AU6664900A - Human antibodies that bind human TNFalpha - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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*9*6 *9 Name of Applicant/s: Actual Inventor/s: Address for Service: BASF Aktiengesellschaft Jochen G. Salfeld and Deborah J Allen and Zehr Kaymakcalan and Boris Labkovsky and John A. Mankovich and Brian T. McGuiness and Andrew J. Roberts and Paul Sakorafas and Hendricus R.J.M.

Hoogenboom and David Schoenhaut and Tristan J. Vaughan and Michael White and Alison J. Wilton BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 'HUMAN ANTIBODIES THAT BIND HUMAN TNFalpha' Invention Title: Details of Original Application No. 21229/97 dated 10 February 1997 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 29786AUP00 la HUMAN ANTIBODIES THAT BIND HUMAN TNFc The present invention relates to TNFa ane more particularly to antibodies binding to human TNFa. This application is a divisional of AU 21229/97 which is incorporated in its entirety herein by reference.

Tumor necrosis factor a (TNFa) is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its capacity to induce the necrosis of certain mouse tumors (see Old, L. (1985) Science 230:630-632). Subsequently, a factor termed cachectin, associated with cachexia, was shown to be the same molecule as TNFa. TNFa has been implicated in mediating shock (see Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518; Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655). Furthermore, 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 Moeller, et al. (1990) Cytokine 2:162-169; 5 U.S. Patent No. 5,231,024 to Moeller et al.; European Patent Publication No. 260 610 BI by Moeller, et al.Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).

Because of the harmful role of human TNFa (hTNFa) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract hTNFa 2 activity. In particular. antibodies that bind to, and neutralize, hTNFa have been sought as a means to inhibit hTNFa activity. Some of the earliest of such antibodies were "i mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNFc (see Hahn T; et al., (1985) Proc Nail Acad Sci USA 82: 3814-3818: Liang, et al. (1986) Biochem. Biophys. Res.

Commun. 137:847-854; Hirai, et al. (1987) J. Immunol. Methods 96:57-62; Fendly, et al. (1987) Hybridoma 6:359-370; Moeller, et al. (1990) Cytokine 2:162- 169; U.S. Patent No. 5,231,024 to Moeller et al.; European Patent Publication No. 186 833 BI by Wallach. European Patent Application Publication No. 218 868 Al by Old et al.: European Patent Publication No. 260 610 Bi by Moeller, et While 3~ these mouse anti-hTNFa antibodies often displayed high affinity for hTNFac Kd 10-9M) and were able to neutralize hTNFa activity, their use in vivo may be limited by problems associated with administration of mouse antibodies to humans, such as short serum half life. an inability to trigger certain human effector functions and elicitation of an unwanted immune response against the mouse antibody in a human (the "human antimouse antibody" (HAMA) reaction).

In an attempt to overcome the problems associated with use of fuliv-murine antibodies in humans. murine anti-hTNFa antibodies have been geneticallv ensineered to be more "human-like." For example, chimeric antibodies, in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived, have been prepared (Knight, D.M, et al. (1993) Mol.

Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 by Daddona, et al.).

Additionally, humanized antibodies, in which the hypervariable domains of the antibody variable regions are murine-derived but the remainder of the variable regions and the antibody constant regions are human-derived, have also been prepared (PCT Publication No. WO 92/11383 by Adair, et However, because these chimeric and humanized antibodies still retain some murine sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged periods, for chronic indications, such as rheumatoid arthritis (see Elliott, et al. (1994) Lancet 344:1125-1127; Elliot.

et al. (1994) Lancet 344:1105-1110).

A preferred hTNFa inhibitory agent to murine mAbs or derivatives thereof 15 chimeric or humanized antibodies) would be an entirely human anti-hTNFa. antibody, since such an agent should not elicit the HAMA reaction, even if used for prolonged *periods. Human monoclonal autoantibodies against hTNFa have been prepared using human hybridoma techniques (Boyle, et al. (1993) Cell. Immunol. 152:556-568; Boyle, et al. (1993) Cell. Immunol. 152:569-581; European Patent Application Publication No. 614 984 A2 by Boyle, et However, these hybridoma-derived monoclonal autoantibodies were reported to have an affinity for hTNFa that was too low to calculate by conventional methods, were unable to bind soluble hTNFa and were S unable to neutralize hTNFa-induced cytotoxicity (see Boyle, et al.; supra). Moreover, the success of the human hybridoma technique depends upon the natural presence in S. 25 human peripheral blood of lymphocytes producing autoantibodies specific for hTNFoa.

Certain studies have detected serum autoantibodies against hTNF( in human subjects (Fomsgaard, et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, et al.

(1990) Prog. Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, et al. (1991)J. Immunol. Methods 139:145-147).

Alternative to naturally-occurring human anti-hTNFa antibodies would be a recombinant hTNFa antibody. Recombinant human antibodies that bind hTNFa with relatively low affinity Kd -10- 7 M) and a fast off rate Koff 10-2 sec- 1 have been described (Griffiths, et al. (1993) EMBO J. 12:725-734). However. because of their relatively fast dissociation kinetics, these antibodies may not be suitable for therapeutic use. Additionally, a recombinant human anti-hTNFa has been described that does not neutralize hTNFa activity, but rather enhances binding of hTNFa to the surface of cells and enhances internalization of hTNFa (Lidbury, et al. (1994) Biotechnol. Ther. 5:27-45; PCT Publication No. WO 92/03145 by Aston, R. et al.) Accordingly, human antibodies, such as recombinant human antibodies, that bind soluble hTNFa with high affinity and slow dissociation kinetics and that have the capacity to neutralize hTNFc activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and hTNFa-induced cell activation, are still needed.

Summarv of the Invention This invention provides human antibodies, preferably recombinant human antibodies, that specifically bind to human TNFa. The antibodies of the invention are characterized by binding to hTNFa with high affinity and slow dissociation kinetics and by neutralizing hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and hTNFa-induced cellular activation. Antibodies of the invention are further characterized by binding to hTNFa but not hTNFp (lymphotoxin) and by having the 15 ability to bind to other primate TNFas and non-primate TNFas in addition to human TNFa.

The antibodies of the invention can be full-length an IgGI or IgG4 antibody) or can comprise only an antigen-binding portion a Fab, F(ab') 2 or scFv fragment). The most preferred recombinant antibody of the invention, termed D2E7, has 20 a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR3 domain comprising the 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 (HCVR) comprising the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFca with a Kd of 1 x 8 M or less and a Koff rate constant of I x 10- 3 s- 1 or less, both determined by surface plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC 50 of 1 x 10- 7 M or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koff of 5 x 10- 4 s- 1 or less, or even more preferably, with a Koff of I x 10- 4 s-1 or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC 50 of I x 10- 8

M

or less, even more preferably with an IC 50 of 1 x 10-9 M or less and still more preferably with an IC 50 of 5 x 10- 10 M or less.

