, J. P. L. and others. (1994)?A Directory of Human Genem-line V78 Segments Reveals Strong Bias in Their Use? Eur. J. Immunol. 24:827-836. The contents of each are included herein by reference. Standard PCR is used to amplify a DNA fragment that encodes the D2E7 heavy chain variable region or an antibody related to D2E7. Amplification is most preferred for the DP-31 VH gene sequence. Standard PCR is used to amplify a DNA fragment that encodes the D2E7 light chain variable region or an antibody related to D2E7. Amplification of the A20 VL germline sequence would be preferred. Using standard methods, you can create PCR primers that are suitable for amplifying the DP31 germline VH or A20 germline L sequences.

These sequences can be mutated once the germline VH or VL fragments have been obtained to encode the D2E7 and D2E7-related sequences of amino acids disclosed herein. First, the VH and VL sequences of the amino acids are compared with the D2E7 and D2E7-related VH or VL amino sequences to identify any amino acid residues that are different from the germline D2E7 and D2E7-related sequence. Next, the appropriate nucleotides from the germline DNA sequences must be mutated so that the D2E7/D2E7-related sequence of amino acids is encoded. The genetic code will determine the nucleotide modifications to be made. The standard methods for mutagenesis of germline sequences include PCR-mediated mutagenesis (where the mutated nucleotides in the PCR primers are incorporated so that the PCR product contains mutations), and site-directed mutagenesis.

“Once DNA fragments encoding D2E7 and D2E7-related DH and D2E7 VL segments have been obtained (by amplification of germline VH or VL genes, described above), these DNA pieces can be further modified by standard recombinant techniques, such as to convert variable region genes into full-length antibody chains genes, Fab fragment genes, or to a SCFv gene. A VL- or VH encoding DNA fragment can be operatively linked with another DNA fragment, such as an antibody constant area or a flexible connecter, in these manipulations. In this context, the term “operatively linked” is used to refer to the joining of two DNA fragments so that their amino acid sequences are preserved in-frame.

“The VH region’s DNA can be isolated and converted into a full-length, heavy-chain gene by linking it to another DNA molecule that encodes heavy chain constant regions (CH1,CH2 or CH3). The sequences of human genes encoding the heavy chain constant regions are well-known (see E. A. Kabat et al. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. Standard PCR amplification can yield DNA fragments encompassing these areas. You can choose to have the heavy chain constant area as an IgG1, IgG2, IgG3, IgG4, IgA or IgE constant region. However, it is preferred that you have an IgG1 constant region. The VH-encoding DNA of a Fab fragment can be operatively linked with another DNA molecule encoding the heavy chain CH1 constant area.

“The VL region-encoding DNA can be transformed to a full-length light-chain gene (as a well as a Fab light chain gene) by linking the VL-encoding DNA with another DNA molecule that encodes the light-chain constant region, CL. The sequences of human light-chain constant region genes are well known (see E. A. Kabat et al. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. Standard PCR amplification can yield DNA fragments encompassing these areas. You can choose to have the light chain constant region as either a lambda or kappa region, but it is more preferred to be a region that is kappa.

“To create a SCFv gene, VH- and VL encoding DNA fragments must be operatively linked with another fragment encoding an amino acid sequence (Gly4Ser)3. This allows the VH and VL sequences to be expressed as a single-chain contiguous protein, with the VL, and VH regions joining by the flexible linker (see Bird et. al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).”

