MXPA98006347A - Human antibodies that link human tnfalfa - Google Patents

Abstract

The present invention relates to human antibodies, preferably human recombinant antibodies that specifically bind to a facor of human tumor necrosis alpha (hTNFalfa). These antibodies have high affinity for hTNFalpha (eg Kd = 10-8 M or less), a slow dissociation regime for dissociation of hTBFalpha (eg Koff = 10-3 sec-1 or less) and neutralize the activity of hTNFalpha in vitro and in vivo An antibody of the invention can be a full length antibody or an antigen binding portion thereof. The antibodies or antibody portions of the invention are useful for detecting hTNFalpha and for inhibiting the activity of hTNFalpha e.g. in human patients suffering from a disorder in which the activity of hTNFalfa is detrimental. The nucleic acids, vectors and host cells for expressing the recombinant human antibodies of the invention and the methods for sintering recombinant human antibodies are also encompassed by the invention.

Description

'HUMAN ANTIBODIES THAT LINK HUMAN TNFa' BACKGROUND OF THE INVENTION Tumor necrosis factor a (TNFa) is a cytokine produced by numerous cell types, including monocytes and macrophages, which was originally identified based on its ability to induce necrosis of certain mouse tumors (see eg L. Oíd (1985)). ) Science 230: 630-632). Subsequently, a factor called cachectin, associated with cachexia, was shown to be the same molecule as TNFa. TNFa has been implicated in intervening in the shock (see eg B. Beutler and A. Cerami (1988) Annu Rev. Biochem 57: 505-518; B. Beutler and A. Cerami (1989) Annu. Rev. Immunol 7: 625-655). In addition, TNFa has been implicated in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease (see, eg, A. Moeller et al. ) Cytokine 2: 162-169, US Patent Number 5,231,024 issued to Moeller et al, European Patent Publication No. 260 610 Bl of A. Moeller et al., P. Vasilli (1992) Annu. Rev. Immunol., 10: 411 -452; KJ and A. Cerami (1994) Annu. Rev. Med. 45: 491-503).

Due to the deleterious role of human TNFα (hTNFα) in a variety of human disorders, therapeutic strategies have been designed to inhibit and counteract the activity of hTNFα. In particular, antibodies that bind to, and neutralize the hTNFa that they have sought as a means of inhibiting the activity of hTNFa. Some of the first of these antibodies were mouse monoclonal antibodies (mAbs) secreted by hybridomas prepared from mouse lymphocytes immunized with hTNFa (see, eg, T. Hahn et al. (1985) Proc Nati Acad Sci USA 82_: 3814-3818 CM Liang et al. (1986) Biochem. Biophys. Res. Commun. 13: 847-854; M. Hirai et al. (1987) J. Immunol. Methods £ 6: 57-62; BM Fendly et al. (1987) Hybridoma. 6: 359-370: A. Moeller et al. (1990) Cytokine 2: 162-169; US Patent Number 5,231,024 issued to Moeller et al .; European Patent Publication No. 186 833 Bl of D. Wallach; of European Patent Number 218 868 Al de Oíd et al, European Patent Publication Number 260 610 Bl of A. Moeller et al.). Even though these mouse anti-hTNFα antibodies have frequently exhibited high affinity for hTNFα (eg Kd = 10-9M) and were able to neutralize the activity of hTNFα, their use in vivo may be limited by the problems associated with the administration of antibodies. from mouse to beings human, such as half life of short serious, an inability to activate certain functions of the human effector and produce an unwanted immune response against a mouse antibody in a human (the reaction "of the human anti-mouse antibody" (HAMA) ). In an attempt to overcome the problems associated with the use of fully murine antibodies in humans, the anti-hTNFα murine antibodies have been genetically modified to be more "humanlike". For example, chimeric antibodies wherein the variable regions in the antibody chains are derived from murine and the constant regions of the antibody chains are derived from humans have been prepared (DM Knight et al. (1993) Mol. Immunol. : 1443-1453; PCT Publication Number WO 92/16553 by PE Daddona and others). In addition humanized antibodies wherein the hypervariable domains of the variable regions of the antibody are derived from murine, but the rest of the variable regions and constant regions of the antibody are derived from humans, have also been prepared (PCT Publication Number WO 92 / 11383 by JR Adair and others). However, because these chimeric and humanized antibodies still retain certain murine sequences, they can still provide an unwanted immune reaction, the anti-chimeric antibody reaction human (HACA), especially when administered for prolonged periods e.g. for chronic indications, such as rheumatoid arthritis (see, e.g., M.J. Elliott et al. (1994) Lancet 344: 1125-1127; M.J. Elliot et al. (1994) Lancet 344: 1105-1110). A preferred hTNFoc inhibitory agent for murine mAbs or derivatives thereof (e.g., chimeric or humanized antibodies) would be an entirely human anti-hTNFcc antibody, since this agent should not provide the HAMA reaction even if it were used for prolonged periods. Human monoclonal antibodies against hTNFa have been prepared using human hybridoma techniques (P. Boyle et al. (1993) Cell, Immunol., 152: 556-568 P. Boyle et al. (1993) Cell. Immunol., 152: 569-581; European Patent Application Publication Number 614 984 A2 by Boyle et al.). However, these hybridoma-derived monoclonal antibodies were disclosed as having an affinity for hTNFa that was too low to be calculated by conventional methods, and that were unable to bind to soluble hTNFa and were unable to neutralize the cytotoxicity induced by hTNFa ( see eg Boyle et al., supra). In addition, the success of the human hybridoma technique depends on the natural presence in human peripheral blood of lymphocytes that produce specific autoantibodies for hTNFa. Certain studies of serum autoantibodies against hTNFa have been detected in human patients (A. Fomsgaard et al. (1989) Scand. J. Immunol., 30: 219-223; K. Bendtzen et al. (1990) Prog. Leukocyte Biol. 10B : 447-452), while others do not (HG Leusch et al. (1991) J. Immunol. Ethods 139: 145-147). Alternatively, to naturally occurring anti-hTNFα antibodies will be a recombinant hTNFα antibody. Recombinant human antibodies that bind hTNFa with relatively low affinity (ie, Kd ~ 10 ~ 'M) and a rapid dissociation regime (ie, Kotf ~ 10"2 sec"!) Have been described (AD Griffiths et al. (1993) EMBO J. 12: 725-734). However, due to their relatively rapid dissociation kinetics these antibodies may not be suitable for therapeutic use. In addition, a recombinant human anti-hTNFα has been described which does not neutralize the activity of hTNFα but rather improves the binding of hTNFα to the cell surface and improves the internalization of hTNFα (A. Lidbury et al. (1994) Biotechnol. 5: 27-45; PCT Publication Number WO 92/03145 by R. Aston et al.). Accordingly, human antibodies, such as recombinant human antibodies that bind soluble hTNFa with high affinity and slow dissociation kinetics having the ability to neutralize the hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and cell activation induced by hTNFa, are still needed.

COMPENDIUM OF THE INVENTION This invention provides human antibodies, preferably recombinant human antibodies, which specifically bind to human TNFα. The antibodies of the invention are characterized by binding to hTNFα with high affinity and slow dissociation kinetics by neutralizing the activity of hTNFα, including hTNFα-induced cytotoxicity (in vitro and in vivo) and cellular activation induced by hTNFα. The antibodies of the invention are further characterized by binding to hTNFa, but not to hTNFp (lymphotoxin) and having the ability to bind to other TNFα primates and TNFα non-primates in addition to human TNFα. The antibodies of the invention may be full length (eg, an IgG1 or IgG4 antibody) or may comprise only a portion of antigen binding (eg, a fragment of Fab, F (ab or scFv). Especially preferred recombinant of the invention, designated D2E7, has a light chain CDR3 domain comprising the acidic amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR3 domain comprising an amino acid sequence of SEQ ID NO: 4. Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region of (HCVR). ) comprising the amino acid sequence SEQ ID NO: 2. In one embodiment, the invention provides an isolated human antibody or an antigen binding portion thereof, which is dissociated from human TNFa with a K of 1 x 10"° M. or less and a Koff regime constant of 1 x 10 ~ 3 s "1 or less, both determined by surface plasmon resonance and neutralizes the cytotoxicity of human TNFa in a normal in vitro L929 assay with an ICso of 1 x 1 CT7 M or less. More preferably, the human antibody isolated, or the antigen binding portion thereof, dissociates from TNFa with a Koff of 5 x 1 CT4 s "1 or less, or even more preferably, with an off of 1 x 10 ~ 4 s "1 or less. More preferably, the human antibody isolated from the antigen binding portion thereof neutralizes the cytotoxicity of human TNFa in a normal in vitro L929 assay with an IC50 of 1 x 10"μM or less, even more preferably with an ICso of 1 x 10"9 M or less and still especially preferably with an ICso of 5 x 10 ~ 10 M or less.

In another embodiment, the invention provides a human antibody, or an antigen binding portion thereof, with the following characteristics: a) it is dissociated from human TNFa with a Kotf of 1 x 10 ~ 3 s "1 or less, as determined by surface plasmon resonance, b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 or modified from SEQ ID NO: 3 by a single substitution of alanine at position 1, 4, 5, 7 or 8, by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9, c) has a heavy chain CDR3 domain comprising the sequence of amino acid of SEQ ID NO: 4, or that is modified of SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and / or 12. Most preferably the antibody, or the p The antigen binding site thereof is dissociated from human TNFa with a Kotf of 5 x 10"" s "1 or less. Still more preferably, the antibody, or the antigen binding portion thereof, is dissociated from human TNFa with a Koff of 1 x 10"4-s" 1 or less.

In yet another embodiment, the invention provides a human antibody or an antigen binding portion thereof with an LCVR having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified of SEQ ID NO: 3 by a single substitution of alanine at position 1, 4, 5, 7 or 8 and with an HDVR having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified of CDR3 SEQ ID NO: , by a single substitution of alanine at the 2, 3, 4, 5, 6, 8, 9, 10 or 11 position. Particularly preferably, LCVR also has a CDR2 domain comprising the amino acid sequence of SEQ ID NO. : 5 and additional HCVR has a CDR2 domain comprising the amino acid sequence SEQ ID NO: 6. Still especially preferred LCVR further has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 and HCVR has a domain of CDR1 comprising the amino acid sequence of SE Q ID NO: 8. In yet another embodiment, the invention provides an isolated human antibody, or an antigen binding portion thereof, with LCVR comprising the amino acid sequence of SEQ ID NO: l and HCVR comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the antibody has a heavy chain constant region of IgGl a heavy chain constant region IgG4. In still other modalities, the antibody is a fab of Fab, a F (ab) fragment or a single chain Fv fragment In still other embodiments, the invention provides antibodies, or antigen binding portions thereof, with LCVR having a CDR3 domain comprising an amino acid residue which is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or with an HCVR having a CDR3 domain comprising an amino acid sequence that is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28 ,, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. In yet another embodiment, the invention provides an isolated human antibody, or an antigen binding portion thereof, that neutralizes TNFc activity c human but not human TNFp (lymphotoxin). In a preferred embodiment, the human antibody or antigen binding portion thereof, neutralizes the activity of human TNF, chimpac TNFa and at least one additional primate TNF that is selected from the group consisting of of mandrel TNF, TNFa of titi, TNF cynomolgus and TNFa of rhesus monkeys. Preferably, the antibody also neutralizes the activity of at least one TNFα that is not a primate. For example, in a sdality, the isolated human antibody, or the antigen binding portion thereof, also neutralizes canine TNFa activity. In another sdality, the isolated human antibody or antigen binding portion thereof also neutralizes the activity of the pig TNFa. In yet another sdality, the isolated human antibody, or the antigen binding portion thereof also neutralizes the activity of mouse TNFa. Another aspect of the invention relates to nucleic acid molecules encoding the antibodies, or the antigen binding portions of the invention. A preferred nucleic acid of the invention encoding a D2E7 LCVR has the nucleotide sequence shown in Figure 7 and SEQ ID NO 36. Another preferred nucleic acid of the invention, which encodes a D2E7 HCVR, has the nucleotide sequence shown in Figure 8 and SEQ Id NO 37. The recombinant expression vectors carrying the nucleic acids encoding the antibody of the invention and the host cells into which these vectors have been introduced are also encompassed by the invention and are methods for producing the antibodies of the invention by culturing the host cells of the invention. Yet another aspect of the invention relates to methods for inhibiting human TNFα activity using an antibody, or the antigen binding portion thereof, of the invention. In one embodiment, the method comprises contacting human TNFα while the anti-serum of the invention or the antigen binding portion thereof such as the activity of human TNFα is inhibited. In another embodiment, the method comprises administering an antibody of the invention, or a binding portion of antigen thereof to a human patient suffering from a disorder wherein the activity of TNFa is deleterious such that the activity of human TNFa in the human patient it is inhibited. The disorder may be for example, sepsis, an autoimmune disease (e.g., rheumatoid arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome), an infectious disease, a malignant disease, transplant rejection or inj erto-versus-host, a pulmonary disorder, a bone disorder, an intestinal disorder or a cardiac disorder.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB show the amino acid sequences of the light chain variable region D2E7 (D2E7 VL shown also in SEQ ID N0: 1), the alanine scanning mutants of D2E7 VL (LD2E7 * .Al, LD2E7 *. A3 , LD2E7 *. A4, LD2E7 * .A5, LD2E7 * .A7 and LD2E7 * .A8), the light chain variable region of the D2E7-related antibody to 2SD4 (2SD4 VL, also shown in SEQ ID NO: 9) and other light chain variable regions related to D2E7 ((EP B12, VL10E4, VL100A9, VL100D2, VL10F4, LOE5, VLLOF9, VLL0F10, VLLOG7, VLLOG9, VLLOH1, VLLOH10, VL1B7, VL1C1, VL1C7, VL0.1F4, VL0.1H8) , LOE7.A and LOE7.T) Figure 1A shows the domains of FR1, CDR1, FR2 and CDR2 Figure IB shows the domains of FR3, CDR3 and FR4 The domains of light chain CDR1 ("CDR Ll") , CDR2 ("CDR L2") and CDR3 ("CDR L3") are in boxes Figures 2A and 2B show the amino acid sequences of the heavy chain variable region of D2E7 (D2E7 VH, also shown in SEQ ID NO. : 2), the alanine scanning mutants of D2E7 VH (HD2E7 * .A1, HD2E7 * .A2, HD2E7 * .A3, HD2E7 * .A4, HD2E7 * .A5, HD2E7 * .A6, HD2E7 * .A7, HD2E7 * .A8 and HD2E7 * .A9), the heavy chain variable region of the antibody related to D2E7, 2SD4 (2SD4 VH; also shown in SEQ ID NO: 10) and other heavy chain variable regions related to D2E7 (VH1B11, VH1D8, VH1A11, VH1B12, VH1-D2, VH1E, VH1F6, VH1G1, 3C-H2, VH1-D2.N and VH1-D2.Y). Figure 2A shows the domains of FR1, CDR1, FR2 and CDR2. Figure 2B shows the domains of FR3, CDR3 and FR4. The heavy chain domains CDR1 ("CDR Hl"), CDR2 ("CDR H2") and CDR3 ("CDR H3") are in boxes. Figure 3 is a graph illustrating the inhibition of L929 cytoxicity induced by TNFα by the human anti-hNFα antibody D2E7, as compared to MAK 195 of the murine anti-hTNFα antibody. Figure 4 is a graph illustrating inhibition of the binding of rhTNF to hTNFa receptors in U-937 cells by the human anti-hTNFa antibody D2E7, as compared to murine anti-hTNFa MAK 195 antibody. Figure 5 is a graph illustrating the inhibition of TNFα-induced ELAM-1 expression in HUVEC by the D2E7 antibody of the human anti-h TNFa compared to the MAK 195 antibody of murine anti-hTNFa.

Figure 6 is a bar graph illustrating the protection of TNFα-induced lethality in mice sensitized with D-galactosamine by administration of the human anti-hTNFa antibody D2E7. (black bars), in comparison with the MAK 195 antibody of murine anti-h TNFa (shaded bars). Figure 7 shows the nucleotide sequence of the light chain variable region of D2E7, with the amino acid sequence predicted below the nucleotide sequence. The CDR Ll, CDR L2 and CDR L3 regions are underlined. Figure 8 shows the nucleotide sequence of the heavy chain variable region of D2E7, with the amino acid sequence predicted below the nucleotide sequence. The CDR Hl, CDR H2 and CDR H3 regions are underlined. Figure 9 is a graph illustrating the effect of treatment of the D2E7 antibody on the average bound size of the transgenic mouse Tg 197 as a polyarthritis model.

DETAILED DESCRIPTION OF THE INVENTION This invention relates to isolated human antibodies, or antigen binding portions thereof which bind to human TNFa with high affinity, low outward regimen and high neutralizing capacity. Several aspects of the invention relate to antibodies and antibody fragments, and the pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells to produce these antibodies and fragments. Methods of using the antibodies of the invention to detect human TNFα or to inhibit human TNFα activity, either in vitro or in vivo, are also encompassed by the invention. In order that the present invention can be more easily understood, certain terms are defined first. The term "human TNFa" (abbreviated herein as hTNFa, or simply hTNF), as used herein, is intended to refer to a human cytokine that exists as a secreted form of 17kD in a manner associated with the 26kD membrane , the biologically active form of which is composed of a trimer of 17 kD molecules bound non-covalently. The structure of hTNFa is further described in for example, the article by D. Pennica et al (1984) Nature 312: 724-729; J.M. Davis et al. (1987) Biochemistry 26: 1322-1326; and E.Y. Jones, et al. (1989) Nature 328-225-228. The term "human TNFα" is intended to include recombinant human TNFα (rhTNFα), which can be prepared by normal recombinant expression methods or purchased commercially (R & D Systems, Catalog No. 210-TA, Minneapolis, MN).