In another embodiment, the invention provides a human antibody, or antigenbinding portion thereof, with the following characteristics: a) dissociates from human TNFc with a Koff 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 alanine substitution 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 alanine substitution 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.

*:**More preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koff of 5 x 10' 4 s-I or less. Still more preferably, the 15 antibody, or antigen-binding portion thereof, dissociates 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 CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with an HCVR having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. More preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6. Still more preferably, the LCVR further has CDRI domain comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8.

In still another embodiment, the invention provides an isolated human antibody, or an antigen binding portion thereof, with an LCVR comprising the amino acid sequence of SEQ ID NO: 1 and an HCVR comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the antibody has an IgGI heavy chain constant region or an IgG4 heavy chain constant region. In yet other embodiments, the antibody is a Fab fragment, an F(ab') 2 fragment or a single chain Fv fragment.

In still other embodiments, the invention provides antibodies, or antigen-binding portions thereof, with an LCVR having CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12. SEQ ID NO: 13, SEQ ID NO: 14. SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17. 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 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: In yet another embodiment, the invention provides an isolated human antibody, or antigen-binding portion thereof, that neutralizes the activity of human TNFa but not human TNFP (lymphotoxin). In a preferred embodiment, the human antibody, or antigen-binding portion thereof,-neutralizes the activity of human TNFa, chimpanzee

TNF

a c and at least one additional primate TNFa selected from the group consisting of baboon TNFa, marmoset TNFa, cynomolgus TNFo and rhesus TNFa. Preferably, the antibody also neutralizes the activity of at least one non-primate TNFa. For example, in 15 one subembodiment, the isolated human antibody, or antigen-binding portion thereof, also neutralizes the activity of canine TNFca. In another subembodiment, the isolated human antibody, or antigen-binding portion thereof, also neutralizes the activity of pig TNFa. In yet another subembodiment, the isolated human antibody, or antigen-binding portion thereof, also neutralizes the activity of mouse TNFa.

20 Another aspect of the invention pertains to nucleic acid molecules encoding the antibodies, or 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, encoding a D2E7 HCVR, has the nucleotide sequence shown in Figure 8 and SEQ ID NO 37.

25 Recombinant expression vectors carrying the antibody-encoding nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing the host cells of the invention.

Yet another aspect of the invention pertains to methods for inhibiting human TNFa activity using an antibody, or antigen-binding portion thereof, of the invention.

In one embodiment, the method comprises contacting human TNFa with the antibody of the invention, or antigen-binding portion thereof, such that human TNFa activity is inhibited. In another embodiment, the method comprises administering an antibody of the invention, or antigen-binding portion thereof, to a human subject suffering from a disorder in which TNFa activity is detrimental such that human TNFa activity in the human subject is inhibited. The disorder can be, for example, sepsis, an autoimmune disease rheumatoid arthritis. allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome), an infectious disease, a malignancy, transplant rejection or graft-versus-host disease, a pulmonary disorder, a bone disorder, an intestinal disorder or a cardiac disorder.

Brief Description of the Drawings Figures 1A and 1B show the amino acid sequences of the light chain variable region of D2E7 (D2E7 VL; also shown in SEQ ID NO: alanine-scan 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 2SD4 (2SD4 VL; also shown in SEQ ID NO.9) and other D2E7-related light chain variable regions (EP B12, VLIOE4, VL.100A9, VL100D2, VL10F4, LOE5, VLLOF9, VLLOG7, VLLOG9, VLLOH1, VLLOH10, VLIB7, VLIC1, VL1C7, VLO.1F4, VLO.1H8, LOE7, LOE7.A and LOE7.T). Figure 1A shows the FRI, CDR1, FR2 and 15 CDR2 domains. Figure 1B shows the FR3, CDR3 and FR4 domains. The light chain CDRI ("CDR L1 CDR2 ("CDR L2") and CDR3 ("CDR L3") domains are boxed.

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: alanine-scan 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 D2E7-related antibody 2SD4 (2SD4 VH; also shown in SEQ ID NO: and other D2E7-related heavy chain variable regions (VHIB11, VH1D8, VHAI A1, VH1B12, VH1-D2, VH1E4, VH1F6, VH1G1, 3C-H2, VHI-D2.N and VHI-D2.Y).

Figure 2A shows the FRI, CDRI, FR2 and CDR2 domains. Figure 2B shows the FR3, 25 CDR3 and FR4 domains. The heavy chain CDR1 ("CDR CDR2 ("CDR H2") and CDR3 ("CDR H3") domains are boxed.

Figure 3 is a graph depicting the inhibition of TNFa-induced L929 cytotoxicity by the human anti-hTNFa antibody D2E7, as compared to the murine anti-hTNFa antibody MAK 195.

Figure 4 is a graph depicting the inhibition of rhTNFa binding to hTNFa receptors on U-937 cells by the human anti-hTNFa antibody D2E7, as compared to the murine anti-hTNFa antibody MAK 195.

Figure 5 is a graph depicting the inhibition ofTNFa-induced

ELAM-

expression on HUVEC by the human anti-hTNFa antibody D2E7, as compared to the murine anti-hTNFa antibody MAK 195.

Figure 6 is a bar graph depicting protection from TNFa-induced lethality in D-galactosamine-sensitized mice by administration of the human anti-hTNFac antibody D2E7 (black bars), as compared to the murine anti-hTNFa antibody MAK 195 (hatched bars).

Figure 7 shows the nucleotide sequence of the light chain variable region of D2E7, with the predicted amino acid sequence below the nucleotide sequence. The CDR L1, CDR L2 and CDR L3 regions are underlined.

Figure 8 shows the nucleotide sequence of the heavy chain variable region of D2E7, with the predicted amino acid sequence below the nucleotide sequence. The CDR H1, CDR H2 and CDR H3 regions are underlined.

Figure 9 is a graph depicting the effect of D2E7 antibody treatment on the mean joint size of Tg197 transgenic mice as a polyarthritis model.

Detailed Description of the Invention This invention pertains to isolated human antibodies, or antigen-binding portions 15 thereof, that bind to human TNFa with high affinity, a low off rate and high neutralizing capacity. Various aspects of the invention relate to antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. Methods of using the antibodies of the invention to detect human TNFa or to inhibit human TNFa activity, either in vitro or in vivo, are also encompassed by the invention.