“To express antibodies, or the invention’s antibody portions, DNAs encoding partial- or full-length light or heavy chains are inserted into expression vectors so that they are operatively linked with transcriptional and translational control sequencings. The term “operatively linked” is used in this context. The term?operatively linked? refers to the fusion of an antibody gene into a vector in order that the transcriptional and translational control sequences contained within the vector fulfill their intended functions of controlling the transcription and expression of the antibody gene. The expression vector and control sequences must be compatible with the host cell being used. Either the antibody light chain gene or the antibody heavy chain genes can be placed in a separate vector, or both genes can be placed into the same vector. Standard methods are used to insert the antibody genes into the expression vector. These include ligation of complementary restriction sites on the fragment or vector of the antibody gene, or blunt end-ligation if there are no restriction sites. The expression vector may contain existing antibody constant region sequences before the D2E7 and D2E7-related light chain or heavy chain sequences are inserted. One way to convert the D2E7 and D2E7-related VH or VL sequences into full-length antibodies genes is to insert them in expression vectors that already encode heavy chain constant and/or light chain constant regions. The VH segment should be operatively linked with the CH segment(s) within vector, and the VL segment to the CL segment within vector. The signal peptide can be encoded in the recombinant expression vector to facilitate the secretion from a host cells. The vector can be cloned with the antibody chain gene so that the signal protein is linked in-frame to its amino terminus. You can choose to clone the signal peptide as an immunoglobulin signal protein or as a heterologous signalpeptide (i.e. a signal from a non-immunoglobulin peptide).

“In addition to the antibody chains genes, the regulatory sequences in the invention recombinant expression channels contain regulatory sequences that regulate the expression of antibody chain genes within a host cell. “Regulatory sequence” is a term that refers to the following: The term “regulatory sequence” refers to promoters, enhancers, and other expression control elements (e.g. polyadenylation signal) that regulate the translation or transcription of the antibody chain gene genes. These regulatory sequences can be found in Goeddel’s Gene Expression Technology: Methods in Enzymology, 185, Academic Press. San Diego, Calif. (1990). The art of designing expression vectors, including selecting regulatory sequences, is dependent on factors such as the type of host cell being transformed and the desired level of protein expression. The preferred regulatory sequences for mammalian hosts include viral elements that can direct high levels in protein expression in mammalian cell. These include promoters and/or enhancements derived from cytomegalovirus CMV (such the CMV promoter/enhancer), Simian Virus40 (SV40), adenovirus (e.g. the adenovirus major later promoter (AdMLP), and polyoma. U.S. Pat. provides more information about viral regulatory elements and their sequences. No. No. No. 4,510,245 as reported by Bell et.al. U.S. Pat. No. 4,968,615 by Schaffner et al.”

“In addition to regulatory sequences and antibody chain genes, recombinant expression viruses of the invention can contain additional sequences such as sequences that regulate the replication of the vector within host cells (e.g. origins of replication) or selectable marker genes. The selectionable marker gene allows for the selection of host cells in which the vector can be introduced (see, e.g. U.S. Pat. Nos. Nos. The selectable marker gene is usually able to confer resistance to drugs such as G418, methotrexate, hygromycin, or G418, on the host cell in which it has been introduced. The preferred selectable marker genes are the dihydrofolate reducetase gene (DHFR) (for use with dhfr). host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).”

“For expression of light and heavy chains, standard methods are used to transfect the expression vector(s), encoding heavy and light chains into host cells. There are many ways to define the term “transfection”. The various forms of the term “transfection” are meant to cover a variety of methods used for the introduction exogenous DNA into prokaryotic and eukaryotic host cells. Though it is theoretically possible for the invention to be expressed in prokaryotic and eukaryotic host cell, the preferred method of expression is in eukaryotic, most preferably mammalian, host cells. This is because eukaryotic, in particular mammalian, cells are more likely to produce and secrete an immunologically active, properly folded antibody. According to Wood, C.R. (1985), Immunology Today 6-13, prokaryotic expressions of antibodies genes have been shown to not be effective in producing high yields active antibodies.

“Chinese Hamster Ovary (CHO) cells are the preferred mammalian host cell for the expression of the recombinant antibody of the invention (including dhfr CHO cells, as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77.4216-4220, used in conjunction with a DHFR selectable markers, e.g. as described by R. J. Kaufman, and P. A. Sharp (1982). Mol. Biol. Biol. Recombinant expression vectors that encode antibody genes are introduced to mammalian host cell cells. The antibodies are made by cultivating the host cells for a sufficient time to allow the expression or secretion of the antibody within the culture medium. Standard protein purification methods can be used to recover antibodies from culture medium.