The term "antibody", as used herein, is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of a CL domain. The VH and VL regions can further be subdivided into regions of hypervariability, termed regions of complementary determination (CDR), interdispersed with regions that are more conserved called framework regions (FR). Each VH and VL is composed of three CDRs and four Frs, placed in amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antigen binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen ( eg, hTNFa). It has been demonstrated that the antigen binding function of an anti-body can be carried out by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL domains VH, CL and CH1; (ii) a fragment of F (ab a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the articulated region; (iii) a Fd fragment consisting of the VH and CH1 domains; a fragment of Fv consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341-544-546), which consists of a domain of VH; and (vi) an isolated complementary determination region (CDR) .In addition, even when the two domains of the Fv, VL and VH fragment are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that allows they are prepared as a single protein chain in which the VL and VH regions form a pair in order to form the monovalent molecules (known as the individual Fv (scFv) chain; see, e.g., of Bird et al. (1988) Science 242: 423-426; and Houston et al. (1988) Proc. Nati. Acad. Sci. USA 85: 5879-5883). These single-antibodies The chain is also intended to be encompassed within the term "antigen binding portion" of an antibody. Other forms of single chain antibodies such as diabodies are also encompassed. The diabodies are bivalent, bi-specific antibodies in which the VH and VL domains are expressed in a single polypeptide chain, but using a linker that is too short to allow pairs between two domains of the same chain, thus forcing the domains to form the pair of complementary condominiums of another chain and creating two antigen binding sites (see, eg, P. Holliger et al. (1993) Proc. Nati. Acad. Sci. USA 90: 6444-6448; RJ Poljak et al. (1994) Structure 2: 1121-1123). Still further, an antibody or an antigen binding portion thereof can be part of larger immunoadhesion molecules, formed by covalent or non-covalent association of the antibody or the antibody portion with one or more of the other proteins or peptides. Examples of these immunoadherent molecules include the use of a streptavidin core region to produce a tetrameric scFv molecule (SM Kipriyanov, et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to produce bivalent and biotinylated scFv molecules (S.M. Kipriyanov et al. (1994) Mol.Immunol., 31: 1047-1058). Antibody portions, such as Fab and F (ab) fragments can be prepared from whole antibodies using conventional techniques, such as digestion with papain or pepsin, respectively, of whole antibodies, in addition, antibodies, antibody portions. and immunoadhesion molecules can be obtained using normal recombinant DNA techniques as described herein The term "human antibody", as used herein, is intended to include antibodies that have variable and constant regions derived from immunoglobulin sequences. of the human germline (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in CDRs and in particular CDR3, however, the term "human antibody", as used in the present, it is not intended to include antibodies wherein the CDR sequences derived from the germline of another species of mammal, such as a mouse, have been grafted onto human framework sequences. The term "recombinant human antibody" as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolate by recombinant means such as antibodies expressed using a recombinant expression vector transfected into a host cell (which is described in Section II, below), antibodies isolated from a recombinant combination human antibody library (further described in Section III, given below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, eg, LD Taylor, et al. (1992) Nuci Acids Res. 20: 6287-6295) or antibodies prepared, expressed, created or isolated by any other means involving the splicing of human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies have variable and constant regions that are derived from the human germline immunoglobulin sequences. In certain embodiments, however, these recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, somatic mutagenesis in vivo) and therefore the amino acid sequences of the VH and VL of the recombinant antibodies are sequences that, even though they are derived from and are related to the human germline VH and VL sequences, may not exist naturally within the repertoire of the line of human antibody germs in vivo. An "isolated antibody" as used herein, is intended to refer to an antibody that is essentially free of other antibodies that have different antigenic specificities (eg, an isolated antibody that specifically binds hTNFα is essentially free of antibodies that specifically bind antigens that are not hTNFa). An isolated antibody that specifically binds hTNFa may however have cross-reactivity for other antigens such as the TNFa molecules of other species (to be discussed in further detail below). In addition, an isolated antibody may be essentially free of other cellular material and / or chemical substances. A "neutralization antibody" as used herein (or an "antibody that neutralizes the activity of hTNFa"), is intended to refer to an antibody whose binding to hTNFa results in the inhibition of a biological activity of hTNFa. This inhibition of the biological activity of hTNFa can be assessed by measuring one or more indicators of the biological activity of hTNFa such as the cytotoxity induced by hTNFa (either in vitro or in vivo), the cellular activation induced by hTNFa and the binding of hTNFa to the hTNFa receptors. These Indicators of the biological activity of hTNFoc can be assessed by one or more of the various normal in vitro or in vivo assays known in the art (see Example 4). Preferably, the ability of an antibody to neutralize the activity of hTNFa is enhanced by the inhibition of hTNFa-induced cytotoxicity of L929 cells. As an additional or alternative parameter of hTNFa activity, the ability of an antibody to inhibit hTNFa-induced expression of ELAM-1 in HUVEC, as a measure of cellular activation induced by hTNFa can be assessed. The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that permits the analysis of bispecific interactions in real time by detecting alterations in protein concentrations within a biosensor matrix, for example using the BIAnucleus system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For additional descriptions, see Example 1 and the article by U Jónsson et al. (1993) Ann. Biol. Cli. 51: 19-26; U. Jónsson (1991) Biotechniques 11: 620-627; B. Johnsson et al. (1995) J. Mol. Recognit .8: 125-131; and B.Johnnson, et al. (1991) Anal. Bioichem. 198: 268-277. The term "Koff", as used herein, is intended to refer to the constant of the low regime of dissociation of an antibody from an antibody / antigen complex. The term "kd", as used herein, is intended to refer to the dissociation constant of an interaction of a specific antibody / antigen. The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single chain or double chain, but preferably it is a double stranded DNA. The term "isolated nucleic acid molecule", as used herein with reference to the nucleic acids encoding the antibodies or the portions of the antibody (eg, VH, VL, CDR3) that bind hTNFa, is intended to be referenced to a nucleic acid molecule wherein the nucleotide sequences encoding the antibody or the antibody portion are free of other nucleotide sequences, encoding antibodies or portions of antibody that bind antigens other than hTNFa, which other sequences may naturally flank the nucleic acid in human genomic DNA. Thus, for example, an isolated nucleic acid of the invention encoding a VH region of an anti-TNFce antibody does not contain other sequences encoding other VH regions that bind antigens other than TNFa.

The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to where it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA circuit in which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous duplication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of duplication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell during introduction into the host cell, and thus are duplicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are linked functionally. These vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, the term "plasmid" and "vector" can be used interchangeably since the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors, (e.g., replication defective retroviruses, adenoviruses and adeno associated viruses), which serve equivalent functions. The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that these terms are intended to refer not only to the specific cell but to the progeny of this cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, this progeny may in fact not be identical to the original cell, but is still included within the scope of the term "host cell" as used at the moment. More detailed aspects of the invention are described in greater detail in the following subsections.

I. Human Antibodies Linking Human TNFg This invention provides isolated human antibodies, or antigen binding portions thereof, which bind human TNFα with high affinity, low outward regimen and high neutralizing capacity. From Preferably, the human antibodies of the invention are recombinant human anti-hTNFα neutralizing antibodies. The especially preferred recombinant neutralizing antibody of the invention is referred to herein as D2E7 and has VL and VH sequences as shown in Figure 1A, 2B and Figure 2A, 2B, respectively (the amino acid sequence of the region of D2E7 VL is also shown in SEQ ID NO: 1, the amino acid sequence of the D2E7 VH region is also shown in SEQ ID NO: 2). The binding properties of D2E7, compared to mAb, MAK 195 of the murine anti-hTNFa exhibiting high affinity and slow dissociation kinetics and another human anti-hTNFa antibody related in sequence with D2E7, 2SD4, are summarized below: Antibody koff kon Kd Estequio-sec-1 M "1 sec" 1 M metria D2E7 IgGl 8.81 X I-5 1.91 x 10s 6.09 x 10"10 1.2 2SD4 IgG4 8.4 X io-3 4.20 x 105 2.00 x 10"8 0.8 MAK 195 F (ab ') 2 8.70 X io-5 1.90 x 105 4.60 x 10"lü 1.4 The anticuepro D2E7, and the related antibodies also exhibit an intense ability to neutralize hTNFa activity as assessed by various in vitro and in vivo assays (see Example 4). For example, these antibodies neutralize the hTNFα-induced cytotoxicity of L929 cells with IC 50 values within the range of about 10 ~ 7 M to about 10"lü M. D2E7, when expressing a full-length IgGl antibody, neutralizes the hTNFa-induced cytotoxicity of L929 cells within IC50 of approximately 1.25 x 10"1? M. In addition, the neutralization capacity of D2E7 is maintained when the antibody is expressed as a fragment of Fab, F (ab ') 2 or scFv. D2E7 also inhibits cellular activation induced by TNFa, as measured by the ELAM-1 expression induced by hTNFa in HUVEC (IC50 = approximately 1.85 x 1CT10 M), and the binding of hTNFa to the hTNFa receptors in U-937 cells (ICso = at approximately 1.56 x 10 ~ 10 M). With respect to the latter, D2E7 inhibits the binding of hTNFa to both p55 and p75 hTNFa receptors. In addition, the antibody inhibits the lethality induced by hTNFa in vivo in mice (DEso = 1-2.5 micrograms per mouse). With respect to the binding specificity of D2E7, this antibody binds to human TNFa in various forms including soluble hTNFa, transmembrane hTNFa and hTNFa bound to cellular receptors. D2E7 does not bind specifically to other cytokines, such as lymphotoxin (TNFP), IL-? A, IL-? B, IL-2, IL-4, IL-6, IL-8, IFNy and TGFP. However, D2E7 exhibits cross-reactivity to tumor necrosis factors of other species. For example, the antibody neutralizes the activity of at least five primate TNFa (chimpanzee, mandrill, titi, cynomolgus and macaque from India) with approximately equivalent IC.¾D values such as for the neutralization of hTNFa, (see Example 4, subsection E). D2E7 also neutralizes the activity of mouse TNFα, even when approximately 1000 times less than human TNFα (see example 4, subsection E). D2E7 is also linked to canine and porcine TNFa. In one aspect, the invention relates to antibody D2E7 portions of antibody, antibodies related to D2E7, portions of antibody and other human antibodies and antibody portions with properties equivalent to D2E7, such as high affinity binding to hTNFa with kinetics of low dissociation and high neutralization capacity. In one embodiment, the invention provides an isolated human antibody or an antigen binding portion thereof, which is dissociated from human TNFa with a Ka of 1 x 10 ~ 9 M or less and a Kofi regime constant of 1 x 10. "J s" 1 or less, both determined by surface plasmon resonance and neutralizes the cytotoxicity of human TNFa in an in vitro L929 assay normal with an ICso of 1 x 10"7 M or less.Preferably, the human antibody isolated, or the antigen binding portion thereof, is dissociated from TNFa with a 5 x 1CT4 s" 1 or less, or even more preferred, with a 1 x 10 ~ 4 s-1 or less. More preferably, the isolated human antibody or the antigen binding portion thereof neutralizes the cytotoxicity of human TNFa in a normal in vitro L929 assay with an ICso of 1 x 10"8 M or less, even more preferably with an IC50. of 1 x 10 ~ 9 M or less and still especially preferably with an ICso of 5 x 10 ~ 10 M or less.In a preferred embodiment, the antibody is a recombinant and more isolated antibody or an antigen binding portion of the In another preferred embodiment, the antibody also neutralizes cellular activity induced by TNFα, as assessed using a normal in vitro assay for the expression of ELAM-1 induced by TNFα in endothelial cells of the human umbilical vein (HUVEC). Resonance analysis of surface plasmen to determine and can be carried out as described in Example 1. A normal in vitro assay L929 for determining the ICsc values is described in Example 4, subsection A. A normal in vitro assay for the expression of ELAM-1 induced by TNFa in human umbilical vein endothelial cells (HUVEC) is described in Example 4, subsection C. Examples of recombinant human antibodies that are pooled or predicted to be pooled, the aforementioned kinetic and neutralization criteria include antibodies having the following pairs of [VH / VL], the sequences of which are shown in Figures 1A and IB, 2A and 2B (see also Examples 2, 3 and 4 for kinetic and neutralization analysis): [D2E7 VH / D2E7 VL]; HD2E7 * .A1 / D2E7 VL], [HD2E7 * .A2 / D2E7 VL], [HD2E7 * .A3 / D2E7 VL], [HD2E7 * .A4 / D2E7 VL], [HD2E7 * .A5 / D2E7 VL], [HD2E7 * .A6 / D2E7 VL], [HD2E7 * .A7 / D2E7 VL], [HD2E7 * .A8 / D2E7 VL], [HD2E7 * .A9 / D2E7 VL], [D2E7 VH / LD2E7 * .Al], [D2E7 VH / LD2E7 * .A4], [D2E7 VH / LD2E7 * .A5], [D2E7 VH / LD2E7 *, A7], [D2E7 VH / LD2E7 * .A8], [HD2E7 * .A9 / LD2E7 * .Al ], [VH1-D2 / LOE7], [VH1-D2.N / LOE7.T], [VH1-D2. Y / LOE7.A], [VH1-D2.N / LOE7.A], [VH1-D2 / EP B12] and [3C-H2 / LOE7]. It is well known in the art that light and heavy chain CDR3 domains of the antibody have an important role in the specificity / binding affinity of an antibody to an antigen. Accordingly, in another aspect, the invention relates to human antibodies that have slow dissociation kinetics for association with hTNFoc and that have light and heavy chain CDR3 domains that are structurally identical or that are related to those of D2E. As shown in the Example 3, position 9 of D2E7 VI CDR3 can be occupied by Ala or Thr without essentially affecting Koff. Accordingly, a consensus motif for D2E7 VL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y- (T / A) (SEQ ID N0: 3). In addition, position 12 of D2E7 VH CDR3 can be occupied by Tyr or Asn, without essentially affecting Koff. Correspondingly, a consensus motif for 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). In addition, as demonstrated in Example 2, the CDR3 domain of the heavy and light chains of D2E7 is subject to substitution with an individual alanine residue (at position 1, 4, 5, 7 or 8 within VL CDR3 or in position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within VL CDR3) without essentially affecting Koff. Still further, the skilled artisan will appreciate that, given the ability to subject alanine substitutions to the D2E7 VL and VH CDR3 domains, the substitution of other amino acids within the CDR3 domains may be possible, while still retaining the constant of low outward antibody regimen, particularly substitutions with conservative amino acids. A "substitution of the conservative amino acid" as used herein is one in which an amino acid residue is replaced with another amino acid residue having a secondary chain or the like. Waste families of amino acids having secondary chains or the like have been defined in the art, including basic secondary chains (eg lysine, arginine, histidine), acidic secondary chains (eg aspartic acid, glutamic acid), polar uncharged secondary chains (eg glycine, aspargin, glutamine, serine, threonine, tyrosine, cysteine), non-polar secondary chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched secondary chains (eg threonine, valine, isoleucine) and secondary chains aromatics (eg tyrosine, phenylalanine, tryptophan, histidine). Preferably, no more than one to five conservative amino acid substitutions are made within the domains of D2E7 VL and / or VH CDR3. Most preferably, no more than one to three conservative amino acid substitutions are made within the domains of D2E7 VL and / or VH CDR3. In addition, the amino acid substitutions must be made at critical amino acid positions for binding to hTNFa. As shown in Example 3, positions 2 and 5 of D2E7 VL CDR3 and positions 1 and 7 of D2E7 VH CDR3 appear to be critical for the interaction with hTNFa, and therefore, conservative amino acid substitions preferably do not are made in these positions (even though a substitution of alanine at position 5 of D2E7 VL CDR3, as described above. Accordingly, in another embodiment, the invention provides an isolated human antibody, an antigen binding portion thereof with the following characteristics: a) it is dissociated from human TNF with a steady state constant of 1 x 10 ~ 3 s_1 or less , as determined by surface plasmon resonance; b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 or modified of SEQ ID NO: 3 by a single substitution of alanine at position 1, 4, 5, 7 or 8, or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9; c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and / or 12. Most preferably the antibody , or the antigen binding portion thereof is dissociated from human TNFa with a Koft of 5 x 10"* s" 1 or less. Still preferably, the antibody or the antigen binding portion thereof, is dissociated from human TNF with a Koff of 1 x 10"4 s" 1 or less. In still another embodiment, the invention provides an isolated human antibody or an antigen binding portion thereof with a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or which is modified from SEQ ID NO: 3 by a single substitution of alanine at position 1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the sequence of amino acid of SEQ ID NO: 4, or that is modified of SEQ ID NO: 4, by a single substitution of alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 (ie, D2E7 VL CDR2) and HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 (ie , D2E7 VH CDR2). Still especially preferred LCVR further has the CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (ie, D2E7 VL CDR1) and HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 (ie, D2E7 VH CDR1). The frame regions for VL are preferably from the line family human germline VKI, more preferably the Vk gene of the human germline A20 and particularly preferably the framework sequences of D2E7 VL shown in Figures 1A and IB. The framework regions for VH are preferably of the human germ line VH3 family, most preferably of the VH gene of the human germ line of DP-31, more preferably of the framework sequences of D2E7 VH shown in Figures 2A and 2B. In yet another embodiment, the invention provides an isolated human antibody, or an antigen binding portion thereof, with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 (ie, the D2E7 VL) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 (ie, D2E7 VH). In certain embodiments, the antibody comprises a heavy chain constant region such as a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD. Preferably, the heavy chain constant region is a heavy chain constant region of IgG1 or a heavy chain constant region of IgG4. In addition, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a light chain lam region. Preferably, the The antibody comprises a constant region of the light chain of kappa. Alternatively, the antibody portion, for example, may be a Fab fragment or a single chain Fv fragment. In still other embodiments, the invention provides an isolated human antibody, or antigen binding portions thereof, that have VL and VH CDR3 domains related to D2E7, eg, antibodies or antigen binding portions thereof with a specific region. light chain variable (LCVR) having a CDR3 domain comprising an amino acid sequence that is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID N0: 16, SEQ ID N0: 17, SEQ ID NO: 18, SEQ ID N0: 19, SEQ ID N0: 20, SEQ ID N0: 21, SEQ ID N0: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or with a heavy chain variable region (HCVR) having a CDR3 domain comprising a sequence of amino acid that is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 , SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. In still In another embodiment, the invention provides a recombinant human antibody or an antigen binding portion thereof, which neutralizes the activity of human TNFα but not of human TNFp. Preferably, the antibody or antigen binding portion thereof also neutralizes the activity of chimpace TNFa and at least one TNFa of an additional primate that is selected from the group consisting of the TNFa of the baboon, the TNFa of the titi, TNFa from cynomolgus and TNFa from rhesus monkeys. Preferably the antibody or the antigen binding portion thereof, neutralizes the human TNFa of the chimpanzee and / or of additional primates in a normal in vitro assay L929 with an ICso of 1 x 1CT8 M or less, more preferably 1 x 10"9 M or less, and even more preferably 5 x 10 ~ 1 C M or less In a submodality, the antibody also neutralizes the activity of canine TNFα, preferably in a normal L929 assay in vitro with an ICsc of 1 x 10 ~ 7 M or less, more preferably 1 x 10 ~ 8 M or less and even more preferably 5 x 10 ~ 9 M or less.In another submodality, the antibody also neutralizes the TNFα activity of the pig, preferably with an ICso of 1 x 10 ~ b M or less, more preferably of 1 x 10"6 or less and even more preferably 5 x 1CT7 M or less. In still another embodiment, the antibody also neutralizes the activity of mouse TNFα, preferably with an IC 50 of 1 x 10"4 M or less, more preferably 1 x 10" 5 M or less and even more preferred 5. x 10 ~ 6 M or less.