:In order that the present invention may be more readily understood, certain terms are first defined.

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 17 kD secreted form 25 and a 26 kD membrane associated form, the biologically active form of which is composed of a trimer of noncovalently bound 17 kD molecules. The structure of hTNFa is described further in, for example, Pennica, et al. (1984) Nature 312:724- 729; Davis, et al. (1987) Biochemistry 26:1322-1326; and Jones, et al.

(1989) Nature 338:225-228. The term human TNFa is intended to include recombinant human TNFa (rhTNFa), which can be prepared by standard 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 comprised of four polypeptide chains, two heavy chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant -8region. The heavy chain constant region is comprised of three domains. CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain. CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).

Each VH and VL is composed of three CDRs and four FRs. arranged from aminoterminus 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 o one or more fragments of an antibody that retain the ability to specifically bind to an antigen hTNFa). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen- 15 binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH 1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et 20 al., (1989) Nature 341:544-546 which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) Such single chain antibodies are 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. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see Holliger. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 6448; Poljak, er al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments. can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoelobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences mutations introduced by 15 random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

20 The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II. below), antibodies isolated from a recombinant. combinatorial human antibody library (described-further in Section III, below), antibodies isolated from an animal a mouse) that is transgenic for human immunoglobulin genes (see Taylor, et al. (1992) Nucl. Acids Res. 20:6287- 6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.

Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that. while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities an isolated antibody that specifically binds hTNFa is substantially free of antibodies that specificallybind antigens other than hTNFa). An isolated antibody that specifically binds hTNFa may, however, have cross-reactivity to other antigens, such as TNFa molecules from other species (discussed in further detail below). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

A "neutralizing antibody", as used herein (or an "antibody that neutralized hTNFa activity"), is intended to refer to an antibody whose binding to hTNFa results in 10 inhibition of the biological activity of hTNFa. This inhibition of the biological activity of hTNFa can be assessed by tneasuring one or more indicators of hTNFa biological activity, such as hTNFa-induced cytotoxicity (either in vitro or in vivo), hTNFa-induced cellular activation and hTNFa binding to hTNFa receptors. These indicators of hTNFa biological activity can be assessed by one or more of several standard in vitro or in vivo 15 assays known in the art (see Example Preferably, the ability of an antibody to neutralize hTNFa activity is assessed by 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-I on HUVEC, as a measure of hTNFa-induced cellular activation, can be assessed.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example S .using the BlAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Example 1 and Jonsson, U, et al. (1993) Ann. Biol.

Clin. 51:19-26; Jonsson, et al. (1991) Biotechniques 11:620-627; Johnsson, et al.

(1995) J. Mol. Recognit. 8:125-131; and Johnnson, et al. (1991) Anal. Biochem.

198:268-277.

The term "Koff", as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.

The term as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.

The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded

DNA.

The term "isolated nucleic acid molecule", as used herein in reference to nucleic acids encoding antibodies or antibody portions VH, VL, CDR3) that bind hTNFa.

1 is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions 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- TNFct antibody contains no 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 which it has been linked. One type of 10 vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector.

wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced bacterial vectors having a bacterial origin of replication and episomal mammalian 15 vectors). Other vectors non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such 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, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors 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 such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Various aspects of the invention are described in further detail in the following subsections.

12- 1. Human Antibodies that Bind Human TNFa This invention provides isolated human antibodies, or antigen-binding portions thereof, that bind to human TNFa with high affinity, a low off rate and high neutralizing capacity. Preferably, the human antibodies of the invention are recombinant, neutralizing human anti-hTNFa antibodies. The most preferred recombinant, neutralizing antibody of the invention is referred to herein as D2E7 and has VL and VH sequences as shown in Figure 1A, IB and Figure 2A, 2B, respectively (the amino acid sequence of the D2E7 VL region is also shown in SEQ ID NO: 1; the amino acid sequence of the D2E7 VH region is also shown in SEQ ID NO: The binding properties of D2E7, as compared to the murine anti-hTNFa MAK 195 mAb that :exhibits high affinity and slow dissociation kinetics and another human anti-hTNFa antibody related in sequence to D2E7, 2SD4, are summarized below: Koff kon Kd Stoichio- Antibody sec-1 M-I sec- 1 M metry D2E7IgGI 8.81 x 10-5 1.91 x 105 6.09 x 10-1o 1.2 2SD4 IgG4 8.4 x 10-3 4.20 x 105 2.00 x 10-8 0.8 MAK 195 8.70 x 10-5 1.90 x 105 4.60 x 10-10 1.4 15s The D2E7 antibody, and related antibodies, also exhibit a strong capacity to neutralize hTNFa activity, as assessed by several in vitro and in vivo assays (see Example For example, these antibodies neutralize hTNFa-induced cytotoxicity of S L929 cells with IC 50 values in the range of about 10- 7 M to about 10-10 M. D2E7, when expressed as a full-length IgGI antibody, neutralizes hTNFa-induced cytotoxicity of L929 cells with IC 50 of about 1.25 x 10-10 M. Moreover, the neutralizing capacity of D2E7 is maintained when the antibody is expressed as a Fab, F(ab') 2 or scFv fragment.

D2E7 also inhibits TNFa-induced cellular activation, as measured by hTNFa-induced ELAM-I expression on HUVEC (IC 50 about 1.85 x 10-10 and binding of hTNFa to hTNFa receptors on U-937 cells (IC 50 about 1.56 x 10-10 Regarding the latter, D2E7 inhibits the binding of hTNFa to both the p55 and p75 hTNFa receptors.

Furthermore, the antibody inhibits hTNFa-induced lethality in vivo in mice (ED 50 1-2.5 pg/mouse).

Regarding 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 specifically bind to other cytokines, such as lymphotoxin (TNFP), IL-la, IL-11, IL-2, IL-4, IL-6, IL-8, IFNy and TGF3. However, 13 D2E7 does exhibit crossreactivity to tumor necrosis factors from other species. For example, the antibody neutralizes the activity of at least five primate TNFas (chimpanzee, baboon, marmoset, cynomolgus and rhesus) with approximately equivalent IC 50 values as for neutralization of hTNFa (see Example 4, subsection E).

D2E7 also neutralizes the activity of mouse TNFa, although approximately 1000-fold less well than human TNFa (see Example 4, subsection D2E7 also binds to canine and porcine TNFa.