“Host cells may also be used to make intact antibodies. You will understand that there are many variations to the above process. It may be possible to transfect host cells with DNA that encodes either the light or heavy chains (but not both) for an antibody of the invention. Recombinant DNA technology can also be used to eliminate some or all the DNA that encodes either one or both of the heavy and light chains. This is not required for binding to the hTNF?. The antibodies of the invention also include molecules expressed from these truncated DNA molecules. Bifunctional antibodies can also be made in which one of the heavy and one of the light chains are an antibody according to the invention, and the other is specific for another antigen than hTNF. Crosslinking an invention antibody to another antibody using standard chemical crosslinking techniques.

“In a preferred method for recombinantly expression of an antigen, or antigen binding portion thereof, a recombinant vector encoding both of the antibody heavy chains and the antibody light chains is introduced into dhfrCHO cells via calcium phosphate-mediated transcription. The recombinant vector contains both the heavy and light chains genes. This allows for high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. Transformed host cells from selected recipients are grown to express the antibody heavy and lightweight chains. The culture medium is then used to recover the intact antibody. Standard molecular biology methods are used to create the recombinant expression virus, transfect host cells, select transformants, culture host cells, and then recover the antibody from culture medium.

“III. “III. Selection of Recombinant Human Antibodies

“Recombinant human antibody of the invention, in addition to D2E7, an antigen binding section thereof, or D2E7 related antibodies, can be isolated by screening a recombinant combinatorial library, preferably one scFv, made using VL and VH DNAs prepared from mRNA obtained from human lymphocytes. These libraries can be prepared and screened using known methods. There are also commercially available kits that can be used to generate phage display libraries, such as the Pharmacia Recombinant Phage Antibody System (catalog no. 27-9400-01; Stratagene SurfZAP phage display kit, catalog no. 240612), are examples of methods and reagents that can be used in generating or screening antibody display libraries. See, for instance, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. Publication no. WO 92/18619; Dower et al. PCT Publication Number. WO 91/17271; Winter et al. No. WO 92/20791; Markland et al. PCT Publication Number. WO 92/15679; Breitling et al. PCT Publication Number. WO 93/01288; McCafferty et al. No. WO 92/01047; Garrard et al. PCT Publication Number. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; 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; Barbas et.al. (1991) PNAS:7978-7982

“In a preferred embodiment to isolate human antibodies with high affinity? and a low rate constant for hTNF?, we use a murine anti hTNF? antibody with high affinity and low off rate constants for hTNF (e.g. MAK 195 hybridoma, deposit number ECACC 87 050801), is used first to select human heavy- and light-chain sequences with similar binding activity towards hTNF? using the epitope printing methods described in Hoogenboom et. al., PCT Publication Number. WO 93/06213. Preferably, the antibody libraries used in this procedure are scFv libraries that have been prepared and screened according to McCafferty et., PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990) 348:552-554; and Griffiths et al., (1993) EMBO J. 12:725-734. The scFv antibodies libraries should be screened with recombinant human TN? as the antigen.”

“Once the initial human VL segment and VH segments have been selected, mix and match? Different pairs of VL and H segments from the initial selection are tested for hTNF in experiments. To select the most desirable VLNH pairing combinations, binding is performed. To further increase the affinity and/or lower down the off rate constant of hTNF, The VL and/or VH segments of the preferred pair(s of VLNH) can be randomly modified, preferably in the CDR3 region or VL. This process is analogous to in vivo somatic mutation processes that result in affinity maturation. In vitro affinity maturation can also be achieved by amplifying the VH and L regions with complementary PCR primers to the VH CDR3 and VL CDR3, respectively. These primers have been “spiked?” With a random mix of the four nucleotide base bases at specific positions so that the PCR products encode VH or VL segments into the which random mutations were introduced into the VH or VL CDR3 areas. These VH and L segments randomly mutated can be screened for binding to the hTNF. sequences with high affinity and low off rates for hTNF? You can choose to bind.”