An antibody or antibody portion of the invention can be derived or linked to another functional molecule (e.g., another peptide or protein). Accordingly, the antibodies and antibody portions of the invention are intended to include forms derived and otherwise modified from the human anti-h TNF antibodies described herein including immunoadhesion molecules. For example, an antibody or antibody portion of the invention can be functionally linked (via chemical coupling, genetic fusion, non-covalent association or otherwise) with one or more other molecular entities, such as another antibody (eg a bi-antibody). specific or a dia-body), a detectable agent, a phytotoxic agent, a pharmaceutical agent and / or a protein or peptide that can intervene to associate the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine label). A type of derived antibody is produced by crosslinking two or more antibodies (of the same type of different types e.g. to create bi-specific antibodies). Suitable crosslinking agents include those which are heterobifunctional having two distinctly reactive groups separated by an appropriate separator (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homogenous bifunctional (e.g., disuccinimidyl suberate). These linkers can be obtained from Pierce Chemical Company, of Rockford, IL. Useful detectable agents with which an antibody or antibody portion of the invention can be derived include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiolate, rhodamine, 5-dimethylamin-1-naphthalenesulfonyl chloride, phycoerythrin and the like. An antibody can also be derived with detectable enzymes such as alkaline phosphatase, strong horseradish peroxidase, glucose oxidase and the like. When an antibody is derived with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent, strong horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product that is detectable. An antibody can also be derived with biotin, and detected through indirect measurement of avidin binding or streptavidin.

II. Expression of Antibodies An antibody, or antibody portion of the invention can be prepared by the recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and are preferably secreted to the medium in which the host cells are cultured, from which medium the antibodies can be recovered. The normal recombinant DNA methodologies are used and obtain the heavy and light chain genes of the antibody, incorporate these genes into the recombinant expression vectors and introduce the vectors into the host cells, such as those described in the articles by Sambrook, Fritsch and Maniatis (editors), Molecular Cloning: A Laboratory Manual, Second Edition, Cold. Spring Harbor, N. Y. (1989), F.M. Ausubel et al. (Editors) Currents Protocols in Molecular Biology, Greene Publishing Associates (1989) and in U.S. Patent Number 4,816,397, to Boss et al. To express D2E7 or an antibody related to D2E7, the DNA fragments that code the regions Light and heavy chain variables are obtained first. These DNAs can be obtained by amplification and modification of the variable sequences of the light and heavy chain of the germline using the polymerase chain reaction (PCR). The germline DNA sequences for the heavy and light chain variable region genes are known in the art (see eg the human geminal line sequence database "Vbase", see also EA Kabat et al. 1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publications No. 91-3242, IM Tomlinson et al. (1992) "The Repertoire of Human Germline VH Sequences Reveal about Fifty Groups of VH Segments with Different Hypervariable Loops "J. Mol. Biol. 227: 776-798; and JPL Cox et al. (1994)" A Directory of Human Germ-line VK Segments Reveal Strong Bias in their Usage "Eur. J. Immunol. : 827-836, the content of each of which is expressly incorporated herein by reference). To obtain a DNA fragment encoding the heavy chain variable region of D2E7, or an antibody related to D2E7, a member of the VH3 family of the human germline VH genes is amplified by PCR. More preferably, the germline sequence of DP-31 VH is amplified. To get a DNA fragment encoding the light chain variable region of D2E7, or an antibody related to D2E7, a member of the VKI family of human germline VL genes is amplified by non-malignant PCR. More preferably, the germline sequence of A20 VL is amplified. PCR primers suitable for use to amplify DP-31 from the VH and A20 germline of the VL germ line can be designed based on the nucleotide sequences disclosed in the references cited above, using standard methods. Once the fragments of the germ line VH and VL are obtained, this sequence can be mutated to encode the amino acid sequences of D2E7 or related to D2E7, disclosed herein. The amino acid sequences encoded by the germline VH and VL DNA sequences are first compared to the VH and VL amino acid sequences of D2E7 or related to D2E7 to identify the amino acid residues in the D2E7 or D2E7 related sequence. which differ from the germ line. Then, the appropriate nucleotides of the germline DNA sequences are mutated in such a way that the mutated germline sequence encodes the amino acid sequence of D2E7 or related to D2E7 using the genetic code to determine which nucleotide changes must be made.

Mutagenesis of the germline sequences is carried out by normal methods such as PCR-mediated mutagenesis (in which the imitated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or mutagenesis directed to the site. In addition, it should be noted that if the "germline" sequences obtained by the PCR amplification encode the amino acid differences in the framework regions from the true germline configuration (i.e., the differences in the amplified sequence in comparison). with the true germline sequence, for example, as a result of somatic mutation), it may be desirable to change these amino acid differences back to the true germline sequences (i.e., "countermutation" of the frame residues towards the configuration of the germinal line). Once the DNA fragments encoding the D2E7 or D2E7 related segments VH and VL are obtained (by amplification in mutagenesis of the germline genes VH and VL, as described above), these fragments of DNA can also be manipulated by normal recombinant DNA techniques, for example to convert the genes of the variable region into long-chain antibody genes. complete, to genes from the Fab fragment or to a scFv gene. In these manipulations, a VL- or VH encoding the DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked", as used in this context, is intended to imply that the two DNA fragments are joined in such a manner that the amino acid sequences encoded by the two DNA fragments remain in the framework. The DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH encoding the DNA into another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3). The sequences of the human heavy chain constant region genes are known in the art (see, eg, EA Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments spanning these regions can be obtained by normal PCR amplification. The heavy chain constant region may be a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but more preferably it is a constant region of IgG1 or IgG4. For a chain gene heavy of Fab fragment, the VH encoding the DNA can be operably linked to another DNA molecule that encodes only the heavy chain CH1 constant region. The isolated DNA encoding the VL region can be converted to a full length light chain gene (as well as a Fab light chain gene) by operably linking the VL encoding the DNA with another DNA molecule encoding the constant region of light chain, CL. The sequences of the human light chain constant region genes are known in the art (see, eg, EA Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments spanning these regions can be obtained by normal PCR amplification. The light chain constant region may be a kappa or lamda constant region, but more preferably it is a kappa constant region. To create a scFv gene, VH and VL encoding the DNA fragments are operably linked to another fragment encoding a flexible linker e.g. encoding the amino acid sequence (Gly4-Ser) J, such that the VH and VL sequences can be expressed as a single-chain protein contiguous with the regions VL and VH joined by a flexible linker (see eg Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Nati. Acad. Sci. USA 85: 5879-5883; McCafferty et al. Nature (1990) 348: 552-554). To express the antibodies, or portions of the antibody of the invention, the DNAs encoding the light and heavy, partial or full-length chains obtained as described above, are inserted into the expression vectors such that the genes they are linked functionally with the transcription and transfer control sequences. In this context, the term "functionally linked" is intended to imply that an antibody gene is bound in a vector such that the transcriptional and translational control sequences within the vector serve their intended purpose. to regulate the transcription and transfer of the antibody gene. The expression vector and the expression control sequences are selected to be compatible with the expression host cell used. The light chain gene of the antibody and the heavy chain gene of the antibody can be inserted into a separate vector or more typically both genes are inserted into the same expression vector. Antibody genes are inserted into the expression vector by normal methods (e.g., ligation of restriction sites complementary to the fragment of the antibody gene and the vector, or blunt-end ligation if no restriction sites are present). Before the insertion of the light or heavy chain sequences of D2E7 or related to D2E7, the expression vector can already carry the sequences of the constant region of the antibody. For example, one approach to converting the D2E7 or D2E7, VH and VL sequences to the full-length antibody genes is to insert the same into the expression vectors that already encode the heavy chain constant regions and the constant regions. of light chain respectively in such a way that the VH segment is operably linked to the segment (s) of CH within the vector and the VL segment is operably linked to the segment of CL within the vector. In addition or alternatively, the recombinant expression vector can encode a signal peptide that facilitates the secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry the regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody's chain genes. These regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It will be appreciated by those skilled in the art that the design of expression vector, including the selection of regulatory sequences may depend on such factors as the selection of the host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for expression of the mammalian host cell include viral elements that direct high levels of protein expression in mammalian cells such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the promoter / enhancer CMV), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (eg the adenovirus major promoter (AdMLP)) and polyoma. For Further description of the viral regulatory elements and sequences thereof, see e.g. U.S. Patent Number 5,168,062 to Stinski, U.S. Patent Number 4,510,245 to Bell et al., and U.S. Patent Number 4,968,615 to Schaffner et al. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can lead to additional sequences, such as the sequences that regulate vector duplication in host cells (eg, duplication origins) and genes. selectable markers The selectable marker gene facilitates the selection of the host cells into which the vector has been introduced (see e.g. US Patent Number 4, 399,216, Number 4,634,665 and Number 5,179,017, all by Axel and others). For example, typically the selectable marker gene confers resistance to drugs such as G418, hygromycin or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells - with methotrexate selection / amplification) and the neo gene (for G418 selection).

For expression of light and heavy chains, the expression vector (s) encoding the heavy and light chains is transfected into the host cell by standard techniques. The various forms of the term "transiection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g. electroporation, calcium-phosphate precipitation, transfection of DEAE-dextran and the like before. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, the expression of the antibodies in the eukaryotic cells and, more preferably, the mammalian host cells, is especially preferred because these eukaryotic and in particular mammalian cells are more likely than prokaryotic cells to assemble and secrete an immunologically active and properly folded antibody. Prokaryotic expression of the antibody genes has been reported to be ineffective for the production of high yields of the active antibody (M. A. Boss and C.R. Wood (1985) Immunology Today 6: 12-13). Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Marmot Ovary (CHO cells) (including dhfr-CHO cells, which are described in the article by Urlaub and Chasin (1980) Proc. Nati. Acad. Sci. USA 7_7 · 4216-4220, used as the DHFR selectable marker, eg as described in article by RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. When the recombinant expression vectors encoding the antibody genes are introduced into the mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow the expression of the antibody in the host cells, or more preferably the secretion of the antibody into the culture medium where the host cells are grown. The antibodies can be recovered from the culture medium using normal protein purification methods. The host cells can also be used to produce portions of the intact antibodies such as Fab fragments or scFv molecules. It will be understood that variations of the aforementioned method are within the scope of the present invention. For example, it may be desirable to transfer a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. Recombinant DNA technology can also be used to remove some or all of the DNAs encoding any or both of the light and heavy chains that are not necessary to bind hTNFa. The expressed molecules of the truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies can be produced wherein the heavy chain and the light chain are an antibody of the invention and the other heavy chain and light chain are specific for an antigen other than hTNFa by crosslinking an antibody of the invention with a second antibody by means of normal chemical cross-linking methods. In a preferred system for the recombinant expression of an antibody, or an antigen binding portion thereof, of the invention, a recombinant expression vector encoding both the heavy chain of the antibody and the light chain of the antibody is introduced into the cells dhrf- CHO by means of transfection mediated with calcium phosphate. Within the recombinant expression vector, the heavy and light chain genes of the antibody each are operably linked to the enhancer / promoter regulatory elements (eg SV40, C Vm adenovirus derivatives and the like, such as the regulatory element. of the CMV enhancer / AdNLP promoter or a regulatory element of the SV40 enhancer / AdMLP promoter) to boost high levels of transcription of genes. The recombinant expression vector also carries a DHFR gene that allows the selection of CHO cells that have been transfected with the vector using methotrexate / amplification selection. The selected transformant host cells are activated to allow the expression of the heavy and light chains of the antibody and the intact antibody to be recovered from the culture medium. In normal molecular biology techniques, they are used to prepare the recombinant expression vector, to transfect the host cells, select for transformants, host cell culture and to recover the antibody from the culture medium. In view of the foregoing, another aspect of the invention relates to the nucleic acid, the vector and the host cell compositions that can be used for recombinant expression of the antibodies and antibody portions of the invention. The nucleotide sequence encoding the light chain variable region of D2E7 is shown in Figure 7 and in SEQ ID NO: 36. The CDR1 domain of LCVR encompasses nucleotides 70-102, the CDR2 domain encompasses nucleotides 148- 168, and the CDR3 domain encompasses the nucleotides 265-291. The nucleotide sequence that codes the regions Heavy chain variables of D2E7 are shown in Figure 8 and in SEQ ID NO: 37. The CDR1 domain of HCVR encompasses nucleotides 91-105, the CDR2 domain encompasses nucleotides 148-198 and the CDR3 domain encompasses nucleotides 295-330. It will be appreciated by a skilled artisan that the nucleotide sequences encoding the D2E7-related antibodies or portions thereof (eg, a CDR domain, such as the CDR3 domain) can be derived from the nucleotide sequences encoding D2E7 LCVR and HCVR, using the genetic code and normal molecular biology techniques. In one embodiment, the invention provides an isolated nucleic acid encoding a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 (ie, D2E7 VL CDR3) or modified from SEQ ID NO: 3 by a single substitution of alanine at positions 1, 4, 5, 7 or 8, or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9. This acid The nucleic acid may encode only the CDR3 region or, more preferably, encodes an entire light chain variable region of the antibody (LCVR). For example, the nucleic acid can encode an LCVR having a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 (ie, D2E7 VL CDR2) and a CDR1 domain that it comprises the amino acid sequence of SEQ ID NO: 7 (ie, D2E7 VL CDR1). In another embodiment, the invention provides an isolated nucleic acid encoding a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4 (ie, D2E7 VH CDR3) or modified from SEQ ID NO: 4 by a single substitution of alanine in the 2, 3, 4, 5, 6, 8, 9, 10 or 11 position, or by one to five conservative amino acid substitutions in positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and / or 12. This nucelic acid may only encode the CDR3 region or more preferably encode an entire heavy chain variable region of the antibody (HCVR). For example, the nucleic acid may encode HCVR having a CVR2 domain comprising the amino acid sequence of SEQ ID NO: 6 (ie, D2E7 VH CDR2) or a CDR1 domain comprising the amino acid sequence of SEQ ID NO. : 8 (that is, D2E7 VH CDR1). In still another embodiment, the invention provides isolated nucleic acids encoding a CDR3 domain related to D2E7, e.g. comprising an amino acid sequence that is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO:, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. In still another embodiment, the invention provides an isolated nucleic acid encoding a light chain variable region of the antibody comprising the amino acid sequence of SEQ ID NO: 1 (ie, D2E7 LCVR). Preferably, this nucleic acid comprises the nucleotide sequence of SEQ ID NO: 36 even though a skilled artisan will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences may encode the amino acid sequence of SEQ ID NO: 1. The nucleic acid can encode only LCVR or can also encode an antibody light chain constant region, operably linked with LCVR. In one embodiment, this nucleic acid is a recombinant expression vector. In still another embodiment, the invention provides an isolated nucleic acid encoding a heavy chain variable region of the antibody comprising the amino acid sequence of SEQ ID NO: 2 (ie, D2E7 HCVR). Preferably, this nucleic acid comprises the nucleotide sequence of SEQ ID NO: 37, although the skilled artisan will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences may encode the amino acid sequence of SEQ ID NO: 2. The nucleic acid may encode only HCVR or may also encode a heavy chain constant region, operably linked with HCVR, For example, the acid nucleic acid may comprise a constant region of IgG1 or IgG4. In one embodiment, this nucleic acid is a recombinant expression vector. The invention also provides recombinant expression vectors that encode both an antibody heavy chain and an antibody light chain. For example, in one embodiment, the invention provides a recombinant expression vector encoding: a) an antibody light chain having a variable region comprising the amino acid sequence of SEQ ID NO: 1 (ie, D2E7 LCVR); and b) an antibody heavy chain having a variable region comprising the amino acid sequence of SEQ ID NO: 2 (ie, D2E7 HCVR). The invention also provides host cells wherein one or more of the recombinant expression vectors of the invention have been introduced. Preferably, the host cell is a mammalian host cell, of greater Preferably the host cell is a CHO cell, an NSO cell or a COS cell. Still further, the invention provides a method for synthesizing a recombinant human antibody of the invention, by culturing a host cell of the invention in an appropriate culture medium until a recombinant human antibody of the invention is synthesized. The method may further comprise isolating the recombinant human antibody from the culture medium.