In one aspect, the invention pertains to D2E7 antibodies and antibody portions, D2E7-related antibodies and antibody portions, and other human antibodies and 10 antibody portions with equivalent properties to D2E7, such as high affinity binding to hTNFa with low dissociation kinetics and high neutralizing capacity. In one embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFa with a Kd of 1 x 10- 8 M or less and a Koff rate constant of 1 x 10- 3 s- 1 or less, both determined by surface plasmon resonance, 15 and neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC 50 of 1 x 10- 7 M or less. More preferably, the isolated human antibody, or antigen-binding 9. portion thereof, dissociates from human TNFa with a Koff of 5 x 10 4 s-1 or less, or even more preferably, with a Koff of 1 x 10- 4 s-1 or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes human TNFa cytotoxicity in a 20 standard in vitro L929 assay with an IC 50 of 1 x 10- 8 M or less, even more preferably with an IC 50 of 1 x 10- 9 M or less and still morepreferably with an IC 50 of 5 x 10- 10

M

or less. In a preferred embodiment, the antibody is an isolated human recombinant antibody, or an antigen-binding portion thereof. In another preferred embodiment, the "i antibody also neutralizes TNFa-induced cellular activation, as assessed using a standard in vitro assay for TNFa-induced ELAM-1 expression on human umbilical vein endothelial cells (HUVEC).

Surface plasmon resonance analysis for determining Kd and Koff can be performed as described in Example 1. A standard in vitro L929 assay for determining

IC

5 o values is described in Example 4, subsection A. A standard in vitro assay for TNFa-induced ELAM-1 expression on human umbilical vein endothelial cells (HUVEC) is described in Example 4, subsection C. Examples of recombinant human antibodies that meet. or are predicted to meet, the aforementioned kinetic and neutralization criteria include antibodies having the following [VH/VL] pairs, the sequences of which are shown in Figures 1A, IB, 2A and 2B (see also Examples 2, 3 and 4 for kinetic and neutralization analyses): [D2E7 VH/D2E7 VL]; [HD2E7*.A1/D2E7 VL], [HD2E7'.A2/D2E7 VL], [HD2E7*.A3/D2E7

VL],

14 [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*.A1], [D2E7 VH/LD2E7*.A4], [D2E7 VH/LD2E7*.A5], [D2E7 VH/LD2E7*.A7], [D2E7 VH/LD2E7*.A8], [HD2E7*.A9/LD2E7*.A1],

[VHI-

D2/LOE7], [VH 1-D2.N/LOE7.T], [VH 1-D2.Y/LOE7.A], [VH 1-D2.N/LOE7.A], [VH I- D2/EP B12] and [3C-H2/LOE7].

It is well known in the art that antibody heavy and light chain CDR3 domains play an important role in the binding specificity/affinity of an antibody for an antigen.

Accordingly, in another aspect, the invention pertains to human antibodies that have *10 slow dissociation kinetics for association with hTNFa and that have light and heavy *chain CDR3 domains that structurally are identical to or related to those of D2E7. As demonstrated in Example 3, position 9 of the D2E7 VL CDR3 can be occupied by Ala or Thr without substantially affecting the Koff. Accordingly, a consensus motif for the D2E7 VL CDR3 comprises the amino acid sequence:

(SEQ

15 ID NO: Additionally, position 12 of the D2E7 VH CDR3 can be occupied by Tyr or Asn, without substantially affecting the Koff. Accordingly, a consensus motif for the D2E7 VH CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: Moreover, as demonstrated in Example 2, the CDR3 domain of the D2E7 heavy and light chains is amenable to substitution with a single alanine residue (at position 1,4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without substantially affecting the Koff. Still further, the skilled artisan will appreciate that, given the amenability of the D2E7 VL and VH CDR3 domains to substitutions by alanine, substitution of other amino acids within the CDR3 domains may be possible while still retaining the low off rate constant of the antibody, in particular substitutions with conservative amino acids. A "conservative amino acid substitution", as used herein, is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains lysine, arginine, histidine), acidic side chains aspartic acid, glutamic acid), uncharged polar side chains glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains threonine, valine, isoleucine) and aromatic side chains tyrosine, phenylalanine.

tryptophan. histidine). Preferably, no more than one to five conservative amino acid substitutions are made within the D2E7 VL and/or VH CDR3 domains. More preferably, no more than one to three conservative amino acid substitutions are made 15 within the D2E7 VL and/or VH CDR3 domains. Additionally, conservative amino acid substitutions should not be made at amino acid positions critical for binding to hTNFa.

As shown in Example 3, positions 2 and 5 of the D2E7 VL CDR3 and positions I and 7 of the D2E7 VH CDR3 appear to be critical for interaction with hTNFa and thus, conservative amino acid substitutions preferably are not made at these positions (although an alanine substitution at position 5 of the D2E7 VL CDR3 is acceptable, as described above).

Accordingly, in another embodiment, the invention provides an isolated human antibody, or antigen-binding portion thereof, with the following characteristics: 10 a) dissociates from human TNFa with a Koff rate constant of I 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 alanine substitution 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 alanine substitution 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.

20 More preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFca with a Koff of 5 x 10 4 s- 1 or less. Even more preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koff of I x 10 4 s- or less.

In yet 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 modified from SEQ ID NO: 3 by a single alanine substitution 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 of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 the D2E7 VL CDR2) and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 the D2E7 VH CDR2). Even more preferably, the LCVR further has CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 the D2E7 VL CDR1) and the HCVR has a CDRI domain comprising the amino acid sequence of SEQ ID NO: 8 the D2E7 VH CDRI). The framework regions for VL preferably 16are from the V\l human germline family, more preferably from the A20 human germline Vk gene and most preferably from the D2E7 VL framework sequences shown in Figures 1A and IB. The framework regions for VH preferably are from the VH 3 human germline family, more preferably from the DP-31 human germline VH gene and most preferably from the D2E7 VH framework sequences shown in Figures 2A and 2B.

In still 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 the D2E7 VL) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 10 the D2E7 VH). In certain embodiments. the antibody comprises a heavy chain constant region. such as an IgGI, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.