“Following screening, isolation of an anti-hTNF The invention can be obtained from a recombinant immuneglobulin display database. Nucleic acid encoding selected antibody can also be extracted from the display package (e.g. from the phage genome). Subcloning into other expression vectors can be done using standard recombinant DNA methods. The nucleic acids can be further modified to create additional antibody forms. The DNA encoding an antibody from a human is cloned into the recombinant expression vector. This vector can then be introduced into mammalian host cells as described in Section II.

“IV. “IV.

“The invention’s antibodies and portion of the invention may be included in pharmaceutical compositions that can be administered to a subject according to the methods described herein. The pharmaceutical composition typically contains an antibody (or portion thereof) from the invention, methotrexate, and a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” is defined herein. Any and all solvents and dispersion media, coatings and antibacterial and/or fungal agents, as well as isotonic or absorption delay agents and the like, that are physiologically compatible, are acceptable for administration to a subject in accordance with the methods described herein. One or more of the following pharmaceutically acceptable carriers are examples: water, saline and phosphate buffered saltsaline; dextrose; glycerol; ethanol, and combinations thereof. It is preferable to include isotonic substances, such as sugars, polyalcohols like mannitol or sorbitol in many cases. A small amount of pharmaceutically acceptable carriers can also contain auxiliary substances, such as preservatives or buffers, wetting agents, emulsifying or buffers that enhance the shelf-life or effectiveness of the antibody.

“The compositions described in this invention can be made in many forms. These can include liquid, semi-solid, and solid dosage forms such as dispersions, suspensions, dispersions, tablets, pills and powders. The intended use and therapeutic purpose of the preferred form will determine which one is chosen. The preferred compositions are either injectable or infusible, similar to compositions used for passive immunization. Parenteral administration is preferred (e.g. intravenous subcutaneous, intraperitoneal and intramuscular). Preferably, the antibody can be administered intravenous injection or infusion. Another preferred embodiment is intramuscular injection. A preferred embodiment of the antibody is administered subcutaneously (e.g., biweekly subcutaneous injection).

“Therapeutic compositions must be stable and sterile under all conditions. You can formulate the composition as a microemulsion or dispersion, liposome or other orderly structure that is suitable for high drug concentrations. You can prepare sterile injectable solutions by combining the active compound (i.e. the antibody or portion thereof) with the appropriate solvent. Then, filter sterilization is performed. Dispersions can be prepared by adding the active compound to a sterile medium that includes the basic dispersion medium as well as the other required ingredients. For the preparation of sterile injection solutions, vacuum drying and freeze drying are preferred methods. This produces a powder of active ingredient as well as any desired ingredients from a previously sterilized solution. A coating, such as lecithin or surfactants, can help maintain the fluidity of a solution. You can prevent prolonged absorption of injectable formulations by including an agent that delays absorption (e.g. monostearate salts or gelatin).

Although there are many methods for administering the antibodies and their portions, subcutaneous injection is the preferred method of administration. The route and/or method of administration will depend on the desired results. The active compound can be prepared in certain embodiments with a carrier to prevent rapid release. This could include implants, microencapsulated delivery systems, or transdermal patches. You can use biodegradable and biocompatible polymers such as ethylene vinyl, polyethylene glycol, (PEG), polyanhydrides or polyglycolic acids, collagen, polyorthoesters and polylactic acid. Many of the methods used to prepare such formulations have been patented. See, e.g., Sustained Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

“In some embodiments, the antibody or portion of an invention can be administered orally, for example with an inert or edible carrier. You can also include the compound and other ingredients in a capsule or tablet, or compress them into tablets. The compounds can be combined with excipients to be used for oral therapeutic administration. They may be taken as tablets, buccal tablets or capsules. It may be necessary to co-administer a compound of invention with a material to inhibit its inactivation to administer the compound other than via parenteral administration.