III. Selection of Recombinant Human Antibodies The recombinant human antibodies of the invention in addition to the D2E7 or D2E7-related antibodies disclosed herein may be isolated by the selection of a recombinant combination antibody library, preferably a macrophage display library. scFv, prepared using the human VL and VH cDNAs, prepared from mRNAs derived from human lymphocytes. The methodologies for preparing and selecting these libraries are known in the art. In addition, of the kits commercially available to generate macrophage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01, and the macrophage presentation kit Stratagene SurfZAP ™, catalog number 240612), Examples of particularly special methods and reagents for use in generating and selecting antibody display libraries can be found, for example, in the US Patent Number of Ladner and others No. 5,223,409, Kang PCT Publication and others No. WO 92/18619; PCT Publication of Dower and others WO 91/17271; Winter PCT Publication and other WO 92/20791; PCT Publication Markland et al. WO 92/15679; PCT Publication of Breitling and others WO 93/01288; MaCafferty PCT Publication Number WO 92/01047; PCT Publication of Garrard et al. WO 92/09690; the article by Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay and others (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; McCafferty et al., Nature (1990) 3 8: 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; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 1_9: 4133-4137; and Barbas et al. (1991) PNAS 88: 7978-7982. In a preferred embodiment, to isolate human antibodies with high affinity and a low low dissociation constant for hTNFa, an antibody of murine anti-hTNFa having high affinity and a low dissociation constant for hTNFa (eg MAK 195, the hybridoma for which a deposit number ECACC 87 050801 is available) is first used to select the heavy chain sequences and light human having similar binding activity towards hTNFa, using epitope printing, or guided screening, the methods described in the PCT Publication of Hoogenboom and others WO 93/06213. The antibody libraries used in this method are preferably scFv libraries prepared and selected as described in the PCT Publication of McCafferty et al., Nature (1990) 348: 552-554; and Griffiths and others (1993) EMBO J 12: 725-734. The scFv antibody libraries are preferably selected using recombinant human TNFα, as the antigen. Once the initial human VL and VH segments are selected, the "mix and match" experiments in which the different pairs of initially selected VL and VH segments are selected for hTNFa binding, are carried out to select the combinations of the preferred VL / VH pair. In addition, in order to further improve the affinity and / or decrease the dissociation rate constant for hTNFa binding, the VL and VH segments of the preferred VL / VH pair (s) can be mutated randomly, preferably within the CDR3 region of VH and / or VL in a process analogous to the somatic mutation process in vivo which is responsible for the maturation of affinity of the antibodies during a natural immune response. This in vitro affinity maturation can be achieved by amplifying the VH and VL regions using PCR primers complementary to VH CDR3 or VL CDR3, respectively, whose primers have been "scavenged" with a random mixture of four nucleotide bases in certain positions in such a way that the resulting PCR products encode the VH and VL segments to which random mutations have been introduced in the VH and VL CDR3 regions. These VH and VL segments that have been randomly mutated can be re-selected to bind to hTNFa and sequences that exhibit high affinity and a low dissociation rate so that the binding of hTNFa can be selected. The amino acid sequences of the selected heavy and light chains of the antibody can be compared to the amino acid sequences of the heavy and light chain of the germ line. In cases where certain framework residues of the selected VL and / or VH chains differ from the germline configuration (e.g., as a result of the somatic mutation of the immunoglobulin genes used to prepare the bacteriophage library), it may be desirable to "reverse" the altered framework residues of the selected antibodies to the germline configuration (i.e., changing the amino acid sequences of the selected antibody frameworks so that they are the same as the sequences). of the amino acid structure of the germ line). This "back-mutation" (or "germline") of framework residues can be achieved by normal molecular biology methods to introduce specific mutations (e.g., site-directed mutagenesis, PCR-mediated mutagenesis and the like). After selection and isolation of the anti-hTNFα antibody of the invention from a recombinant immunoglobulin display library, the nucleic acid encoding the selected antibody can be recovered from the presentation package (eg, from the bacteriophage genome) and subcloned to other expression vectors by normal recombinant DNA techniques. If desired, the nucleic acid can further be manipulated to create other forms of antibodies of the invention, (e.g., bind to the nucleic acid encoding the additional immunoglobulin domains, such as the additional constant regions). To express a recombinant human antibody isolated by selection of a combination library, the DNA encoding the antibody is cloned into a recombinant expression vector is introduced into the host cells of the mammal as described in greater detail in Section II above.

IV. Pharmaceutical Compositions and Pharmaceutical Administration The antibodies and antibody portions of the invention can be incorporated into the appropriate pharmaceutical compositions for administration to a patient. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all of the solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption and isotonic delay agents, and the like which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate stabilized saline, dextrose, glycerol, ethanol and the like as well as combinations thereof. In any of the cases, preference is given to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. The pharmaceutically acceptable carriers may further comprise small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or stabilizing agents, which improve the shelf life or effectiveness of the antibody or antibody portion. The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, pills, pills, powders, liposomes and suppositories. The preferred form depends on the mode that is intended for administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions such as compositions similar to those used for the passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure appropriate for high concentration of drug. Sterile injectable solutions can be prepared by incorporating the active compound (ie, the antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients listed above as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient of a previously filtered solution thereto until sterile . The proper fluidity of a solution can be maintained for example by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. Prolonged absorption of the injectable compositions can be carried out by including in the composition an agent that retards absorption, for example, salts of monoetherate and gelatin.

The antibodies and portions of the antibody of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is intravenous injection or infusion. It will be appreciated by an expert artisan that the route and / or mode of administration will vary depending on the desired results. In certain embodiments, the active compound can be prepared with a carrier that will protect the compound against rapid release such as a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used such as ethyl vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The many methods for the preparation of these formulations are patented or are generally known to those skilled in the art. See, e.g., Sustained and Controlled Relay Drug Delivery Systems, J.R. Robinson, editors, Marcel Dekker, Inc. New York, 1978.

In certain embodiments, an antibody or antibody portion of the invention can be administered orally, for example, with an inert diluent or an edible assimilable carrier. The compound (and the other ingredients if desired) can also be enclosed in a hard or soft shell gelatin capsule, compressed into pellets, or incorporated directly into the patient's diet. For oral therapeutic administration, the compounds can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixiris, suspensions, syrups, wafers and the like. To administer a compound of the invention by means of another than parenteral administration, it may be necessary to confine the compound with or co-administer the compound with a material to prevent its inactivation. Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an antibody or a portion of the antibody of the invention is co-formulated with and / or co-administered with one or more additional therapeutic agents that are useful for treating disorders wherein TNFa activity is detrimental. For example, an antibody or antibody portion of the anti-hTNFa of the invention can be co-formulated and / or co-administered with one or more additional antibodies that are link to other cytokines and that bind molecules on the surface of the cell), one or more cytokines, the soluble TNFa receptor (see, e.g., PCT Publication Number WO 94/06476) and / or one or more chemical agents that inhibit the production or activity of hTNFa (such as the cyclohexane-ylidene derivatives as described in PCT Publication Number WO 93/19751). In addition, one or more antibodies of the invention can be used in combination with two or more of the aforementioned therapeutic agents. These combination therapies can advantageously use lower dosages of the therapeutic agents administered thus avoiding toxicities or possible complications associated with the different monotherapies. Non-limiting examples of the therapeutic agents for rheumatoid arthritis with which an antibody or antibody portion of the invention can be combined, include the following: the non-steroidal anti-inflammatory drug (s) (NSAIDs), the drug (s) anti-inflammatory cytokine suppressant (CSAIDs); CDP-571 / BAY-10-3356 (the humanized anti-TNFa antibody; Celltech / Bayer; cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion proteins; Immunex see, eg, Arthritis &Rheumatism (1994) Volume 37, S295; J. Invest. Med. (1996) Volume 44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-Hoffmann-La Roche IgG fusion protein); IDEC-CE9.1 / SB 210396 (primed non-depleting anti-CD4 antibody, IDEC / SmithKline, see e.g., Arthritis &Rheumatism (1995) Volume 38, S185); DAB 486-IL-2 and / or DAB 389-IL-2 (IL-2 fusion proteins, Seragen, see, e.g., Arthritis &Rheumatism (1993) Volume 36, 1223); Anti-Tac (humanized anti-IL-2Ra; Protein Design of Laboratories / Roche); IL-4 (anti-inflammatory cytokine; DNAX / Schering); IL-10 (SCH 52000, recombinant IL-10, anti-inflammatory cytokine, DNAX / Schering); IL-4; IL-10 and / or IL-4 agonites (e.g., agonist antibodies); IL-1RA (IL-1 receptor antagonist, Synergen / Amgen); TNF-bp / s-TNFR (soluble TNF-binding protein, see, e.g., Arthristis &Rheumatism (1996) Volume 39; number 9 (supplement), S284; Amer. J. Physiol. - Heart and Circulatory Physiology (1995) Volume 268, pages 37 to 42); R973401 (Type IV phosphodiesterase inhibitor, see, v. G., Arthritis &Rheumatism (1996) Volume 3_9, number 9 (supplement), S282); M-966 (COX-2 inhibitor, see eg Arthritis &Rheumatism (1996) Volume 39, number 9 (supplement), S281; Iloprost (eg, Arthritis &Rheumatism (1996) Volume 3_9, number 9 (supplement), S282), methotrexate, thalidomide (see, eg, Arthritis &Rheumatism (1996) Volume 39, number 9 (supplement), S282; and drugs related to thalidomide (eg, Celgen); leflunomide (cytokine and anti-inflammatory inhibitor; see, e.g., Arthritis &Rheumatism (1996) Volume 39, number 9 (supplement), S131 Inflammation Research (1996) volume 45, pages 103-107); tranexamic acid (inhibitor of plasminogen activation, see, e.g., Arthritis &Rheumatism (1996) Volume 3_9, number 9 (supplement), S284); T-614 (cytokine inhibitor, see, eg, Arthritis &Rheumatism (1996) Volume 39, number 9 (supplement), S282) Prostaglandin El (see, eg, Arthritis &Rheumatism (1996) Volume 39, number 9 (supplement), S282); Tenidap (non-steroidal anti-inflammatory drug, see Arthritis &Rheumatism (1996) Volume 39, number 9 (supplement), S280); Naproxen (non-steroidal anti-inflammatory drug, see Neuro Report (1996) Volume 7, pages 1209 to 1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalizine (see, eg, Arthritis &Rheumatism (1996) Volume 39, Number 9 (supplement), S281; Azathioprine (see, v.gr, Arthritis &Rheumatism (1996) Volume 39, Number 9 (supplement), S281) ICE inhibitor (enzyme interleukin-? ß inhibitor that converts the enzyme), zap-70 inhibitor and / or lck (inhibitor of tyrosine kinase zap-70 or lck) inhibitor of VEGF and / or inhibitor of VEGF-R (inhibitors of endothelial cell growth factor or vascular endothelial cell growth factor receptor, angiogenesis inhibitors, anti-inflammatory drugs corticosteroids (eg, SB203580), TNF-convertase inhibitors, anti-IL-12 antibodies, interleukin-11 (see, eg, Arthritis &Rhematism (1996) Volume 39, Number 9 (supplement), S296); 13 (see, eg, Arthritis &Rheumatism (1996) Volume 39, Number 9 (supplement), S308); interleukin-17 inhibitors (see, eg, Arthritis &Rheumatism (1996) Volume 39, Number 9 (supplement), S120), gold, penicillamine, chloroquine, hydroxychloroquine, chlorambucil, cyclophosphamide, cyclosporine, total lymphoid irradiation, anti-thymocyte globulin, anti-CD4 anti bodies, CD5 toxins, orally administered peptides and collagen, lobenzarit disodium; of Cytokine Regulation (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); oligodeoxynucleotides of antisense phosphorothioate ICAM-1 (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc); prednisone; orgotein; glycosaminoglycan polysulfate; Minocycline; anti-IL2R antibodies; marine and botanical lipids (fatty acids of plant and fish seed, see e.g., DeLuca et al. (1995) Rheum.

Dis. Clin. North Am. 21: 759-777); auranofin; phenylbutazone; mefenophenamic acid flufenamic acid; intravenous immune globulin; zileuton; Mycophenolic acid (RS-61443); tacrolimus (F-506); silolimus (rapamycin); amiprilose (terafectin); cladribine (2-chlorodeoxyadenosine); and azaribine. Non-limiting examples of the therapeutic agents for inflammatory bowel disease with which an antibody or antibody portions of the invention can be combined include the following: budenoside; epidermal growth factor; corticosteroids, cyclosporine, sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-? ß monoclonal antibodies; anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl imidazole compounds; CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody, Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD, kD receptor TNF-IgG fusion protein; Immunex; see Arthritis &Rheumatism (1994) Volume 37, S295; J. Invest. Med. (1996) Volume 44, 235A); 55 kdTNFR-IgG (55kD TNF receptor-fusion protein IgG; Hoffmann-LaRoche); interleukin-10 (SCH 52000; Sc ering Plow); IL-4 agonists; IL-10 and / or IL-4 (e.g., agonist antibodies); interleukin-11; prednisolone prodrugs conjugated with glucuronide or dextran, dexamethasone or budesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISI 2302; Isis Pharmaceuticals, Inc.); soluble complementary receptor 1. { TP10; TP10; T Cell Sciences, Inc.); mesalazine slow release; methotrexate; antagonists of the Platelet Activating Factor (PAF); ciprofloxaxin; and lignocaine.

Non-limiting examples of the therapeutic agents for multiple sclerosis with which an antibody or antibody portion of the invention can be combined include the following corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporin; methotrexate; 4-aminopyridine; tizanidine; interferon-pla (Avonex ™; Biogen); interferon-ß? > (Betaseron ™, Chiron / Berlex); Copolymer 1 (Cop-1; Copaxone ™; Teva Pharmaceutical Industries Inc.); hyperbaric oxygen; intravenous immunoglobulin; Clabribine; CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody, Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD of the TNF receptor-IgG fusion protein Immunex, see e.g. Arthritis &Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD of the TNF receptor-LgG fusion protein, Hoffmann-LaRoche); IL-10 agonists; IL-4 and IL-10 and / or IL-4 (e.g., agonist antibodies).

Non-limiting examples of therapeutic agents for sepsis with which an antibody or antibody portion of the invention can be combined include the following: hypertonic saline solutions, antibiotics; intravenous gamma globulin; continuous hemofiltration; carbapenems (e.g., meropenem); cytokine antagonists such as TNFa, IL-? β, IL-6 and / or IL-8; CDP-571 / BA-10-3356 (humanized anti-TNFa antibody, Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD of the TNF receptor-IgG fusion protein); Immunex; see Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD of the TNF receptor-IgG fusion protein, Ho fmann-LaRoche); Cytokine Regulation Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc); SK & F 107647 (low molecular peptide; SmithKline Beecham); tetravalent guanilhidrazona CNI-1493 (Picower Institute); Tissue Factor Trajectory Inhibitor (TFPI, Chiron); PHP (chemically modified hemoglobin; APEX Bioscience); chelators and iron chelates, including the iron complex of diethylenetriamine (III) pentaacetic acid (DTPA (III) iron; Molichem Medicines); Lyophilin (synthetic small molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan (glucanpi, soluble 3 aqueous, Alpha-Beta Technology); apolipoprotein A-1 reconstituted with liquids; chiral hydroxamic acids; (synthetic antibacterials that inhibit the biosynthesis of lipid A); anti-endotoxin antibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.); rBPl2i (recombinant N-terminal fragment of the Human Bactericidal Protein / Which Increases Permeability); Synthetic peptides of Anti-Endotoxin. { SAEP; Bios Ynth Research Laboratories); Non-limiting examples of the therapeutic agents for adult respiratory syndrome (ARDS) with which it can be combined in an antibody or antibody portion of the invention include the following: anti-IL-8 antibodies; surfactant replacement therapy; CDP-571 / BAY-10-3356 (humanized anti-TNFa antibody, Celltech / Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG. { 75 kD of the TNF receptor-IgG fusion protein; Immunex; see e.g. Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD of the TNF receptor-IgG fusion protein, Hoffmann-LaRoche).

The use of the antibodies or portions of antibodies of the invention in combination with other therapeutic agents is discussed further in subsection IV. The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" refers to an effective amount, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the antibody or antibody portion to provide a desired response in the individual . A therapeutically effective amount is also one in which any of the toxic or detrimental effects of the antibody or antibody portion are counteracted by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an effective amount, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in patients before of or in an initial stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount. Dosage regimens can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single large pill can be administered, several divided doses can be administered over time or the dose can be reduced or increased in proportion, as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in the form of a dosage unit for ease of administration and uniformity of dosage. The dosage unit form, as used herein, refers to physically discrete units suitable as unit dosages for the mammalian subjects to be treated, each unit containing a predetermined amount of the active compound that is calculated to produce the desired therapeutic effect. in association with the required pharmaceutical carrier. The specification for dosage unit forms of the invention are regulated by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technique from stir this active compound for the treatment of sensitivity in individuals. An exemplary non-limiting scale for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.1 to 20 milligrams per kilogram, more preferably from 1 to 10 milligrams per kilogram. It may be noted that the dosage values may vary with the type of seriousness of the condition to be alleviated. In addition, it will be understood that for any specific patient, specific dosage regimens should be adjusted over time according to the individual's need and the professional judgment of the person administering or supervising the administration of the compositions, and that the Dosages noted herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

IV. Uses of the Antibodies of the Invention Given their ability to bind to hTNF, the anti-hTNFa antibodies or portions thereof of the invention can be used to detect hTNFa (eg in a biological sample such as a serum or plasma), using a conventional immunoassay such as the immunosorbent assays bound above (ELISA), a radioimmunoassay (RIA) or a tissue immunohistochemistry. The invention provides a method for detecting hTNFa in a biological sample comprising contacting a biological sample with an antibody or portion of the antibody of the invention, and detecting either the antibody (or antibody portion) linked to hTNFa or the non-antibody. bound (or antibody portion) to thereby detect hTNFa in the biological sample. The antibody is irradiated directly or indirectly with a detectable substance to facilitate the detection of bound or unbound antibody. Appropriate detectable substances include the various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include strong horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase examples of appropriate prosthetic group complexes include streptavidin / biotin and avidin / biotin examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate , rhodamine, dichlorotriacylamine fluorescein, dansyl chloride or phycoerythrin; an example of luminescent materials that include luminol; and examples of suitable radioactive materials include: 25Y, I31Y, 35S or 3H.