In still other embodiments, the invention provides an isolated human antibody, or an antigen-binding portions thereof, having D2E7-related VL and VH CDR3 domains, for example, antibodies, or antigen-binding portions thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID .NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, 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 and SEQ ID NO: 26 or with a heavy chain variable region (HCVR) having a CDR3 domain comprising an amino acid sequence 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: In yet another embodiment, the invention provides a recombinant human antibody, or antigen-binding portion thereof, that neutralizes the activity of human TNFa but not human TNFP. Preferably, antibody, or antigen-binding portion thereof.

also neutralizes the activity of chimpanzee TNFa and at least one additional primate TNFc selected from the group consisting of baboon TNFa, marmoset TNFa, cynomolgus TNFa and rhesus TNFc. Preferably, the antibody, or antigen-binding portion thereof, neutralizes the human, chimpanzee and/or additional primate TNFa in a 17standard in vitro L929 assay with an IC 50 of 1 x 10- 8 M or less, more preferably Ix 10- 9 M or less, and even more preferably 5 x 10- 10 M or less. In one subembodiment. the antibody also neutralizes the activity of canine TNFa, preferably in a standard in vitro L929 assay with an IC 50 of I x 10- 7 M or less, more preferably Ix 10-8 M or less and even more preferably 5 x 10-9 M or less. In another subembodiment, the antibody also neutralizes the activity of pig TNFa, preferably with an IC 50 of 1 x 10-5 M or less, more preferably lx 10- 6 M or less and even more preferably 5 x 10-7 M or less. In yet another embodiment, the antibody also neutralizes the activity of mouse TNFa, preferably with an IC 50 of I x 10- 4 M or less, more preferably lx 10- 5 M or less and even more 10 preferably 5 x 10-6 M or less.

An antibody or antibody portion of the invention can be derivatized or linked to another functional molecule another peptide or protein). Accordingly, the antibodies and antibody portions of the invention are intended to include derivatized and otherwise modified forms of the human anti-hTNFa antibodies described herein, 0 15 including immunoadhesion molecules. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate e.0e 20 associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, to create bispecific antibodies).

Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer m-maleimidobenzoyl-Nhydroxysuccinimide ester) or homobifunctional disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL.

Useful detectable agents with which an antibody or antibody portion of the invention may be derivatized include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized 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 horseradish peroxidase is present, the addition of hydrogen peroxide and 18.

diaminobenzidine leads to a colored reaction product. which is detectable. An antibody may also be derivatized with biotin. and detected through indirect measurement of avidin or streptavidin binding.

II. Expression of Antibodies An antibody, or antibody portion. of the invention can be prepared by 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 10 light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, (1989), Ausubel, F.M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Patent No. 4,816,397 by Boss et al.

To express D2E7 or a D2E7-related antibody, DNA fragments encoding the light 20 and heavy chain variable regions are first obtained. These DNAs can be obtained by 0 amplification and modification of germline light and heavy chain variable sequences using the polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see the "Vbase" human o* germline sequence database; see also Kabat, et al. (1991 )-Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services.

NIH Publication No. 91-3242; Tomlinson, et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, J.P.L. et al. (1994) "A Directory of Human Germ-line VK Segments Reveals a Strong Bias in their Usage" Eur.

J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference). To obtain a DNA fragment encoding the heavy chain variable region of D2E7, or a D2E7-related antibody, a member of the VH3 family of human germline VH genes is amplified by standard PCR. Most preferably, the DP-31 VH germline sequence is amplified. To obtain a DNA fragment encoding the light chain variable region of D2E7, or a D2E7-related antibody, a member of the V-I family of human germline VL genes is amplified by standard PCR. Most preferably, the A20 VL germline sequence is -19amplified. PCR primers suitable for use in amplifying the DP-31 germline VH and germline VL sequences can be designed based on the nucleotide sequences disclosed in the references cited supra, using standard methods.

Once the germline VH and VL fragments are obtained, these sequences can be mutated to encode the D2E7 or D2E7-related amino acid sequences disclosed herein.

The amino acid sequences encoded by the germline VH and VL DNA sequences are first compared to the D2E7 or D2E7-related VH and VL amino acid sequences to identify amino acid residues in the D2E7 or D2E7-related sequence that differ from germline.

Then. the appropriate nucleotides of the germline DNA sequences are mutated such that 10 the mutated germline sequence encodes the D2E7 or D2E7-related amino acid sequence, using the genetic code to determine which nucleotide changes should be made.

Mutagenesis of the germline sequences is carried out by standard methods, such as PCRmediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis.

Moreover, it should be noted that if the "germline" sequences obtained by PCR amplification encode amino acid differences in the framework regions from the true germline configuration differences in the amplified sequence as compared to the true germline sequence, for example as a result of somatic mutation), it may be desireable to change these amino acid differences back to the true germline sequences 20 "backmutation" of framework residues to the germline configuration).

Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments are obtained (by amplification and mutagenesis of germline VH and VL genes, as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see Kabat. E.A., et al. (1991) Sequences of Proteins of Immunological Interest. Fifth Edition. U.S.

Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.

The heavy chain constant region can be an IgGI, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG 1 or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI 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 operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., 10 Kabat, et al. (1991) Sequences ofProteins of Immunological Interest, Fifth "Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

but most preferably is a kappa constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, encoding the amino acid sequence (Gly 4 -Ser) 3 such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl.

20 Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions of the invention, DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended ,to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the D2E7 or D2E7-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting the D2E7 or D2E7-related VH and VL sequences to full-length -21 antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions. respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or altematively, the recombinant expression vector can encode a signal peptide that facilitates 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 in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide a signal peptide from a non-immunoglobulin protein).

10 In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such 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 the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.

20 Preferred regulatory sequences for mammalian host cell expression 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 CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see U.S.

Patent No. 5,168,062 by Stinski, U.S. Patent No. 4,510,245 by Bell et al. and U.S.

Patent No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see U.S. Patents Nos. 4,399,216, 4,634.665 and 5,179,017, all by Axel et For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate. on a host cell into which the vector has been introduced. Preferred selectable marker genes 22include 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 the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokarvotic or eukaryotic host cell, electroporation. calcium-phosphate precipitation, DEAEdextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of 10 antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M.A.

and Wood, C. R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, as described in R.J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or. more preferably, secretion of the antibody into the culture medium in which the host cells are grown.

.Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect 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 may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to hTNFa. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than hTNFa by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, or antigenbinding portion thereof, of the invention, a recombinant expression vector encoding both antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an 10 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are culture to allow for expression of the antibody heavy and light chains and intact antibody is recovered from 15 the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium.