Supplementary active compounds may also be included in the compositions. An antibody or a portion thereof may be coformulated with or coadministered by one or more therapeutic agents in certain embodiments. An example of this is an anti-hTNF. An anti-hTNF antibody may be combined with methotrexate and/or administered in combination with one or more other antibodies (e.g. antibodies that bind to other cytokines or cell surface molecules), one cytokines, or soluble TNF? See e.g. PCT Publication Number. WO 94/06476), and/or one or several chemical agents that inhibit the hTNF. Production or activity (e.g. cyclohexaneylidene derivatives, as described in PCT Publication No. WO 93/19751. One or more of these therapeutic agents may also be combined with one or more of the antibodies described in the invention. Combination therapies can be advantageous because they use lower doses of therapeutic agents. This avoids potential toxicities and complications that may arise from monotherapies. In subsection IV, we discuss the use of the antibody, or portions thereof, in combination with other therapeutic drugs.

Non-limiting examples of therapeutic agents to treat rheumatoidarthritis with which an antibody or a portion of an invention can be combined are: non-steroidal antiinflammatory drug(s), cytokine suppressive drug(s), CSAIDs; CDP-571/BAY-10-33556 (humanized antiTNF). Celltech/Bayer; cA2 [chimeric anti-TNF antibody; Centocor] 75 kdTNFRIgG (75 kD IgG fusion protein, Immunex; Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. (1996) Vol. 38, S 185; DAB 486 -IL-2 and/or DAB389 -IL-2 (IL-2 fusion protein; Seragen); see e.g. Arthritis & Rheumatism (1993). Vol. 36, 1223); Anti-Tac (humanized anti-IL-2R? ; Protein Design Labs/Roche; IL-4 (antiinflammatory cytokine, DNAX/Schering); IL-10 SCH 52000 (recombinant IL-10; DNAX/Schering); IL-1RA; IL-1 receptor antagonist, Synergen/Amgen; TNF-bp/sTNFR (soluble TNF binding proteins; see e.g. Arthritis & Rheumatism, Vol. 39, No. 9 (supplement), 5284; Amer. J. Physiol. Physiol. 268, pp. 268 pp. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic (inhibitor plasminogen activation; e.g. Arthritis & Rheumatism (1996) Vol. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 39, No. 7, pp. 12, pp. 39, No. 39, No. 39, No. 39, No. converting enzyme; zap70 and/or lck inhibit (inhibitor the tyrosine kinase zap70 or lck); VEGF inhibition and/or VEGFR inhibitor (inhibitors or receptors for vascular endothelial cells growth factor; inhibitors of angiogenesis); corticosteroid antiinflammatory drugs (e.g. SB203580); anti-IL-12 antibody; TNF-convertase inhibitors; TNF-convertase; IL-12; interleukin-11; Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S120); gold; penicillamine; chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; orally-administered peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.”

Non-limiting examples of therapeutic agents to treat inflammatory bowel diseases with which an antibody or a portion of the invention could be combined include: budenoside, epidermal growth factors; corticosteroids, cyclosporins, sulfasalazine, aminosalicylates, 6-mercaptopurine, metronidazole, lipoxygenase inhibitions, mesalamine, olsalazine, balsalazine, IL-1 receptor antagonists, anti-IL-1 monoclonal antibody; anti-IL-6 monoclonal antibody; growth factors; elastase inhibitors; pyridinyl?imidazole compounds CDP-571/BAY-10-333356 (humanized antiTNF). antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kdTNFRIgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 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 agonists (e.g., agonist antibodies); interleukin-11; glucuronide- or dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); slow-release mesalazine; methotrexate; antagonists of Platelet Activating Factor (PAF); ciprofloxacin; and lignocaine.”

“Non-limiting examples of therapeutic agents to multiple sclerosis with the which an antibody or a portion of an invention can be combined include: prednisolone, methylprednisolone, azathioprine, cyclophosphamide, methotrexate, cyclophosphamide, cyclosporine, cyclophosphamide, cyclosporine, methotrexate; 4-aminopyridine, tizanidine, interferon-?1a (Avonex?) ; Biogen); interferon-?1b (Betaseron? ; Chiron/Berlex; Copolymer 1; Copaxone; Copolymer 2? Teva Pharmaceutical Industries Inc.; hyperbaric oxygen; intravenous immuneglobulin; Clabribine; CDP-571/BAY-10-33556 (humanized antiTNF). antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kDTNFR-IgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. Vol.