Alternatively to the irradiation of the antibody, hTNFo can be assayed in biological fluids by a competition immunoassay using the rhTNFoc standards irradiated with a detectable substance and a non-irradiated anti-hTNFa antibody. In this assay, the biological sample, the irradiated rhTNFoc standards and the anti-hTNFα antibody are combined and the amount of the irradiated rhTNFα standard bound to the non-irradiated antibody is determined. The amount of hTNFa in the biological sample is inversely proportional to the amount of the irradiated rhTNFa standard bound to the anti-hTNFa antibody. A D2E7 antibody of the invention can also be used to detect TNFa from the non-human species, in particular TNFa from primates (eg chimpanzee, mandrill, titi, cynomolgus and macaque from India), pig and mouse, since D2E7 can be linked to each of these TNFa (which are further discussed in Example 4, subsection E). The antibodies and the antibody portions of the invention are capable of neutralizing the activity of hTNFa both in vitro and in vivo (see Example 4). In addition, at least some of the antibodies of the invention, such as D2E7, can neutralize the TNFa activity of other species. Therefore, the antibodies and antibody portions of the invention can be used to inhibit TNFa activity, e.g. in a cell culture containing TNFa in human patients or in other mammalian patients having TNFα with which the antibody of the invention (eg chimpanzee, mandrel, titi, cynomolgus or macaque from India, pig or mouse) cross-reacts. . In one embodiment, the invention provides a method for inhibiting TNFa activity comprising contacting TNFa with an antibody or antibody portion of the invention in such a manner that TNFa activity is inhibited. Preferably, TNFa is human TNFa. For example, in a cell culture containing or suspected to contain TNFa, an antibody or antibody portion of the invention can be added to the culture medium to inhibit the activity of TNFa in the culture. In another embodiment, the invention provides a method for inhibiting TNFa activity in a patient suffering from a disorder in which TNFa activity is detrimental. TNFa has been implicated in the pathophysiology of a wide variety of disorders (see, eg, A. oeller et al. (1990) Cytokine 2: 162-169; US Patent Number 5,231,014 issued to A. Moeller et al., European Patent Publication Number. 260 610 Bl of A. Moeller). The invention provides methods for the activity of TNFa in a patient suffering from this disorder whose method comprises administering to the patient an antibody or antibody portion of the invention in such a way that the activity of TNFa in the patient is inhibited. Preferably, TNFa is human TNFa and the patient is a human patient. Alternatively, the patient may be a mammal expressing a TNFα with which the antibody of the invention cross-reacts. Still further the patient may be a mammal in which TNFa has been introduced (e.g., by administration of hTNFa or by expression of a hTNFa transgene). An antibody of the invention can be administered to a human patient for therapeutic purposes (which will be discussed further below). In addition, an antibody of the invention can be administered to a non-human mammal expressing a TNFα with which the antibody cross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. With respect to the latter, these animal models may be useful for evaluating the therapeutic efficacy of the antibodies of the invention (e.g., the dosages test and courses of administration time). As used herein, the term "a disorder wherein the activity of TNFa is detrimental" it is intended to include diseases and other disorders where the presence of TNFo in a patient suffering from the disorder has been shown to be or is suspected to be either responsible for the pathophysiology of the disorder or a contributing factor to the worsening of the disorder. Accordingly, a disorder in which TNFα activity is detrimental is a disorder wherein inhibition of TNFα activity is expected to relieve symptoms and / or advance the disorder. These disorders can be evidenced, for example, by an increase in the concentration of TNFa in a biological fluid of a patient overcoming the disorder (eg an increase in the concentration of TNFα in the patient's serum, plasma, synovial fluid, etc.) it can detect for example, using an anti-TNFα antibody, as described above. There are numerous examples of disorders where the activity of TNFa is detrimental. The use of the antibodies and portions of the antibody of the invention in the treatment of specific disorders will be further discussed below: A. Sepsis Tumor necrosis factor has an established role in the pathophysiology of sepsis, with biological effects including hypotension, myocardial suppression, vascular leak syndrome, organ necrosis, stimulation of release of toxic secondary mediators and activation of the coagulation cascade (see eg A. Moeller et al. (1990) Citokine 2: 162-169, US Patent Number 5,231,024 issued to Moeller and others; European Patent Publication Number 260 619 Bl by A. Moeller; KJ Tracey and A. Cerami (1994) Annu. Rev. Med. 5: 491-503; D. Russell and RC Thompson (1993) Curr. Opin. Biotech 4: 714-721). Accordingly, the human antibodies and the antibody portions of the invention can be used to treat sepsis in any of its clinical aspects including septic shock, endotoxic shock, gram-negative sepsis and toxic shock syndrome. In addition, to treat sepsis, an anti-hTNFoc antibody or antibody portion of the invention can be co-administered with one or more additional therapeutic agents that can further alleviate sepsis, such as an interleukin-1 inhibitor (such as those described in PCT publications WO 92/16221 and WO 92/17853), cytokine interleukin-6 (see eg PCT Publication Number WO 93/11793) or a platelet activating factor antagonist (see eg Publication of the European Patent Application Number EP 374 510). Other combination therapies for the treatment of sepsis are discussed further in subsection III. In addition, in a preferred embodiment, an antibody or anti-TNFα antibody portion of the invention is administered to a human patient within a subgroup of sepsis patients having a serum or plasma concentration of IL-6 above 500. pg / milliliter and preferably 1000 pg per milliliter at the time of treatment (see PCT Publication Number WO 95/20978 by L. Daum et al.).

B. Autoimmune Diseases Tumor necrosis factor has been implicated in a role in the pathophysiology of a variety of autoimmune diseases. For example, TNFa has been implicated in the activation of tissue inflammation and causing joint destruction in rheumatoid arthritis (see, e.g., A. Moeller et al. (1990).

Citokine 2: 162-169, U.S. Patent Number 5,231,024 issued to Moeller et al .; European Patent Publication Number 260 610 Bl by A. Moeller; Tracey and Cerami, supra; W.P. Arend and J-M. Dayer (1995) Arth. Rheum. 38: 151-160; R.A. Fava et al. (1993) Clin. Exp. Immunol. 9: 261-266).

TNFa has also been implicated in promoting the death of islet cells and in mediating resistance to insulin in diabetes (see e.g. Tracey and Cerami, supra PCT Publication Number WO 94/08609). TNFa has also been implicated in mediating cytotoxicity to oligodendrocytes and the induction of inflammatory plaques in multiple sclerosis (see Tracey and Cerami, supra). Antibodies to murine, immunized and chimeric anti-hTNFa have undergone clinical trials for the treatment of rheumatoid arthritis (see eg MJ Elliot et al. (1994) Lancet 344: 1125-1127; MJ Elliot et al. (1994) Lancet 344: 1105 -1110; EC Rankin et al. (1995) Br. Rheumatol. 34: 334-342). The human antibodies and the antibody portions of the invention can be used to treat autoimmune diseases, in particular those associated with inflammation including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gout arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome. . Typically the antibody or portion of the antibody is administered systematically even when for certain disorders, local administration of the antibody or antibody portion at a site of inflammation (eg, local administration to the joints in rheumatoid arthritis or topical application to diabetic ulcers alone or in combination with a cyclohexane-ylidene derivative, as described in PCT Publication Number WO 93/19751). An antibody or antibody portion of the invention can also be administered with one or more additional therapeutic agents useful in the treatment of autoimmune diseases, as further discussed in subsection III.

C. Infectious Diseases The tumor necrosis factor has been implicated in the intervention of the biological effects observed in a variety of infectious diseases. For example, TNFa has been implicated in intervening in brain inflammation and capillary thrombosis and infarction in malaria. TNFa has also been implicated in intervening in brain inflammation by inducing the disintegration of the blood-brain barrier, activating the skeptic shock syndrome and activating venous infarction in meningitis. TNFa has also been implicated to induce cachexia, stimulating viral proliferation by interfering with central nervous system damage to acquire the immune deficiency syndrome (AIDS). Accordingly, the antibodies and antibody portions of the invention can be used in the treatment of infectious diseases, including bacterial meningitis (see eg European Patent Application Publication Number EP 585 705), cerebral malaria, AIDS and related complex. AIDS (ARC) (see e.g. Patent Application Publication Euroepa Number EP 230 574), as well as cytomegalovirus infection secondary to transplantation (see e.g. E. Fietze et al. (1994) Transplatation 58: 675-680). Antibodies and portions of the antibody of the invention can also be used to alleviate symptoms associated with infectious diseases, including fever and myalgia due to infection (such as influenza) and secondary cachexia for infection (e.g., secondary to AIDS or ARC).

D. Transplantation Tumor necrosis factor has been implicated as a key mediator of graft ction and graft versus host disease (GVHD) and to mediate an adverse reaction that has been observed when rat antibody OKT3, directed against the CDR3 complex of the T cell receptor is used to inhibit the ction of renal transplants (see eg JD Eason et al. (1995) Transplantation 59: 300-305; M. Suthanthiran and T.B. Strom (1994) New Engl. J. Med. 331: 365-375). Accordingly, the antibodies and antibody portions of the invention can be used to inhibit rejection of transplantation including rejection of allografts and xenografts and to inhibit GVHD. Even when the antibody or antibody portion it can be used alone, more preferably it is used in combination with one or more of the other agents that inhibit the immune response against the allograft or inhibit GVHD. For example, in one embodiment, an antibody or portion of the antibody of the invention is used in combination with OKT3 to inhibit the reactions induced by OKT3. In another embodiment, an antibody or portion of the antibody of the invention is used in combination with one or more antibodies directed to other targets involved to regulate immune responses, such as the surface molecules of the CD25 cell (interleukin-1 receptor). 2), CD1 (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4, CD80 (B7-1) and / or CD86 (B7-2). In yet another embodiment, an antibody or portion of the antibody of the invention is used in combination with one or more general immunosuppressive agents such as cyclosporin A or FK506.

E. Malignancy The tumor necrosis factor has been implicated to induce cachexia, stimulating tumor growth, improving metastatic potential and mediating cytoxicity in malignancies. Accordingly, the antibodies and portions of the antibody of the invention can be used in the treatment of malignancies, to inhibit the growth or metastasis of the tumor and / or to alleviate the cachexia secondary to malignancy. The antibody or portion of the antibody can be administered systematically or locally at the site of the tumor.

F. Pulmonary Disorders Tumor necrosis factor has been implicated in the pathophysiology of adult respiratory distress syndrome (ARDS) which includes stimulating leukocyte-endothelial activation, directing cytotoxicity to pneumocytes and inducing escape syndrome. Accordingly, the antibodies, and portions of antibodies, of the invention, can be used for pulmonary disorders, including adult repiration distress syndrome (see eg PCT Publication Number O 91/04054), lung shock, inflammatory disease pulmonary sarcomas, pulmonary sarcoidosis, fibrosis and pulmonary silicosis. The antibody or portion of the antibody can be administered systematically or locally to the surface of the lung, for example as an aerosol. An antibody or portion of the antibody of the invention can be administered with one or more additional therapeutic agents useful in the treatment of pulmonary disorders, as discussed further in subsection III.

G. Bowel Disorders Tumor necrosis factor has been implicated in the pathophysiology of inflammatory bowel disorders (see eg KJ Tracey et al. (1986) Science 234: 470-474; XM.Sun et al. (1988) J. Clin. Invest. 1: 1328-1331; TT MacDonald et al. (1990) Clin. Exp. Immunol. £ 1: 301-305). Chimeric murine anti-hTNF antibodies have undergone clinical trials for the treatment of Crohn's disease (HM van Dullemen et al. (1995) Gastroenterology 109: 129-135) Human antibodies and antibody portions of the invention can also be used to treat intestinal disorders such as inflammatory bowel disease iodopathic, which includes two syndromes, Crohn's disease and ulcerative colitis. An antibody or portion of the antibody of the invention can also be administered with one or more additional therapeutic agents useful in the treatment of intestinal disorders, as discussed further in subsection III.

H. Cardiac Disorders The antibodies and antibody portions of the invention can also be used to treat various cardiac disorders, including ischemia of the heart (see e.g. European Patent Application Publication Number EP 453 898) and heart failure. (weakness of the heart muscle) (see e.g. PCT Publication Number WO 94/20139).

I. Others The antibodies and portions of the antibody of the invention can also be used to treat various other disorders where the activity of TNFa is detrimental. Examples of other diseases and disorders where TNFa activity has been implicated in pathophysiology and therefore can be treated using an antibody or antibody portion of the invention, include inflammatory bone disorders and bone reabsorption disease (see eg by DR Bertolini et al. (1986) Nature 319: 516-518; A. Konig et al. (1988) J. Bone Miner. Res. 3: 621-627; U.H. Lerner and A. Ohlin (1993) J. Bone Miner. Res. 8: 147-155; and G. Shankar and P.H. Stern (1993) Bone 14: 871-876), hepatitis, including alcoholic hepatitis (see eg CJ McClain and DA Cohen (1989) Hepatology 9: 349-351; ME Felver et al. (1990) Alcohol, Clin. Exp. Res. : 255-259; and J. Hansen et al. (1994) Hepatology 20_: 461-474), viral hepatitis (N. Sheron et al. (1991) J. Hepatol 12: 241-245; and MJ Hussaimn et al. (1994) J. Clin. Pathol. 47: 1112-1115), and fulminant hepatitis; coagulation disorders (see, e.g., T. van der Poli et al. (1990) N. Engl. J. Med. 322: 1622-1627; T. van der Poli et al. (1991) Prog. Clin. Biol. Res. 367: 55-60), burns (see eg BP Giroir et al. (1994) Am. J. Physiol. 267: H118-12; and XS Liu et al. (1994) Burns 2_0: 20-44), damage of reperfusion (see eg WE Scales et al. (1994) Am. J. Physiol 267: G1122-1127; of C. Serrick et al. (1994) Transplantation 58: 1158-1162; and YM Yao et al. (1995) Resuscitation 29: 157-168), keloid formation (see eg RL McCauley et al. (1992) J. Clin Immunol. 12: 300-308), scar tissue formation; pyrexia; periodontal disease; Obesity and radiation toxicity. This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all patent references and published patent applications cited by this application are incorporated herein by reference.

EXAMPLE 1: Kinetic Analysis of the Ligand of Human Antibodies to hTNFa The real-time binding interactions between the coordinating group (biotinylated recombinant human TNFa (rhTNFa) immobilized in a biosensing matrix) and the result of the analysis (antibodies in solution) were measured by surface plasmon resonance (SPR) using the BIAnucleus system (Pharmacia Biosensor, Piscataway, NJ). The system uses the optical properties of SPR to detect alterations in protein concentrations within a dextran biosensor matrix. The proteins are linked covalently to the dextran matrix at known concentrations. The antibodies are injected through the dextran matrix and the specific binding between the injected antibodies of the immobilized coordinator group results in an increased matrix protein concentration and a resultant change in the SPR signal. These changes in the SPR signal are recorded as resonance units (RU) and are presented with respect to the time course along the "y" axis of a sensorgram. To facilitate immobilization of the biotinylated rhTNFoc in the biosensing matrix, streptavidin is covalently linked through the free amine groups to the dextran matrix by first activating the carboxyl groups in the matrix with 100 mM N-hydroxysuccinimide (NHS) and 400 mM of N-ethyl-N '- (3-diethylaminopropyl) carbodiimide hydrochloride (EDC). Then, streptavidin is injected through the activated matrix, 35 microliters of streptavidin (25 micrograms) are injected per milliliter), diluted in sodium acetate with a pH of 4.5, through the activated biosensor and the free amines in the protein are directly linked to the activated carboxyl groups. The EDC esters of the unreacted matrix are deactivated by injection of 1 M ethanolamine. Biosensor chips coupled with streptavidin can be obtained commercially (Pharmacia BR-1000-16, Pharmacia Biosensor, Piscata ay, NJ). The biotinylated RhTNFa was prepared by first dissolving 5.0 milligrams of biotin (N-hydroxysuccinimide D-biotinyl-8-aminocaproic acid D-ester, Boehringer Mannheim Catátogo Number 1008 960) in 500 microliters of dimethylsulfoxide to prepare a solution of 10 milligrams per milliliter . Ten microliters of biotin per milliliter of rhTNFa (at 2.65 milligrams per milliliter) was added during a 2: 1 molar ratio of biotin to rhTNFa. The reaction was mixed gently and incubated for two hours at room temperature in the dark. A column of A PD-10 Sephadex G-25 (Pharmacia Catalog No. 17-0851-01) was equilibrated with 25 milliliters of cold PBS and loaded with 2 milliliters of rhTNFa-biotin per column. The column was eluted with 10 x 1 milliliter of cold PBS. The fractions were collected and read at OD280 (1.0 OD = 1.25 milligrams per milliliter). The appropriate fractions were moistened and stored at -80 ° C until used. Biotinylated rhTNFa can also be obtained commercially (R & D Systems Catalog Number FTAOO, Minneapolis, MN). The biotinylated rhTNFa to be immobilized in the matrix via streptavidin was diluted in a PBS stabilizer (Gibco Catalog Number 14190-144, Gibco BRL, Grand Island, NY) supplemented with 0.05 percent of a P20 surfactant (BIAnucleus) (Pharmacia BR-1000-54, Pharmacia Biosensor, Piscataway, NJ). To determine the ability of rhTNF-specific antibodies to bind immobilized rhTNFα, a binding assay was carried out in the following manner. Aliquots of the biotinylated rhTNFa (25 nM, 10 microliter aliquots) were injected through the dextran matrix coupled with streptavidin at a flow rate of 5 microliters per minute. Prior to the injection of the protein and immediately after, the PBS stabilizer only flowed through each flow cell. The net difference in signal between the baseline and approximately 30 seconds after the injection of biotinylated rhTNFa was completed was taken to represent the binding value (approximately 500 RU). The binding of the specific antibody to rhTNFa directly to the immobilized biotinylated rhTNFa was then measured. The antibodies (20 micrograms per milliliter) were diluted in a stabilizer from ??? and 25 microliter aliquots were injected through the immobilized protein matrices at a flow rate of 5 microliters per minute. Prior to the injection of the antibody, and immediately afterwards, the PBS stabilizer flowed only through each flow cell. The net difference in the baseline signal and the signal after completion of the antibody injection was taken to represent the binding value of the specific sample. Biosensor matrices were regenerated using 100 M HC1 before injection of the next sample. To determine the rate of dissipation (Koff), connection (on), the association regime (a) and the constants of the dissociation regime (Kd), the BIAnucleus kinetic evaluation software (version 2.1) was used.

Representative results of D2E7 (full-length IgG4 antibody) that binds to biotinylated rhTNFa, compared to the mouse mAb MAK 195 fragment of (F (ab ') 2, are shown below in Table 1.

Table 1: Link of D2E7 IqG4 or MA 195 to Biotinylated rhTNFa In a second series of experiments, molecular kinetic interactions between a form of! of full-length IgGl and biotinylated rhTNFa analyzed quantitatively using the BlAnucleus technology, coso is described above, and derived the constants of the kinetic regime, which will be summarized below in Tables 2, 3 and 4.