In view of the foregoing, another aspect of the invention pertains to nucleic acid, vector and host cell compositions that can be used for recombinant expression of the 20 antibodies and antibody portions of the invention. The nucleotide sequence encoding the D2E7 light chain variable region is shown in Figure 7 and SEQ ID NO: 36. The CDRI domain of the LCVR encompasses nucleotides 70-102, the CDR2 domain encompasses nucleotides 148-168 and the CDR3 domain encompasses nucleotides 265- S. 291. The nucleotide sequence encoding the D2E7 heavy chain variable region is shown in Figure 8 and SEQ ID NO: 37. The CDR1 domain of the 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 the skilled artisan that nucleotide sequences encoding D2E7-related antibodies, or portions thereof a CDR domain, such as a CDR3 domain), can be derived from the nucleotide sequences encoding the D2E7 LCVR and HCVR using the genetic code and standard 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 the D2E7 VL CDR3). or modified from SEQ ID NO: 3 by a single alanine substitution 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. This nucleic acid can encode only the CDR3 region 24or, more preferably, encodes an entire antibody light chain variable region (LCVR). For example, the nucleic acid can encode an LCVR having a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 the D2E7 VL CDR2) and a CDRI domain comprising the amino acid sequence of SEQ ID NO: 7 the D2E7 VL CDRI).

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 the D2E7 VH CDR3), or modified from SEQ ID NO: 4 by a single alanine substitution 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. This nucleic acid can encode only the CDR3 region or, more preferably, encodes an entire antibody heavy chain variable region (HCVR): For example, the nucleic acid can encode a HCVR having a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 the D2E7 VH CDR2) and a CDRI domain comprising the amino acid sequence of SEQ ID NO: 8 the D2E7 VH CDR1).

15 In yet another embodiment, the invention provides isolated nucleic acids encoding a D2E7-related CDR3 domain, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO 4, 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: n still another embodiment, the invention provides an isolated nucleic acid .encoding an antibody light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 1 the D2E7 LCVR). Preferably this nucleic acid comprises the nucleotide sequence of SEQ ID NO: 36, although the skilled artisan will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences can encode the amino acid sequence of SEQ ID NO: 1. The nucleic acid can encode only the LCVR or can also encode an antibody light chain constant region, operatively linked to the LCVR.

In one embodiment, this nucleic acid is in a recombinant expression vector.

In still another embodiment, the invention provides an isolated nucleic acid encoding an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 the D2E7 HCVR). Preferably this nucleic acid comprises the nucieotide sequence of SEQ ID NO: 37, although the skilled artisan will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences can encode the amino acid sequence of SEQ ID NO: 2. The nucleic acid can encode only the HCVR or can also encode a heavy chain constant region. operatively linked to the HCVR. For example. the nucleic acid can comprise an IgGI or IgG4 constant region. In one embodiment, this nucleic acid is in a recombinant expression vector.

The invention also provides recombinant expression vectors encoding 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 the D2E7 LCVR); and b) an antibody heavy chain having a variable region comprising the amino acid 10 sequence of SEQ ID NO: 2 the D2E7 HCVR).

The invention also provides host cells into which one or more of the recombinant expression vectors of the invention have been introduced. Preferably, the host cell is a mammalian host cell. more preferably the host cell is a CHO cell. an NSO cell or a COS S. cell.

Still further the invention provides a method of synthesizing a recombinant human antibody of the invention by culturing a host cell of the invention in a suitable culture medium until a recombinant human antibody of the invention is synthesized.

The method can further comprise isolating the recombinant human antibody from the culture medium.

III. Selection of Recombinant Human Antibodies Recombinant human antibodies of the invention in addition to the D2E7 or .D2E7-related antibodies disclosed herein can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage displaylibrary, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes.

Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene Sur/ZAPTM phage display kit, catalog no. 240612), examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example. Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffeny et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) -26 Hum Antibod Hybridomas 3:81-85: Huse et al. (1989) Science 246:1275-1281; McCaffert et al.. Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725- 734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991)Nuc AcidRes 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with high affinity and a low off rate constant for hTNFa, a murine anti-hTNFa antibody having high affinity and a low off rate constant for hTNFa MAK 195, the hybridoma for which has 10 deposit number ECACC 87 050801) is first used to select human heavy and light chain sequences having similar binding activity toward hTNFa. using the epitope imprinting, or guided selection, methods described in Hoogenboom et al., PCT Publication No. WO 93/06213. The antibody libraries used in this method are preferably scFv libraries prepared and screened as described in McCafferty et PCT Publication No. WO 15 92/01047, McCafferty et al., Nature (1990) 348:552-554; and Griffiths et al., (1993) EMBO J 12:725-734. The scFv antibody libraries preferably are screened using recombinant human TNFa as the antigen.

Once initial human VL and VH segments are selected, "mix and match" experiments, in which different pairs of the initially selected VL and VH segments are 20 screened for hTNFa binding, are performed to select preferred VL/VH pair combinations. Additionally, to further improve the affinity and/or lower the off rate constant for hTNFa binding, the VL and VH segments of the preferred VL/VH pair(s) can be randomly mutated, preferably within the.CDR3 region of VH and/or VL, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complimentary to the VH CDR3 or VL CDR3, respectively, which primers have been "spiked" with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VH and VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be rescreened for binding to hTNFa and sequences that exhibit high affinity and a low off rate for hTNFax binding can be selected.

The amino acid sequences of selected antibody heavy and light chains can be compared to germline heavy and light chain amino acid sequences. In cases where certain framework residues of the selected VL and/or VH chains differ from the germline configuration as a result of somatic mutation of the immunoglobulin 27genes used to prepare the phage library), it may be desireable to "backmutate" the altered framework residues of the selected antibodies to the germline configuration change the framework amino acid sequences of the selected antibodies so that they are the same as the germline framework amino acid sequences). Such "backmutation" (or "germlining") of framework residues can be accomplished by standard molecular biology methods for introducing specific mutations site-directed mutagenesis; PCR-mediated mutagenesis, and the like).

Following screening and isolation of an anti-hTNFa antibody of the invention from a recombinant immunoglobulin display library, nucleic acid encoding the selected 10 antibody can be recovered from the display package from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the invention linked to nucleic acid encoding additional immunoglobulin domains.

such as additional constant regions). To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cells, as described in further detail in Section II above.

IV. Pharmaceutical Compositions and Pharmaceutical Administration 20 The antibodies and antibody-portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. 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 solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance 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 injectable and infusible solutions), dispersions or suspensions. tablets, pills, powders, -28liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral 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 10 of manufacture and storage. The composition can be formulated as a solution, microemulsion. dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the Spreparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 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 dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption. for example, monostearate salts and gelatin.

The antibodies and antibody-portions 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. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may 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 ethylene vinyl acetate, polyanhydrides, polyglycolic acid. collagen.

polyorthoesters. and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g..

-29- Sustained and Controlled Release Drug Delivery Systems. J.R. Robinson. ed.. Marcel Dekker. Inc.. New York, 1978.

In certain embodiments, an antibody or antibody portion of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier.