“Non-limiting examples of therapeutic agents to treat sepsis that an antibody or a portion thereof can be combined with include: antibiotics; hypertonic saline solution; intravenous gamma globulin (IgG); continuous hemofiltration; carbapenems, e.g., meropenem); antagonists cytokines like TNF?, IL-113, IL-6, IL-8; CDP-571/BAY-10-33556 (humanized anti-TNF) antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kdTNFRIgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular peptide; SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (Picower Institute); Tissue Factor Pathway Inhibitor (TFPI; Chiron); PHP (chemically modified hemoglobin; APEX Bioscience); iron chelators and chelates, including diethylenetriamine pentaacetic acid-iron (III) complex (DTPA iron (III); Molichem Medicines); lisofylline (synthetic small molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan (aqeuous soluble ?1,3glucan; Alpha-Beta Technology); apolipoprotein A-1 reconstituted with lipids; chiral hydroxamic acids (synthetic antibacterials that inhibit lipid A biosynthesis); anti-endotoxin antibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.); rBPI21 (recombinant N-terminal fragment of human Bactericidal/Permeability-Increasing Protein); and Synthetic Anti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories);”

Nonlimiting examples of therapeutic agents to treat adult respiratory distress syndrome (ARDS), with which an antibody or a portion of the invention could be combined are: anti-IL-8 antibody; surfactant therapy; CDP-571/BAY-10-33556 (humanized antiTNF). antibody; Celltech/Bayer); cA2 (chimeric anti-TNF? antibody; Centocor; 75 kdTNFRIgG (75 kD IgG TNF receptor-IgG fusion proteins; Immunex; see e.g. Arthritis & Rheumatism (1994), Vol. 37, S295; J. Invest. Med. (1996) Vol. Vol.

“The compositions of the pharmaceutical invention may contain a?therapeutically efficacious amount? Or a?prophylactically efficacious amount? An antibody or a portion of the invention. Therapeutically effective amounts? A therapeutically effective amount is one that is effective at the prescribed dosages and over the required time to achieve the desired therapeutic effect. The therapeutically effective amount or portion of an antibody may differ depending on factors like the person’s age, gender, and health. It also depends on the individual’s ability to produce the desired response. Therapeutically effective amounts are those that have no toxic or harmful effects. They also include therapeutically beneficial effects. What is a ‘prophylactically efficient amount? A prophylactically effective amount is one that can be administered in the dosages and over the time required to produce the desired prophylactic effect. The prophylactically-effective amount is typically lower than the therapeutically-effective amount, as it is usually administered to subjects at an earlier stage of the disease.

The dosage regimens can be altered to achieve the desired result (e.g., therapeutic or prophylactic). One bolus can be given, or several doses may be divided over time. The dose may also be adjusted to meet the needs of the therapeutic situation. For ease of administration and uniform dosage, it is particularly advantageous to create parenteral compositions using dosage unit form. As used herein, dosage unit form refers to physically distinct units that can be used as unitary doses for mammalian subjects. Each unit contains a predetermined amount of active compound to produce the desired therapeutic effect when combined with the appropriate pharmaceutical carrier. Specification for dosage units forms according to the invention is determined by and directly dependent upon (a) the unique characteristics and prophylactic effects of the active compound, and (b), the limitations inherent in the art and science of compounding such active compounds for treatment of sensitive individuals.

“An example, non-limiting range of therapeutically or prophylactically efficacious amount of an antibody/antibody portion of the invention is 10-100 mg, more preferably 20-40 mg, and most preferably around 40 mg. Dosage values can vary depending on the severity and type of the condition being treated. It should also be noted that specific dosage regimens for any subject should be adjusted over time depending on the individual need and professional judgment of those administering or supervising the administration. The dosage ranges herein are only an example and do not limit the practice or scope of the claimed composition.