Table 2: Constants of apparent dissociation regime of the interaction between D2E7 and biotinylated rhTNF Table 4: Constants of apparent kinetic regime and affinity of D2E7 and biotinylated rhTNF Experiment Y '-'. S- ') 1.33 x 10 9.58x 10-5; 7.20 ?? 0-10 2 1.05 105 9.26 x 10-5 8.¡ > 2x! 0-10 3 3.36 x 10s 7.60 x 10-5 2.26 x 10- '° Average! 1.91 ± 1.26 x 10 8.81 ± 1.06 xl ?? 6.09 ± 3.42 x 10- '° The constants of dissociation and association regime are. calculated by analyzing the regions of dissociation and association of the sensorgrams using the BIA analysis software. The conventional chemical reaction kinetics was assumed for the interaction between D2E7 and the biotinylated rhTNF molecule: a zero-order dissociation and first order association kinetics. For analysis purposes, the interaction between only one arm and the bivalent D2E7 antibody and one trimeric biotinylated rhTNF unit was considered when selecting the molecular models for the kinetic data analysis. Three independent experiments were carried out and the results were analyzed separately. The average apparent dissociation rate constant (kd) of the interaction between D2E7 and biotinylated rhTNF was 8.81 + 1.06 x 10-5 s_1, and the average apparent association regime constant, ka was 1.91 + 1.26 x 105 M "1 s'1.The apparent intrinsic dissociation constant (Kd) was then calculated by the formula: Thus, the average Kd of the The D2E7 antibody for rhTNF derived from the kinetic parameters was 6.09 + 3.42 x 10"10 M. The small differences in the kinetic values for the IgG1 form of D2E7 (shown in Tables 2, 3 and 4) and the The IgG4 form of D2E7 (presented in Table 1 and Examples 2 and 3) is not believed to be true differences resulting from the presence of either a constant IgG1 region or the IgG4 constant regions but rather it is believed that they are attributable to the more accurate antibody concentration measurements used for the IgG1 kinetic analysis. Accordingly, the kinetic values for the IgG1 form of D2E7 that are presented here are believed to be the most accurate kinetic parameters for the D2E7 antibody.

EXAMPLE 2: Mutagenesis of Alanine Scan of the Domains of D2E7 CDR3 A series of individual alanine mutations were introduced by the normal methods throughout the CDR3 domain of the D2E7 VL and D2E7 VH regions. The light chain mutations are illustrated in Figure IB (LD2E7 * .A1, LD2E7 * .A3, LD2E7 * .A4, LD2E7 * .A5, LD2E7 * .A7, LD2E7 * .A8, which have an alanine mutation in the position 1, 3, 4, 5, 7 or 8, respectively, of the domain of LD2E7 VL CDR3). Heavy chain mutations are illustrated in Figure 2B (HD2E7 * .Al, HD2E7.A2, HD2E7 * .A3, HD2E7 * .A4, HD2E7 * .A5, HD2E7 * .A6, HD2E7 * .A7, HD2E7 * .A8 , HD2E7 * .A9, having an alanine mutation at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, respectively, of the D2E7 VH CDR3 domain). The kinetics of the interaction of rhTNFa with an antibody composed of wild type D2E7 VL and VH was compared with antibodies composed of 1) a wild type D2E7 VL paired with a D2E7 VH; 2 replaced with alanine) a wild type D2E7 VH paired with a D2E7 VL substituted with alanine; or 3) a D2E7 VL substituted with alanine paired with D2E7 VH substituted with alanine. All antibodies were tested as full-length I gG4 molecules. The kinetics of antibody interaction with rhTNFa was determined by surface plasmon resonance as described in Example 1. The K ft regimens for the different VH / VL pairs are summarized below in Table 5.

Table 5: Linkage of D2E7 Mutants from Alanine Scan to Biotinylated rhTNFa HD2E7 * .A6 D2E7 VL 2.9 x 10-4 HD2E7 * .A7 D2E7 VL 1.0 x 10-4 HD2E7 * .A8 D2E7 VL 3.1 x 10-4 HD2E7 * .A9 D2E7 VL 8.1 x 10-4 D2E7 VH LD2E7 * .A1 6.6 x 10-5 D2E7 VH LD2E7 * .A3 NOT DETECTABLE D2E7 VH LD2E7 * .A4 1.75 x 10"4 D2E7 VH LD2E7 * .A5 1.8 x 10-4 D2E7 VH LD2E7 * .A7 1.4 x 10-4 D2E7 VH LD2E7 * .A8 3.65 x 10-4 HD2E7 * .A9 LD2E7 * .A1 1.05 xl O-4 These results demonstrate that most positions of the CDR3 domains of the D2E7 VL region and the VH region are subject to substitution with a single alanin residue: < . The substitution of a single alanine in position 1, 5 or 7 of the domain D2E7 VL CD 3 or in position 2, 6, 8, 9 or 10 of the domain of D2E7 VH CDR3 does not significantly affect the rate of dissociation of the binding of hTNFa compared to 'D2E7 antibody or wild type origin. the substitution of alanine at position 8 of D2E7 VL CDR3 or position 3 of D2E7 VH CDR3 provides a K.f .; 4 times faster and a substitution of alanine in position 4 or 11 of D2E7 VH CDR3 provides a K «, 8 times faster, indicating that these positions were more critical for the binding to hTNFa. However, a single substitution of alanine in position 1, 4, ¾, 7 or 8 of the D2E7 CDR3 domain or in the position 2, 3, 4, 5, 6, 8, 9, 10 or 11 of the D2E7 VH CDR3 domain still results in an anti-hTNFa antibody having a Koff of 1 x 10-3 sec-1 or less.

EXAMPLE 3: Ligand Analysis of Antibodies Related to D2E7 A series of antibodies related in sequence with D2E7 was analyzed for its binding to rhTNFa, compared to D2E7, by surface plasmon resonance as described in Example 1. The amino acid sequences of the VL regions tested are shown in the Figures 1A and IB. The amino acid sequences of the VH regions were tested as shown in Figures 2A and 2B. The Koff regimens for the different VH / VL pairs (in the indicated format, either as the full-length antibody IgG1 or IgG4 or as scFv) are summarized below in Table 6: Table 6: Ligand of Antibodies Related to D2E7 to Biotinylated rhTNFa VH YL -'ormato K, ff (seg-H D2E7 VH D2E7 VL lgGl lgG4 9.65 x 10-3 VH1-D2 LOE7 IgGl / IgG4 7.7 x 1U-5 VH1-D2 LOE7 scFv 4.6 x 10"4 VH1 -D2.N LOE7.T IgG4 2.1 x 10-5 VH1-D2.Y LOE7.A IgG4 2.7 x 10-5 VH1-D2.N LOE7.A IgG4 3.2 x 10-5 VH1-D2 EP B12 scFv 8.0 x IO-4 VH1-D2 2SD4 VL scFv 1.94 x 10-3 3C-H2 LOE7 scFv 1.5 x 10-3 2SD4 VH LOE7 scFv 6.07 x 10-3 2SD4 VH 2SD4 VL scFv 1.37 x 10-2 VH1AU 2SD4 VL scFv 1.34 x 10-2 VH1B12 2SD4 VL scFv 1.01 x lo-2 VH1B 1 1 2SD4 VL scFv 9.8 x 10-3 VH1E4 2SD4 VL scFv 1.59 x 10-2 VH1 F6 2SD4 VL scFv 2.29 x 10-2 VH1D8 2SD4 VL scFv 9.5 x 10-3 VH1G1 2SD4 VL scFv 2.14 x 10-2 2SD4 VH EP B12 scFv 6.7 x 10-3 2SD4 VH VL10E4 scFv 9.6 x 10-3 2SD4 VH VL100A9 scFv 1.33 x 10-2 2SD4 VH VL100D2 scFv 1 .41 x 10-2 2SD4 VH VL10F4 scFv 1.1 1 x lo-2 2SD4 VH VLLOE5 scFv 1.16 x 10-2 2SD4 VH VLL0F9 scFv 6.09 x 10-3 2SD4 VH VLL0F10 scFv 1.34 x 10-2 2SD4 VH VLLOG7 scFv 1.56 x 10-2 2SD4 VH VLLOG9 scFv 1:46 10-2 ' 2SD4 VH VLLOH I scFv 1.17 x IO-2 2SD4 VH VLLOH10 scFv 1.12 x 10-2 2SD4 VH VL1B7 • scFv 1.3 x 10-2 2SD4 VH VL1C1 scFv 1.36 x 10-2 2SD4 VH VL1C7 scFv 2.0 x 10-2 2SD4 VH VL0.1F4 scFv 1.76 x 10-2 2SD4 VH VL0.1H8 scFv 1.14 x 10-2 Slow dissociation regimens (ie, Koff <1 x 10 ~ 4 sec-1) for full-length antibodies (ie, IgG format) having a VL selected from D2E7, LOE7, LOE7.T and LOE7A , which have either a threonine or an alanine at position 9, indicate that position 9 of D2E7 VL CDR3 can be occupied by any of these two residues without essentially affecting Kof; . Accordingly, a consensus motif for D2E7 VL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y. (T / A) (SEQ ID NO: 3). In addition, the slow dissociation rates (ie, Kctf <1 x 10-sec-1) for antibodies having a VH selected from D2E7, VH1-D2.N and VH1.D2.Y, which have either tyrosine or asparagine at position 12, indicate that position 12 of D2E7 VH CDR3 can be occupied by any of these two residues without essentially affecting Ko.t.Therefore, a consensus motif for D2E7 VH CDR3 comprises the amino acid sequence : VSYLSTASSLD- (Y / N) (SEQ ID N0: 4) The results shown in Table 6 demonstrate that, in the scFv format, antibodies containing the 2SD4 VL or VH CDR3 region exhibit a faster Koff (ie say, Kofr: > 1 x 10 ~ 3 sec-1) compared to antibodies that contain the D2E7 VL or VH CDR3 region.In VL CDR3, 2SD4 differs from D2E7 in position 2, 5, and 9. As is discussed in the foregoing, without However, position 9 can be occupied by Ala (as in 2SD4) or Thr (as in D2E7) without essentially affecting Koff. In this way, by comparison of 2SD4 and D2E7, positions 2 and 5 of D2E7 VL CDR3, both arginines, can be identified as being critical for the association of the antibody with hTNFa. These residues could be directly involved as contact residues at the antibody binding site or could contribute critically to maintain the architecture of the antibody molecule in this region. With respect to the importance of position 2, the replacement of Arg (in L0E7, which has the same VL CDR3 as D2E7) with Lys (in EP B12) accelerates the dissociation rate by a factor of two. With regard to the importance of position 5, the replacement of Arg (in D2E7) with Ala (in LD2E7 * .A5), as described in Example 2, also raises the dissociation rate twice. Furthermore, without either Arg in positions 2 and 5 (in 2SD4), the dissociation rate is five times faster. However, it should be noted that even when position 5 is important for the improved binding to hTNFa, a change in this position can be denied by changes in other positions as can be seen in VLLOE4, VLLOH1 or VL0.1H8. Within VH CDR3, 2SD4 differs from D2E7 in positions 1, 7 and 12. As discussed above. however, position 12 can be occupied by Asñ (as in 2SD4) or Tyr (as in D2E7) without essentially affecting K3ff. In this way, by comparing 2SD4 and D2E7, positions 1 and 7 of D2E7 VH CDR3 can be identified as being critical for binding to hTNFa. As discussed above, these residues could be directly involved as contact residues at the antibody binding site or could critically contribute to maintaining the architecture of the antibody molecule in this region. Both positions are important for binding to hTNFa since when 3C-H2 VH CDR3 (which has a change from valine to alanine at position 1 with respect to D2E7 VH CDR3) is used, the scFv has a dissociation rate 3 times faster than when using D2E7 VH CDR3 but this dissociation rate is still four times slower than when using 2SD4 VH CDR3 (which has changes in both positions 1 and 7 with respect to D2E7 VH CDR3).

EXAMPLE 4: Functional Activity of D2E7 To examine the functional activity of D2E7, the antibody was used in several assays that measure the ability of the antibody to inhibit the activity of hTNFa, either in vitro or in vitro.

A. Neutralization of Cytotoxicity Induced by TNF in L929 Cells Recombinant human TNFα (rhTNFa) causes cell cytotoxity to murine L929 cells after an incubation period of 18 to 24 hours. Human anti-hTNFa antibodies were evaluated in the L929 assays by co-incubation of antibodies with rhTNFa and cells in the following manner. A 96-well microtiter plate containing 100 microliters of the anti-hTNFa Abs was serially diluted one-third down the plate in duplicates using the RPMI medium containing 10 percent fetal bovine serum. { FBS). Fifty microliters of rhTNFa was added for a final concentration of 500 pg per milliliter of each sample well. The plates were then incubated for 30 minutes at room temperature. Then, 50 microliters of TNFα sensitive L929 mouse fibroblast cells were added for a final concentration of 5 x 104 cells per well, including 1 microgram per milliliter of Actinomycin-D. The controls included the medium plus the cells and cells plus rhTNFa. These controls, and a normal TNFα curve ranging from 2 ng per milliliter to 8.2 pg per milliliter, were used to determine the assay quality and provide a window of neutralization. The plates then they were incubated overnight (18 to 24 hours) at 37 ° C in 5 percent CO2. One hundred microliters of the medium from each well was removed and 50 microliters of 5 milligrams per milliliter of 3- (4,4-dimethylthiazol-2-yl), 2,5-diphenyl-tetrazolium bromide (MTT) commercially available from Sigma was added. Chemical Co., of St. Louis, MO in PBS The plates were then incubated for 4 hours at 37 ° C. Fifty microliters of 20% sodium dodecylsulfate (SDS) were then added to each well and the plates were incubated 'overnight at 37 ° C. The optical density at 570/630 nm was measured, the curves plotted for each sample and the I 50 were determined by normal methods.The representative results for the human antibodies having the different pairs of VL and VH, as compared to murine MAK 195 mAb, are shown in Figure 3 and Table 7, which is presented below.

Table 7: Neutralization of L929 Cytotoxicity Induced by TNFa 2SD4 LOE7 scFv 4.3 x Í0-8 VH1-D2 2SD4 scFv 1.0 x 10-8 VH1-D2 L0E7 scFv IgGl IgG4 3.4 x 10 · '° VH1.D2.Y L0E7.T IgG4 8.1 x 10-1 1 VH1-D2.N L0E7.T IgG4 1.3 x 10- '° VH1-D2.Y L0E7.A IgG4 2.8 x 10-11 VH1-D2.N L0E7.A IgG4 6.2 x 10- " MAK 195 MAK 195 scFv 1.9 x 10-8 MAK 195 MAK 195 F (ab ') 2 6.2 x 10- " The results in Figure 3 and Table 7 demonstrate that the human anti-hTNFa antibody D2E7 and the various antibodies related to U2F.1 neutralize the L929 cytotoxicity induced by TNta with a capacity approximately equivalent to that of MAK 195 murine anti-hTNFa mAb. In another series of experiments, the ability of the IgG1 form of D2E7 to neutralize L929 cytotoxicity induced by TNFa was examined as described above. The results of three independent experiments, and the average of them, are summarized below in Table 8: Table 8: Neutralization of Cytoxicity L929 Induced by TOFa by D2E7 IgGl This series of experiments confirmed that D2E7, in the form of full length IgGl, neutralizes L929 cytotoxicity induced by TNFa with an average of IC50 [M] of 1.25 + 0.01 x ?? - "- ':.