The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wqfers, and the like. To administer a compound of the 10 invention by other than parenteral administration, it may be necessary to coat 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 antibody portion of the invention 15 is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which TNFa activity is detrimental. For example, an anti-hTNFa antibody or antibody portion of the invention may be coformulated and/or coadministered with one or more additional antibodies that bind other targets antibodies that bind other cytokines or that bind cell surface molecules), one or more cytokines, soluble TNFa receptor (see PCT Publication No. WO 94/06476) and/or one or more chemical agents that inhibit hTNFa production or activity (such as cyclohexane-ylidene derivatives as described in PCT Publication No.

WO 93/19751). Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

Nonlimiting examples of therapeutic agents for rheumatoid arthritis with which an antibody, or antibody portion, of the invention can be combined include the following: non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer); cA2 (chimeric anti-TNFe antibody; Centocor); 75 kdTNFR- IgG (75 kD 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 TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see e.g., Arthritis Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see Arthritis Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanized anti-IL-2Ra. Protein Design Labs/Roche); IL-4 (antiinflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, antiinflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists agonist antibodies); IL-RA (IL-1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF binding protein; see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer. J. Physiol. Heart and Circulatory Physiology (1995) Vol.

268, pp. 37-42); R973401 (phosphodiesterase Type IV inhibitor; see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g.,Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S81); Iloprost (see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S82); methotrexate: thalidomide (see Arthritis Rheumatism (1996) Vol. 9, No. 9 (supplement), S282) and thalidomide-related drugs Celgen); leflunomide (anti-inflammatory and 15 cytokine inhibitor; see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid (inhibitor of plasminogen activation; see Arthritis Rheumatism (1996) Vol. 39. No. 9 S (supplement), S284); T-614 (cytokine inhibitor; see Arthritis Rheumatism (1996) Vol. 39. No. 9 (supplement), S282); prostaglandin El (see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); Tenidap (non-steroidal anti-inflammatory drug; see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidal anti-inflammatory drug; see Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (nonsteroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal antiinflammatory drug); Sulfasalazine (see Arthritis Rheumatism (1996) Vol. 39, No.

9 (supplement), S281); Azathioprine (see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor (inhibitor of the enzyme interleukin-l convening enzyme); zap-70 and/or Ick inhibitor (inhibitor of the tyrosine kinase or Ick); VEGF inhibitor and/or VEGF-R inhibitor (inhibitos of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); conicosteroid anti-inflammatory drugs SB203580); TNFconvertase inhibitors; anti-IL-12 antibodies; interleukin-l l (see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see e.g., Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S308); interleukin-1 7 inhibitors (see Arthritis Rheumatism (1996) Vol. 39. No. 9 (supplement), S120); -31 gold: penicillamine: chloroquine: hydroxychloroquine: chlorambucil; cyclophosphamide: cyclosporine; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins: orally-administered peptides and collagen; lobenzarit disodium: Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP 10; T Cell Sciences. Inc.); prednisone: orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); **amiprilose (therafectin); cladribine (2-chlorodeoxadenosine); and azaribine.

*Nonlimiting examples of therapeutic agents for inflammatory bowel disease with which an antibody, or antibody portion, of the invention can be combined include the 15 following: budenoside; epidermal growth factor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants: thromboxane inhibitors; IL-I receptor antagonists; anti-IL-lp monoclonal antibodies; anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole 20 compounds; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Baer); cA2 (chimeric anti-TNFc. antibody; Centocor); 75 kdTNFR-IgG kD 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 TNF :receptor-IgG fusion protein; Hoffmann-LaRoche); interleukin-10 (SCH 52000; Schering Plough); IL-4; IL-O10 and/or IL-4 agonists agonist antibodies); interleukin- 11; glucuronide- or dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP 10; T Cell Sciences, Inc.); slow-release mesalazine; methotrexate; antagonists of Platelet Activating Factor (PAF); ciprofloxacin; and lignocaine.

Nonlimiting examples of 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; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-pla (AvonexTM; Biogen); interferon-plb (BetaseronTM; Chiron/Berlex); Copolymer 1 (Cop-l; CopaxoneTM; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous -32immunoglobulin; clabribine; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG kD 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 TNF receptor-lgG fusion protein; Hoffmann-LaRoche); IL-10; IL-4; and IL-10 and/or IL-4 agonists agonist antibodies).

Nonlimiting 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 meropenem); antagonists of cytokines such as TNFa, IL-13, IL-6 and/or IL-8; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD 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 TNF receptor-lgG fusion 15 protein; Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular peptide; SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (Picower Institute); Tissue Factor S Pathway Inhibitor (TFPI; Chiron); PHP (chemically modified hemoglobin; APEX Bioscience); iron chelators and chelates, including diethylenetriamine pentaacetic acid iron (III) complex (DTPA iron (III); Molichem Medicines); lisofylline (synthetic small molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan (aqeuous soluble pl,3glucan; Alpha-Beta Technology); apolipoprotein A-l reconstituted with lipids; chiral hydroxamic acids (synthetic antibacterials that inhibit lipid A biosynthesis); antio. endotoxin antibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.); rBPI 2 1 (recombinant N-terminal fragment of human Bactericidal/Permeability-Increasing Protein); and Synthetic Anti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories); Nonlimiting examples of therapeutic agents for adult respiratory distress syndrome (ARDS) with which an antibody, or antibody portion, of the invention can be combined 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 TNF receptor-IgG fusion protein; Immunex; see Arthritis Rheumatism (1994) Vol. 37, S295; J.

Invest. Med. (1996) Vol. 44, 235A); and 55 kdTNFR-IgG (55 kD TNF receptor-lgG fusion protein; Hoffmann-LaRoche).

The use of the antibodies, or antibody portions. 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 amount effective. 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 elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at 15 an earlier stage of disease. the prophylactically effective amount will be less than the "therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound 25 calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage'unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0. 1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or 34supervising the administration of the compositions, and that dosage ranges set forth 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 hTNFa, the anti-hTNFa antibodies, or portions thereof, of the invention can be used to detect hTNFa in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method for detecting hTNFa in a biological sample comprising contacting a biological sample with an antibody, or antibody portion. of the invention and detecting either the antibody (or antibody portion) bound to hTNFa or unbound antibody (or antibody portion). to thereby detect hTNFa in the biological sample. The antibody is directly or indirectly labeled with a detectable 15 substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes .luminol; and examples of suitable radioactive material include 1251, 1311 3 5 S or 3H.