“V. Uses for the Antibodies to the Invention”

“Given their binding ability to hTNF, the anti-hTNF is? The invention allows for the detection of hTNF using antibodies or portions thereof. (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method of detecting hTNF. In a biological specimen, the method involves contacting the biological sample with an antibody or antibody portion of the invention, and detecting the antibody (or portion) bound to the hTNF? Unbound antibody (or a portion thereof) to detect hTNF. The biological sample. To facilitate detection of bound or unbound antibodies, the antibody can be directly or indirectly labeled using a detectable substance. There are many suitable detectable substances, including prosthetic groups, enzymes, fluorescent materials, luminescent material and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ?-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125I, 131I, 35S or 3H.”

“Alternative to the labeling of the antibody, hTNF?” Can be tested in biological fluids using a competition immunoassay that uses rhTNF Standards labelled with detectable substances and unlabeled anti hTNF? antibody. The biological sample is the one that has been labeled with rhTNF. The standards and the anti -hTNF are combined. The amount of labeled anti-hTNF is determined by combining the two. The standard bound to unlabeled antibodies is determined. How much hTNF is in the biological sample? The amount of hTNF in biological samples is proportional to their labeled levels. What is the standard bound to anti-hTNF antibody.”

“A D2E7 antibody can be used to detect TNF’s from other species, such as primates (e.g., monkey, baboon and marmoset), pig, and mouse. D2E7 is able to bind to each TNF?”

“The invention’s antibodies and anti-tumor components are capable of neutralizing the hTNF?” activity in vitro as well as in vivo (see U.S. Pat. No. 6,090,382). D2E7 and at least some other antibodies in the invention can neutralize hTNF. activity from other species. The invention’s antibody and antibody parts can therefore be used to inhibit the hTNF. activity in cell cultures containing hTNF, or in mammalian subjects with TNF?s with whom an antibody of this invention crosses-reacts (e.g. chimpanzees, marmosets, cynomolgus, rhesus, pig, and mouse. The invention is a method of inhibiting TNF in one embodiment. activation that involves contacting TNF? With an antibody or a portion of the invention so that TNF? activation is blocked. The TNF is preferred. Is human TNF?. To inhibit hTNF, you can add an antibody or portion of the invention to a cell culture that contains, or is suspected to contain, TNF?. “Activity in the culture.”

“In a preferred embodiment of the invention, methods are provided for treating disorders that require anti-TNF administration. It is possible to subcutaneously give the subject an antibody or a portion of the invention biweekly. This will make the disorder easier to treat. The preferred embodiment of the antibody is given subcutaneously on an every other week basis. Another preferred embodiment is to administer the antibody subcutaneously prior, during, or after administrations of methotrexate. The subject should be a human. You can also use a mammal that expresses TNF. An antibody of the invention can cross-react with it. Further, the subject could be a mammal in which hTNF has been introduced? (e.g. by administration of hTNF.c or expression of an hTNF. transgene). The invention allows for the administration of an antibody to human subjects for therapeutic purposes. An antibody of the invention may also be administered to non-human mammals that express TNF. The antibody can cross-react with any animal (e.g., a primate or pig, or mouse) to test for human disease or to veterinary purposes. These animal models can be used to evaluate the therapeutic efficacy and administration times of antibodies.

“A disorder in which an anti-TNF agent is administered?, as used herein. Is antibody beneficial? TNF is present in diseases and other conditions. If a person suffering from the disorder is shown or suspected to be responsible for the disease’s pathophysiology or contributing to its worsening, or where another anti-TNF antibody has been found to be effective, The disease has been successfully treated with an antibody or a biologically active component. TNF-inhibiting disorders are those in which TNF is not present. Activity is considered to be harmful if inhibition of TNF is involved. Activity is expected to reduce the severity and/or progression of the condition. TNF may increase in these cases. In a biological fluid of a person with the disorder (e.g. an increase in TNF concentration). in serum, plasma, synovial fluid, etc. Anti-TNF can detect these antibodies. As described above, TNF can be used to treat many disorders. TNF? activity can be harmful. Below is more information about the use of antibodies and the antibody portions of the invention for the treatment of certain disorders:

“A. Sepsis”

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