B. Inhibition of TNFa Ligand to TNFa Receptors in U-937 Cells The ability of human anti-hTNFα antibodies to inhibit the binding of hTNFα to hTNFα receptors on the surface of cells was examined using a cell line U-937 (ATCC No. CRL 1593), a human histiocytic cell line expressing hTNFa receptors. U-937 cells were grown in a medium of RPMI 1640 supplemented with 10 percent fetal bovine serum (Hyclone A-III, Hyclone Laboratories, Logan, UT), L-glutamine IV4M, HEPES 1-washer solution. (10 mM), penicillin 100 jcicrograms per milliliter) and streptomycin (100 micrograms per milliliter!) To examine the activity of the full-length IgG antibodies, the U-937 cells were pre-incubated with PBS supplemented with 1 milligram per milliliter of human IgG (Sigma 1-4506, Sigma Chemical Co., St. Louis, MO) for 45 minutes on ice and then the cells were washed three times with the binding stabilizer. For the receptor binding assay, U-937 cells (5 x 10 ^ cells per well) were incubated in a binding stabilizer (PBS supplemented with 0.2 percent bovine serum albumin) in 96-well microassay plates ( Costar 3799, Costar Corp., Cambridge, MA) together with rhTNFa irradiated with 125 I (3 x 10"10 M; 25 microCi / milliliter; obtained from NEN Research Products, Wilmington, DE), with or without anti-human antibodies. -hTNFct, in a total volume of 0.2 milliliter The plates were incubated on ice for 1.5 hours, then 75 microliters of each sample were transferred to 1.0 milliliter test tubes (Sarstedt 72,700, Sarstedt Corp., Princeton, NJ) they contain dibutyl phthalate (Sigma D-2270, Sigma Chemical Co., of St. Louis, MO) and dinonyl phthalate (ICN 210733, ICN, Irvine, CA) The test tubes contained a mixture of 300 microliters of phthalate dibutyl and dinonyl phthalate, volume ratio of 2: 1, respectively, rhTNFa irradiates with free 125 I (ie, unbound) was removed by microcentrifugation for five minutes. Then, each end of the tube Test that contains a granule of the cell was cut with the help of a microtube scissor (Bel-Art 210180001, Bel-Art Products, Pequannock, NJ). The cell pellet contains rhTNFoc irradiated with 125 I-linked TNF receptor p60 or p80, while the aqueous phase above the oil mixture contains an excess of rhTNFoc irradiated with 125 I. All cell granules were collected in a counter tube (Falcon 2052, Becton Dickinson Labware, Lincoln Park, NJ) and counted in a scintillation counter. Representative results are shown in Figure 4. The IC50 value for D2E7 inhibition of the binding of hTNFa to hTNFa receptors in U-937 cells is approximately 3 x 10 -10 M in these experiments. These results demonstrate that the human anti-hTNFa antibody D2E7 inhibits the binding of rhTNFa to the hTNFa receptors in U-937 cells at concentrations approximately equivalent to that of the MAK 195 mAb of the murine anti-hTNFa. In another series of experiments, the ability of the IgG1 form of D2E7 to inhibit the binding of rhTNFa to hTNFa receptors in U-937 cells was examined as described above. The results of three independent experiments and the average thereof are summarized in Table 9 below: Table 9: Inhibition of TNF Receptor Ligand in U-937 cells by D2E7 IgGl This series of experiments confirmed that D2E7, in the form of full-length IgG1 inhibits the binding of the TNF receptor in U-937 cells with an average of IC50 [M] of 1.56 + 0.12 x 10-10. To investigate the inhibitory potency of D2E7 in the binding of ": [_ rnTNF to the individual receptors p55 and p75, a solid phase radioimmunoassay was carried out. To measure the IC value of D2E7 for separate TNF receptors, varying antibody concentrations were incubated with 3 x 10"10 concentration of 126I-rhTNF The mixture was then tested on separate plates containing either p55 receptors or p75 of TNF in a manner that depends on the dose.The results are summarized below in Table 10: Table 10: Inhibition of TNF Receptor Ligament to p55 and p75 of TNFR by D2E7 IgGl Inhibition of 12¾I-rhTNF binding to the p55 and p75 TNF receptors in U937 cells by D2E7 followed a simple sigmoidal curve, indicating similar ICSO values for each receptor. In the solid phase radioimmunoassay (RIA) experiments with recombinant TNF receptors, the IC5 values, for inhibition of l: 5I-TNF binding to p55 and p75 receptors, by D2E7 were calculated as 1.47 x 10"' and 1.26 x lO- 'M, respectively The decrease in ICtj0 values in the solid phase was probably due to the higher density of the receptors in the RIA format, since unirradiated rhTNF was also inhibited with values of Similar values The CI values for inhibition of binding of 12¾I-rhTNF towards the p55 and p75 receptors by unradiated rhTNF were 2.31 x 10"and gave 2.70 x 10'9 M, - respectively. C. Inhibition of ELAM-1 Expression in HUVEC Human umbilical vein endothelial cells (HUVEC) can be induced to express the molecule of leukocyte adhesion of endothelial cell 1 (ELAM-1) on its cell surface by treatment with rhTNFa, which can be detected by reacting HUVEC treated with rhTNFa with a mouse anti-human ELAM-1 antibody. The ability of human anti-hTNFα antibodies to inhibit this TNFα-induced expression of ELAM-1 in HUVEC was examined as follows: HUVEC (ATTCC No. CRL 1730) was placed in 96-well plates (5 x 10 4 cells) per well) and incubated overnight at 37 ° C. The next day, serial dilutions of the human anti-hTNFa antibody (1:10) were prepared in a microtiter plate, starting with 20 to 100 micrograms per milliliter of the antibody. A solution of the rhTNFa material was prepared at 4.5 ng per milliliter, aliquots of rhTNFa were added to each well containing the antibody and the contents mixed well. Controls included the medium alone, the medium plus the anti-hTNFa antibody and the medium plus rhTNFa. The HUVEC plates were removed from their incubation overnight at 37 ° C and the medium aspirated gently from each well. Two hundred microliters of the rhTNFa antibody mixture were transferred to each well of the HUVEC plates. The HUVEC plates were then further incubated at 37 ° C for A hours. Then, a material of the murine anti-ELAM-1 antibody was diluted to 1: 1000 in RPMI. The medium in each well of the HUVEC plate it was gently aspirated, 50 microliters was added per well of the anti-ELAM-1 antibody solution and the HUVEC plates were incubated 60 minutes at room temperature. A solution of the anti-mouse Ig antibody irradiated with 125 I-in RPMI (approximately 50,000 cpm in 50 microliters) was prepared. The medium in each well of the HUVEC plates was gently aspirated, the wells were washed twice with RPMI and 50 microliters of the anti-mouse Ig solution irradiated with 125 I was added to each well. The plates were incubated for one hour at room temperature and then each well was washed three times with RPMI. One hundred and eighty microliters of 5 percent SDS were added to each well to lyse the cells. The cell lysate from each well was then transferred to a tube and counted in a scintillation counter. The representative results are shown in Figure 5. The IC50 value for the inhibition of D2E7 of the induced expression of hTNFa of ELAM-1 in HUVEC is approximately 6 x 10 ~ l in these experiments. These results demonstrate that the human anti-hTNF antibody of D2E7 inhibits the TNFα-induced expression of ELAM-1 in HUVEC at concentrations approximately equivalent to that of the MAK 195 mAb of the murine anti-hTNFa.

In another series of experiments, the ability of the IgG1 form of D2E7 to inhibit the hTNFa-induced expression of ELAM-1 in HUVEC was examined as described above, the results of three independent experiments, and the average of the They are summarized below in Table 11: Table 11: Inhibition of ELAM-1 Expression Induced by TNFg by Receptor D2E7 IgGl This series of experiments confirmed that D2E7, in the form of full-length IgG1, inhibits the expression of ELAM-1 induced by TNFa in HUVEC with an average lesion [M] of 1.85 + 0.14 x 10"10. D2E7 IgG1 was also examined for the expression induced by rhTNF of two adhesion molecules, ICAM-1 and VCAM-1, since the rhTNF evaluation curve for ICAM-1 expression at 16 hours was very similar to the curve of expression of ELAM-1-, the same concentration of rhTNF was used in the antibody neutralization tests. HUVEC was incubated with rhTNF in the presence of various concentrations of D2E7 in a C02 incubator at 37 ° C for 16 hours, and the expression of ICAM-1 was measured by the mouse anti-ICAM-1 antibody followed by the sheep antirate irradiated with 125I. Two independent experiments were carried out and the IC 50 values were calculated. An unrelated human IgGl antibody did not inhibit the expression of ICAM-1. The experimental procedure for testing the inhibition of VCAM-1 expression was the same as the procedure for ELAM-1 expression except that anti-VCAM-1 MAb was used instead of anti-ELAM-1 Mab. Three independent experiments were carried out and the IC50 values were calculated. An unrelated human IgGl antibody did not inhibit VCAM-1 expression. The results are summarized below in the Table 12: Table 12: Inhibition of Expression ICAM-1 and VCAM-1 by D2E7 IgGl Inhibition of ICAM-1 Experiment IC50 [MI Experiment IC50 [MI 1 1.84X10-10 1 1.03 x 10-10 2 2.49 x 10- 1 ° 2 9.26 x 10-H 3 1.06 x 10-? Average 2.17 ± 0.46 x 1010 Average 1.01 ± 0.01 x io- '° These experiments demonstrate that treatment of endothelial cells of the primary human umbilical vein with rhTNF led to optimal expression of the adhesion molecules: ELAM-1 and VCAM-1 at four hours; and the up-regulated maximal expression of ICAM-1 at 16 o'clock. D2E7 was able to inhibit the expression of the three adhesion molecules in a way that depends on the dose. The IC50 values for inhibition of ELAM-1, ICAM-1 and VCAM-1 were 1.85 x 10'10, 2.17 x 10"lc and 1.01 x 10 ~ 10 M, respectively.These values were very similar, indicating similar requirements for the dose of the activation signal of rhTNF to induce the expression of ELAM-1, ICAM-1 and VCAM-1 Interestingly, D2E7 was similarly effective in the longer inhibition assay of ICAM-1 expression. The ICAM-1 inhibition assay required 16 hours of co-incubation of rhTNF and D2E7 with HUVEC as opposed to 4 hours required for ELAM-1 and inhibition assays of VCAM-1, since D2E7 has a slow dissociation rate for rhTNF, with conceivable that during the 16-hour co-incubation period there was no significant competition through the TNF receptors in HUVEC.

D. In Vivo Neutralization of hTNFa Three different in vivo systems were used to demonstrate that D2E7 is effective in inhibiting the in vivo activity of hTNFa.

I. Inhibition of TNF-Induced Lethality in D-Galoctosamine Sensitized Mice The injection of recombinant human TNFα (rhTNFa) into mice sensitized with D-galactosamine causes lethality within a period of 24 hours. TNFa neutralizing agents have been shown to prevent lethality in this model. To examine the ability of human anti-hTNFα antibodies to neutralize hTNFα in vivo in this model, C57B1 / 6 mice were injected with different concentrations of D2E7-IgGl, or a control protein, in PBS intraperitoneally (i.p.). The mice were challenged 30 minutes later with one microgram of rhTNFa and 20 milligrams of D-galactosamine in PBS i.p. and they were observed 24 hours later. These amounts of rhTNFa and D-galactosamine were previously determined to achieve 80-90 percent lethality in these mice. The representative results, illustrated in a bar chart, the percentage of survival versus antibody concentration are shown in Figure 6. The black bars represent D2E7, while the bars shaded represent MAK 195. Injection of 2.5-25 micrograms of D2E7 which is an antibody per mouse protected the animals from the lethality induced by TNFa. The ED50 value is approximately 1 to 2.5 micrograms per mouse. The MAK 195 positive control antibody was similar in its protective capacity. Injection of D2E7 in the absence of rhTNFa had no detrimental effect on the mice. Injection of a non-specific human IgGl antibody did not offer any protection from lethality induced by TNFa. In a second experiment, forty-nine mice were divided into 7 equal groups. Each group received different doses of D2E7 thirty minutes before receiving a dose of LD80 of a mixture of rhTNF / D-galactosamine (1.0 microgram of rhTNF and 20 milligrams of D-galactosamine per mouse). Control group 7 received the normal human IgGl kappa antibody at 25 micrograms per mouse dose. The mice were examined 24 hours later. Survival for each group is summarized below in Table 13.

Table 13: 24-hour survival after treatment with D2E7 II. Inhibition of Rabbit Pyrexia Induced by TNF The efficacy of D2E7 to inhibit the response of pyrexia induced rhTNF in rabbits was examined. Groups of three NZW female rabbits weighing approximately 2.5 kilograms each were injected intravenously with D2E7, rhTNF, and immune complexes of D2E7 and rhTNF. The rectal temperatures were measured by thermistor probes and a Kaye thermal recording device every minute lasted * - af._ approximately 4 hours. The human TNF recombine. in saline, injected at 5 micrograms per kilogram, provided a rise in temperature greater than 0.4 ° C to approximately 45 minutes after injection. The preparation of the antibody itself. same, in saline at a dose of 138 micrograms per kilogram did not provide an elevation in temperature in rabbits until 140 minutes after the administration. In all additional experiments, D2E7 or control reagents (human IgGl or a vehicle with saline) were injected i.v. in rabbits followed by 15 minutes later by an injection of rhTNF in a saline solution at 5 micrograms per kilogram intravenously. The representative results of the different experiments are summarized below in Table 14: Table 14: Inhibition of Phyrexia Induced with rhTNF D2E7 in Con ace Maximum Temperature% Inhibition = / ation of temperature with rhTNF and D2E7 / elevation of temperature with rhTNF (only))) x 100.

The intravenous pretreatment with D2E7 at a dose of 14 micrograms per kilogram partially inhibited the pyrogenic response compared to rabbits pretreated with saline alone. D2E7 administered at 137 micrograms per kilogram completely suppressed the pyrogenic response of rhTNF in the same experiment. In a second experiment, D2E7 administered at 24 micrograms per kilogram also partially suppressed the pyrogenic response, compared to rabbits pretreated with a saline solution alone. The molar ratio of D2E7 to rhTNF was 1/6: 1 in this experiment. In a third experiment, D2E7 was injected intravenously at 48 micrograms per kilogram (molar ratio of D2E: hTNF = 3.3: 1) completely suppressed the pyrogenic response compared to rabbits pretreated with human IgGl control in saline at 30 micrograms per kilogram . In the final experiment, rabbits pretreated with D2E7 (792 micrograms per kilogram) at a very high molar ratio to rhTNF (55: 1) did not develop any rise in temperature for any time until 4 hours of observation. The treatment of rabbits with immune complexes generated from a mixture of D2E7 and rhTNF incubated at 37 ° C for 1 hour at a molar ratio of 55: 1, without subsequent administration of rhTNF also did not provide no elevation in temperature in the same experiment.

III. Prevention of Polyarthritis in Transgenic Tgl97 Mice The effect of D2E7 on the development of the disease was investigated in a transgenic murine model of arthritis. Transgenic mice (Tg 197) have been generated which express wild-type human TNF (modified in the 3 'region beyond the coding sequences) and these mice develop chronic polyarthritis with 100% incidence at 4 a 7 weeks of age (see EMBO J (1991) 10: 4025-4031 for further description of the Tgl97 model of polyarthritis). The transgenic animals were identified by PCR at 3 days of age. The baits of transgenic mice were divided into six groups. The transgenic mice were verified by gap-coagulum hybridization analysis at 15 days of age. The treatment protocols for the six groups were as follows: Group l = no treatment; Group 2 = saline (vehicle); Group 3 = D2E7 at 1.5 micrograms per gram: Group 4 = D2E7 at 15 micrograms per gram: Group 5 = D2E7 at 30 micrograms per gram; and Group 6 = control of iso-type of IgGl at 30 micrograms per gram. One litter without transgenic mice was also included in the study to serve as a control (Group 7 - non-transgenic, no treatment). Each group received three intraperitoneal injections per week of the indicated treatments. The injections continued for 10 weeks. Every week there were macroscopic changes in the morphology of the joint for each animal. At 10 weeks, all mice were sacrificed and the mouse tissue was collected in formalin. The macroscopic examination of the tissue was carried out. The weight in grams of the animal was taken for each mouse at the beginning of each week. At the same time measurements of the size of the joint (in millimeters) were also taken as a measure of seriousness of the disease. The size of the joint was established as an average of three measurements on the posterior right ankle using a micrometer device. Arthritic excoriations were recorded weekly as follows: 0 = no arthritis, (normal appearance and flexion) + = mild arthritis (joint distortion); ++ = moderate arthritis (swelling, joint deformation) and +++ = severe arthritis (alkylosis detected in flexion and severely impaired movement) Histopathological scaling based on hematoxylin / eosin staining of the sections of the joint was based as follows; 0 = No detectable disease; 1 = proliferation of the synovial membrane; 2 = heavy synovial thickening; 3 = cartilage destruction and bone erosion. The effect of D2E7 treatment on the average joint size of Tgl97 transgenic arthritic mice is shown in the graph of Figure 9. Histopathological and arthritic excisions of transgenic Tgl97 mice at 11 weeks of age are summarized below in Table 15: Table 15: Effect of D2E7 on Histopathology and Arthritic Cleavage in Tgl97 Mice This experiment showed that the D2E7 antibody had a definite beneficial effect on the transgenic mice expressing wild-type human TNG (Tg 197) without obvious arthritis after the study period.

E. D2E7 Neutralization of TNFa from Other Species The binding specificity of D2E7 was examined by measuring its ability to neutralize the tumor necrosis factors of different primate and mouse species, using an L929 cytotoxicity assay (as described in Example 4, subsection A, in the foregoing). The results are summarized in Table 16, which is presented below: Table 16: Capacity of D2F.7 to Neutralize TNF of Different Species in Test L929 Source or IC60 for D2E7 j TNFa * Provenance Neutralization (M) Recombinant Human 7.8 x 10 · "Chimpace PBMC stimulated with LPS 5.5 x 10-1 1 Recombinant mandrel 6.0 x 10-" titi PBMC stimulated with LPS 4.0 x 10- 10 cinomolgus PBMC stimulated with LPS 8.0 10- "macaque from Indi to PBMC stimulated with LPS 3.0 x 10- "canine WBC stimulated with LPS 2.2 x 10- '° Recombinant porcine 1 .0 x 10-7 murine Recombinant > 1 .0 10-7 The results in Table 16 demonstrate that D2E7 can neutralize the activity of five primate TNFα approximately equivalent to human TNFα and, in addition, can neutralize the activity of canine TNFα (approximately ten times less than human TNFα) and porcine TNFα and mouse (approximately ~ 1000 times less than human TNFa). In addition, the binding of D2E7 to the solution phase of rhTNFa was not inhibited by other cytokines, such as lymphotoxin (NF), IL-? A, IL, IL-2, IL-4, IL-6, IL-8, IFNy and TGFP, indicating that D2E7 is very specific for its TNFa coordinating group.

F. Lack of Cytokine Release by Whole Blood Hunaba Incubated with D2E7 In this example, the ability of D2E7 to induce, by itself, normal human blood cells to secrete cytokines or molecules from the surface of the cell was examined. D2E7 was incubated with diluted whole blood from three different normal donors at varying concentrations for 24 hours. A positive control of LPS was carried out at the same time, at a previously determined concentration to stimulate the immunocompetent blood cells to secrete cytokines. The supernatants were harvested and tested in a panel of ten cytokine ELISA kits soluble, receptor and adhesion molecule: IL-? a, ILP, IL-1 which is the antagonist receptor IL-6, IL-8, TNFa soluble TNF receptor 1, soluble TNF receptor II, soluble ICAM-1 and soluble E-selectin. None of the significant amounts of cytokines or cell surface molecules were measured as a result of co-incubation of the D2E7 antibody at concentrations up to 343 micrograms per milliliter. The control cultures without the addition of the antibody also yielded no measurable amounts of cytokines, while the co-culture control of LPS yielded high values in the high picogram to low manoframa scale. These results indicate that D2E7 did not induce whole blood cells to secrete cytokines or cell surface proteins above normal levels in ex vivo cultures. As part of the present presentation, there is the list of Annexed Sequences, the content of which is summarized in the following table: ^ TYPE CHAIN SEQ ID NO: SEQUENCE REGION ANTIBODY 1 D2E7 VL amino acid 2 D2E7 VH amino acid < 3 D2S7 VL CDR3 amino acid " 4 D2E7 VH CDR3 amino acid D2E7 VL CDR2 amino acid 6 D2E7 VH CDR2 amino acid 7 D2E7 VL CDR1 amino acid 8 D2E7 VH CDR1 aminoacidol 9 2SD4 VL amino acid 2SD4 VH amino acid 11 2SD4 VL CDR3 amino acid 12 EP B12 VL CDR3 amino acid 13 VL10E4 VL CDR3 amino acid 14 VL100A9 VL CDR3 amino acid VLL100D2 VL CDR3 amino acid 16 VLL0F4 VL CDR3 amino acid 17 LOES VL CDR3 amino acid 18 VLLOG7 VL CDR3 amino acid 19 VLLOG9 VL CDR3 aminoacid VLLOH1, VL CDR3 amino acid twenty-one ! VLLOH10 VL CDR3 acid aminc 22 VL1B7 VL CDR3 amino acid 23 VL1C1 VL CDR3 aminoacid 24 VLO .1F4 · VL CDR3 | amino acid VL0.1H8 VL CDR3 amino acid 26 L0E7.A VL CDR3 amyro acid 27 2SD4 VH CDR3 amino acid 28 VH1B11 VH CDR3 amino acid 29 VH1D8 VH CDR3 amino acid 30 VH1A11 VH CDR3 amino acid 31 VH1S12 VH CDR3 aminoacid 32 VH1E4 VH CDR3 amino acid 33 VH1F6 VH CDR3 amino acid ' 34 3C-H2 VH CDR3 amino acid 35 VH1-D2. N VH CDR3 amino acid 36 j D2E7 VL nucleic acid 37 D2E7 VH nucleic acid EQUIVALENTS Those skilled in the art will recognize, or be able to make sure using no more than routine experiments, many of the equivalents for the specific embodiments of the invention described herein. These equivalents are intended to be covered by the following claims: LIST OF SEQUENCES GENERAL INFORMATION (i) APPLICANT: (A) NAME: BASF Aktiengesellschaft (B) STREET: Carl-Bosch Str. 38 (C) CITY: 67056 Ludwigshafen (D) STATE: Rheinland-Pfalz (E) COUNTRY: Federal Republic of Germany < ii) TITLE OF THE INVENTION: Human Antibodies That Link Human TNFa (iiil SEQUENCE NUMBER: 37 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: LAHIVE £ COCKFIELD (B) STREET: 60 State Street, Suite 510 (C) CITY: Boston (D) STATE: Massachusetts (E) COUNTRY: United States of America (F) POSTAL CODE: 02109-1375 (v) COMPUTER LEADING FORM: (A) TYPE OF MEDIA: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS- TWO (D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.25 (i) CURRENT SOLICITATION DATA: (A) APPLICATION NAME: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS SOCLITUDE: (A) APPLICATION NUMBER: US 08 / 599,226 (B) SUBMISSION DATE: February 9, 199 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 60 / 031,476 (B) SUBMISSION DATE: November 25, 1996 (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION : (A) NAME: DeConti, Giulio A., Jr. (B) REGISTRATION NUMBER: 31,503 (C) ATTORNEY'S NUMBER OF REFERENCE / TOCA: BBI-043CPPC (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (617) ) 227-7400 (B) TELEFAX: (617) 227-59 1 INFORMATION FOR SEQ ID NO: l (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: l: sp lie Gln MeC Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg val Thr lie Thr Cys Arg Ala Ser Gln Gly lie Arg Asn Tyr 20 25 30 Leu Wing Trp Tyr Gln Gln Gln Lys Pro Gly Lys Wing Pro Lys Leu Leu lie 35 40 45 Tyr Wing Wing Thr Leu Gln Ser Gly val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gln Pro 65 70 7S 80 Glu Asp Val Wing Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Wing Pro Tyr 85 90 9S Thr Phe Gly Gln Gly Thr Lys Val Glu lie Lys 100 IOS INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 121 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Glu Val Gln Leu V * l Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 5 10 15 Be Leu Arg Leu Be Cys Wing Wing Be Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Wing Met His Trp val Arg Gln Wing Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Wing He Thr Trp Aasa be Gly His He Asp Tyr Wing Asp Ser Val 50 55 60 Glu Gly Arg Pha Thr Ii «Ser Arg Asp Asn Wing Lys Asn Ser Leu Tyr 65 75 80 Leu Gln Met Aan Ser L * u Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys as 90 95 Ala Lys Val Ser Tyr L «u Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly loo ios 110 Gln Gly Thr Leu V «l Thr Val Ser Ser 115 120 INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (AT LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: Modified site (B) LOCATION: 9 (D) OTHER INFORMATION: / note = "Xaa is Thr or Ala" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / note = "Xaa is Tyr or Asn" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: Ala Ala Ser Thr Leu Gln Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Ala lie Thr Trp Asn Ser Gly His lie Asp Tyr Ala Asp Ser Val 1 5 10 15 Glu Gly (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Arg Ala Ser Gln Gly lie Arg Asn Tyr Leu Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Asp Tyr Ala Met His 1 5 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Asp He Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser lie Gly! S 10 - 15 Asp Arg Val Thr lia Thr Cys Arg Ala Ser Gln Gly He Arg Asn Tyr 20 25 30 Leu Wing Trp Tyr Wave Gln Lys Pro Gly Lys Wing Pro Lys Leu Leu He 35 40 45 Tyr Wing Wing Being T¾r Le Gln Being Gly Val Pro Being Arg Phe Ser Gly SO S5 60 Ser Gly Be Gly Thr Asp Phe Thr Leu Thr Be Ser Be Leu Gln Pro 65 70 75 80 Glu Asp Val Wing Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Wing Pro Tyr 85 90 95 Wing Phe Gly Gln Gly Thr Lys Val Glu He Lys 100 IOS (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 121 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Wing Wing Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ser Ala He Thr Trp Asn Ser Gly His Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Ala Val Ser Arg Asp Asn Ala Lys Asn Ala Leu Tyr .65 70 75 80 Leu Gln Met Asn Ser Grape Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Wing Ser Tyr L »u Ser Thr Ser Ser Ser Leu Asp Asn Trp Gly 100 IOS 110 Gln Gly Thr Leu Val Til * Vtl Ser Ser US 120 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: "· 'Gln Lys Tyr Asn Ser Ala Pro Tyr Ala 1 5 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF 'THE SEQUENCE: SEQ ID NO: 12: Gln Lys Tyr Asn Arg Wing Pro Tyr Wing (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: Gln Lys Tyr Gln Arg Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: Gln Lys Tyr Ser Ser Ala Pro Tyr Thr 1 5 INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: Gln Lys Tyr Asn Ser Wing Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: Gln Lys Tyr Asn Arg Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 17: Gln Lys Tyr Asn Ser Ala Pro Tyr Tyr (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: Gln Lys Tyr Asn Ser Ala Pro Tyr Asn 1 5 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: Gln Lys Tyr Thr Ser Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: Gln Lys Tyr Asn Arg Ala Pro Tyr Asn 1 5 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: Gln Lys Tyr Asn Ser Ala Ala Tyr Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: Gln Gln Tyr Asn Ser Ala Pro Asp Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 23: Gln Lys Tyr Asn Ser Asp Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24: Gln Lys Tyr lie Ser Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: Gln Lys Tyr Asn Arg Pro Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: Gln Arg Tyr Asn Arg Ala Pro Tyr Ala (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27: Wing Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 1 5 10 (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28: Wing Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29: Wing Being Tyr Leu Being Thr Being Being Leu Asp Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30: Wing Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp 1 5 10 (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 31 Wing Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32: Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr 1 5 10 INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Wing Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34: Wing Ser Tyr Leu Ser Thr Wing Being Ser Leu Glu Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35: Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Asn i 5 10 (2) INFORMATION FOR SEQ ID NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULA: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 36: GACATCCAGA TGACCCACTC TCCATCCTCC CTGTCTGCAT CTGTAGGGGA CAGAGTCACC 60 ATCACTTGTC GGOCAAGTC * GGGCATCAGA AATTACTTAG GGGAAAGCCC CTAAGCTCCT CCTGGTATCA GCAAAAACCA 120 'ATCTATGCT GCATCCACTT TGCAATCAGG GGTCCCATCT TGGGACAGAT TTCACTCTCA 180 CGGTTCAGTG GCAGTGGAIC CCATCAGCAG CCTACAGCCT 2 0 GAAGATGTTG CAACTTATTA CTGTCAAAGG TATAACCGTG CACCGTATAC TTTTGGCCAG 300 GGGACCAAGG TGGAAATCftA A 321 (2) INFORMATION FOR SEQ ID NO: 37:, (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 363 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 37: GAGGTGCAGC TGGTGGAGTC TGGGGOAGGC TTGGTACAGC CCGGCAGGTC CCTGAGACTC 60 TCCTGTGCGG CCTCTGGATT CACCTTTGAT GATTATGCCA TGCACTGGGT CCGGCAAGCT 120 CCAGGGAAGG GCCTGGAATG QGTCTCAGCT ATCACTTGGA ATAGTGGTCA CATAGACTAT 1B0 GCGGACTCTG TGGAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCCCTGTAT 240 CTGCAAATGA ACAGTCTGAG AGCTQAGGAT ACGGCCGTAT ATTACTGTGC GAAAGTCTCG 300 TACCTTAGCA CCGCGTCCTC CCTTGACTAT TGGGGCCAAG GTACCCTGGT CACCGTCTCG 360

Claims (73)

R E I V I N D I C A C I O N S

1. An isolated human antibody, or an antigen binding portion thereof, which dissociates from human TNFa with a Kd of 1 X lCr8 M or less and a constant of Kof f regime of 1 X 10 ~ 3 s_1 or less, both determined by surface plasmon resonance and neutralizes the cytotoxicity of human TNFa in a normal L929 assay with an ICBO of 1 X 10"7 M or less

2. Human antibody isolated, or antigen binding portion thereof of claim 1, which is dissociated from human TNFa with a Kof f regime constant of 5 X 10"4 s" 1 or less

3. The human antibody isolated or the antigen binding portion thereof of claim 1, which is dissociated from human TNFa with a Kof f regime constant of 1 X 10 -4 s "1 or less.

4. The human antibody isolated or the antigen binding portion thereof of claim 1, which neutralizes the cytotoxicity of human TNFa in a normal L929 assay with an ICBO of 1 X 10 ~ 8 M or less.

The isolated human antibody or antigen binding portion thereof of claim 1, which neutralizes the cytotoxicity of human TNFα in a normal in vitro L929 assay with an IC 50 of 1 X 10 ~ 9 M or less.

6. The isolated human antibody or antigen binding portion thereof of claim 1, which neutralizes the cytotoxicity of human TNFα in a normal in vitro L929 assay with an ICso of 1 X - -10 M or less.

The human antibody isolated or the antigen binding portion thereof of claim 1, which is a recombinant antibody or the antigen binding portion thereof.

The human antibody isolated or the antigen binding portion thereof of claim 1, which inhibits human TNFα-induced expression of ELAM-1 in human umbilical vein endothelial cells.

9. An isolated human antibody or antigen binding portion thereof, having the following characteristics: a) dissociation of human TNFα with a steady-state of 1 X 10 -3 s 1 or less, as determined by surface plasmon resonance; b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or is modified of SEQ ID NO: 3 by a single substitution of alanine at position 1, 4, 5, 7 or 8 , by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9; c) has a heavy chain CDR3 domain comprising the amino acid sequence SEQ ID NO: 4, or is modified from SEQ ID NO: 4 by a single substitution of alanine at the 2, 3, 4, 5, 6 position, 8, 9, 10 or 11, or from one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and / or 12.

10. Human antibody isolated from claim 9, or the antigen binding portion thereof, which dissociates from human TNFa with a Koft regime constant of 5 X 10 ~? s_1 or less.

The isolated human antibody of claim 9, or the antigen binding portion thereof which dissociates from human TNFa at a Korr rate constant of 1 X 10 's "1 or less

12. An isolated human antibody, or an antigen binding portion thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single substitution of alanine at position 1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence SEQ ID NO: 4 or that is modified from SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.

13. The isolated human antibody, or antigen binding portion thereof, of claim 12, wherein LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 and HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6.

14. The human antibody isolated, or an antigen binding portion thereof of claim 13, wherein LCVR further has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 and HCVR has a CDR1 domain comprising the amino acid sequence of SEQ. ID NO:

15. An isolated human antibody, or an antigen binding portion thereof with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2.

16. The isolated human antibody of claim 15, having a heavy chain constant region of IgGl.

17. The isolated human antibody of claim 15, having a heavy chain constant region of IgG.

18. The isolated human antibody of claim 15, which is a fragment of Fab.

19. The isolated human antibody of claim 15, which is a single chain Fv fragment.

20. An isolated human antibody, or the antigen binding portions thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence that is selected from the group consisting of SEQ ID NO : 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 , SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or with a heavy chain variable region (HCVR) having a CDR3 domain comprising an amino acid sequence that is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

21. A recombinant human antibody or an antigen binding portion thereof that neutralizes the activity of TNFcc, but not of the human T FP.

22. The recombinant human antibody or antigen binding portion thereof of claim 21, which also neutralizes the activity of the Chimpanzee TNFa, and at least TNFa from an additional primate that is selected from the group consisting of mandrel TNFa, titi TNFa, cynomolgus TNFa and rhesus monkey TNFa.

23. The recombinant human antibody or an antigen binding portion thereof of claim 22, which also neutralizes canine TNFa activity.

24. The recombinant human antibody or antigen binding portion thereof of claim 22, which also neutralizes pig TNFa activity.

25. An isolated nucleic acid encoding the light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single substitution of alanine at position 1, 4 , 5, 7, or 8, or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9.

26. The isolated nucleic acid of claim 25 which encodes a antibody light chain variable region (LCVR).

27. The isolated nucleic acid of claim 26, wherein the CDR2 domain of the LCVR antibody comprises the amino acid sequence of SEQ ID NO: 5.

28. The isolated nucleic acid of claim 27, wherein the CDR1 domain of the LCVR antibody comprises the amino acid sequence of SEQ ID NO: 7.

29. An acid isolated nucleic acid encoding a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5 , 6, 8, 9, 10 or 11, or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and / or 12.

30. The acid Isolated nucleic of claim 29 which encodes an antibody heavy chain variable region (HCVR).

31. The isolated nucleic acid of claim 30, wherein the CDR2 domain of the HCVR antibody comprises the amino acid sequence of SEQ ID NO: 6.

32. The isolated nucleic acid of claim 31, wherein the CDR1 domain of the HCVR antibody comprises the amino acid sequence of SEQ ID NO: 8.

33. An isolated nucleic acid encoding a CDR3 domain comprising an amino acid sequence that is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: ll, SEQ ID NO: 12, SEQ ID N0: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO : 21, SEQ ID N0: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 , SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.

34. An isolated nucleic acid encoding a light chain variable region of the antibody comprising the amino acid sequence SEQ ID NO: l.

35. The isolated nucleic acid of claim 34, which encodes the light chain variable region of the antibody and a light chain constant region of the antibody.

36. The isolated nucleic acid of claim 35, which is a recombinant expression vector.

37. An isolated nucleic acid encoding a heavy chain variable region of the antibody comprising the amino acid sequence SEQ ID NO: 2.

38. The isolated nucleic acid of claim 37, which encodes the variable region of heavy chain of the antibody and a constant region of the heavy chain of the antibody.

39. The isolated nucleic acid of claim 38, wherein the heavy chain constant region of the antibody is a constant region of IgG1.

40. The isolated nucleic acid of claim 38, wherein the heavy chain constant region of the antibody is a constant region of IgG4.

41. The isolated nucleic acid of claim 38, which is a recombinant expression vector.

42. A recombinant expression vector encoding: a) an antibody light chain having a variable region comprising the amino acid sequence of SEQ ID NO: 1; and b) an antibody heavy chain having a variable region comprising the amino acid sequence of SEQ ID NO: 2.

43. A host cell wherein the recombinant expression vector of claim 42 has been introduced.

44. A method for synthesizing a human antibody that binds human TNFa, which comprises culturing the host cell of claim 43 in a medium of culture until a human antibody that binds human TNFa is synthesized by the cell.

45. A pharmaceutical composition comprising the antibody, or the antigen binding portion thereof of any of claims 1 to 24, and a pharmaceutically acceptable carrier.

46. The pharmaceutical composition of claim 45, further comprising at least one additional therapeutic agent for treating a disorder wherein the activity of TNFa is detrimental.

47. A method for inhibiting human TNFα activity comprising contacting human TNFα with an antibody, or antigen binding portion thereof, of any one of claims 1 to 24 such that the activity of TNFα is inhibited. human.

48. A method for inhibiting human TNFα activity in a human patient suffering from a disorder wherein the activity of TNFα, which comprises administering to the human patient the antibody, or the antigen binding portion thereof, of any claims 1 to 24 in such a manner that the activity of human TNFα in the human patient is inhibited.

49. The method of claim 48, wherein the disorder is sepsis.

50. The method of claim 49, wherein the antibody is administered to the human patient together with cytokine interleukin-6 (IL-6) or is administered to a human patient with a serum or plasma concentration of IL-6 greater than 500. pg per milliliter.

51. The method of claim 48, wherein the disorder is an autoimmune disease.

52. The method of claim 51, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, rheumatoid arthritis, osteoarthritis and gout arthritis.

53. The method of claim 51, wherein the autoimmune disease is selected from the group consisting of an allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome.

54. The method of claim 48, wherein the disorder is an infectious disease.

55. The method of claim 48, wherein the disorder is the rejection of a transplant or a graft versus host disease.

56. The method of claim 48, wherein the disorder is a malignancy.

57. The method of claim 48, wherein the disorder is a pulmonary disorder.

58. The method of claim 48, wherein the disorder is an intestinal disorder.

59. The method of claim 48, wherein it is a cardiac disorder.

60. The method of claim 48, wherein the disorder is selected from the group consisting of inflammatory bone disorders, bone resorption disease, alcoholic hepatitis, viral hepatitis, fulminating hepaticitis, coagulation disorders, burns, reperfusion injury, keloid formation, scar tissue formation, pyrexia, periodontal disease, obesity and radiation toxicity.

61. The use of the antibody or antigen binding portion thereof of any of claims 1 to 24 in the manufacture of a medicament for the treatment of a disorder wherein the activity of TNFa is detrimental.

62. The use of claim 61, wherein the disorder in sepsis.

63. The use of claim 62, wherein the antibody is administered to the human patient together with cytokine interleukin-6 (IL-6) or is administered to a human patient with a serum or plasma concentration of IL-6 greater than 500 pg per milliliter.

64. The use of claim 61, wherein the disorder is an autoimmune disease.

65. The use of claim 64, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gout arthritis.

66. The use of claim 64, wherein the autoimmune disease is selected from the group consisting of an allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome.

67. The use of claim 61, wherein the disorder is an infectious disease.

68. The use of claim 61, wherein the disorder is a transplant rejection or graft versus host disease.

69. The use of claim 61, wherein the disorder is a malignancy.

70. The use of claim 61, wherein the disorder is a pulmonary disorder.

71. The use of claim 61, wherein the disorder is an intestinal disorder.

72. The use of claim 61, wherein is the cardiac disorder.

73. The use of claim 61, wherein the disorder is selected from the group consisting of inflammatory bone disorders, bone resorption disease, alcoholic hepatitis, viral hepatitis, fulminant hepaticitis, coagulation disorders, burns, reperfusion injury, keloid formation, scar tissue formation, pyrexia, periodontal disease, obesity and radicle toxicity .