Alternative to labeling the antibody, hTNFa can be assayed in biological fluids 25 by a competition immunoassay utilizing rhTNFa standards labeled with a detectable substance and an unlabeled anti-hTNFa antibody. In this assay, the biological sample, the labeled rhTNFa standards and the anti-hTNFa antibody are combined and the amount of labeled rhTNFa standard bound to the unlabeled antibody is determined. The amount of hTNFa in the biological sample is inversely proportional to the amount of labeled rhTNFa standard bound to the anti-hTNFa antibody.

A D2E7 antibody of the invention can also be used to detect TNFas from species other than humans, in particular TNFas from primates chimpanzee, baboon, marmoset, cynomolgus and rhesus), pig and mouse, since D2E7 can bind to each of these TNFas (discussed further in Example 4, subsection

E).

The antibodies and antibody portions of the invention are capable of neutralizing hTNFa activity both in vitro and in vivo (see Example Moreover, at least some of the antibodies of the invention, such as D2E7. can neutralize TNFa activity from other species. _Accordingly, the antibodies and antibody portions of the invention can be used to inhibit TNFa activity, in a cell culture containing hTNFoa. in human subjects or in other mammalian subjects having TNFas with which an antibody of the invention cross-reacts chimpanzee, baboon, marmoset. cynomolgus and rhesus, pig or mouse). In one embodiment, the invention provides a method for inhibiting TNFa activity comprising contacting TNFa with an antibody or antibody portion of the invention such that TNFa activity is inhibited. Preferably, the TNFta is human TNFa.

For example, in a cell culture containing, or suspected of containing hTNFa, an antibody or antibody portion of the invention can be added to the culture medium to inhibit hTNFa activity in the culture.

In another embodiment, the invention provides a method for inhibiting TNFa activity in a subject suffering from a disorder in which TNFa activity is detrimental.

TNFa has been implicated in the pathophysiology of a wide variety of disorders (see 15 Moeller, et al. (1990) Cytokine 2:162-169; U.S. Patent No. 5,231,024 to Moeller et al.; European Patent Publication No. 260 610 BI by Moeller, The invention provides methods for TNFa activity in a subject suffering from such a disorder, which method comprises administering to the subject an antibody or antibody portion of the invention such that TNFa activity in the subject is inhibited. Preferably, 20 the TNFa is human TNFa and the subject is a human subject. Alternatively, the subject can be a mammal expressing a TNFa with which an antibody of the invention crossreacts. Still further the subject can be a mammal into which has been introduced hTNFca by administration of hTNFa or by expression of an hTNFa transgene). An antibody of the invention can be administered to a human subject for therapeutic purposes (discussed further below). Moreover, an antibody of the invention can be administered to a non-human mammal expressing a TNFa with which the antibody cross-reacts a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention testing of dosages and time courses of administration).

As used herein, the term "a disorder in which TNFca activity is detrimental" is intended to include diseases and other disorders in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which TNFa activity is detrimental is a disorder in which inhibition of TNFc activity is expected to alleviate the 36symptoms andior progression of the disorder. Such disorders may be evidenced, for example. by an increase in the concentration of TNFa in a biological fluid of a subject suffering from the disorder an increase in the concentration of TNFa in serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti-TNFa antibody as described above. There are numerous examples of disorders in which TNFo activity is detrimental. The use of the antibodies and antibody portions of the invention in the treatment of specific disorders is discussed further below: A. Sepsis Tumor necrosis factor has an established role in the pathophysiology of sepsis, with biological effects that include hypotension, myocardial suppression, vascular leakage syndrome, organ necrosis, stimulation of the release of toxic secondary mediators and activation of the cloting cascade (see Moeller. el al. (1990) Cytokine 2:162-169; U.S. Patent No. 5,231,024 to Moeller et al.; European Patent 15 Publication No. 260 610 B1 by Moeller, Tracey, K.J. and Cerami, A. (1994) Annu.

Rev. Med. 45:491-503; Russell, D and Thompson, R.C. (1993) Curr. Opin. Biotech.

4:714-721). Accordingly, the human antibodies, and antibody portions. of the invention e. can be used to treat sepsis in any of its clinical settings, including septic shock, endotoxic shock, gram negative sepsis and toxic shock syndrome.

Furthermore, to treat sepsis, an anti-hTNFa antibody, or antibody portion. of the invention can be coadministered with one or more additional therapeutic agents that may further alleviate sepsis, such as an interleukin-1 inhibitor (such as those described in PCT Publication Nos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see PCT Publication No. WO 93/11793) or an antagonist of platelet activating factor 25 (see European Patent Application Publication No. EP 374 510). Other combination therapies for the treatment of sepsis are discussed further in subsection III.

Additionally, in a preferred embodiment, an anti-TNFa antibody or antibody portion of the invention is administered to a human subject within a subgroup of sepsis patients having a serum or plasma concentration of IL-6 above 500 pgiml. and more preferably 1000 pg/ml, at the time of treatment (see PCT Publication No. WO 95/20978 by Daum. et al.).

B. Autoimmune Diseases Tumor necrosis factor has been implicated in playing a role in the pathophysiology of a variety of autoimmune diseases. For example, TNFa has been implicated in activating tissue inflammation and causing joint destruction in rheumatoid 37arthritis (see Moeller. et al. (1990) Cwokine 2:162-169; U.S. Patent No.

5,231,024 to Moeller et al.; European Patent Publication No. 260 610 BI by Moeller, Traces and Cerami, supra; Arend, W.P. and Dayer, J-M. (1995) Arth. Rheum.

38:151-160; Fava, et al. (1993) Clin. Exp. Immunol. 94:261-266). TNFa also has been implicated in promoting the death of islet cells and in mediating insulin resistance in diabetes (see Tracey and Cerami, supra; PCT Publication No. WO 94/08609).

TNFa also has been implicated in mediating cytotoxicity to oligodendrocytes and induction of inflammatory plaques in multiple sclerosis (see Tracey and Cerami, supra). Chimeric and humanized murine anti-hTNFcx antibodies have undergone clinical testing for treatment of rheumatoid arthritis (see Elliott, et al. (1994) Lancet 344:1125-1127; Elliot, KM.J., et al. (1994) Lancet 344:1105-1110; Rankin, E.C., et al. (1995) Br. J. Rheumatol. 34:334-342).

The human antibodies, and antibody portions of the invention can be used to treat autoimmune diseases, in particular those associated with inflammation, including 15 rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome.

Typically, the antibody, or antibody portion, is adriinistered systemically, although for certain disorders, local administration of the antibody or antibody portion at a site of inflammation may be beneficial local administration in the joints in rheumatoid 20 arthritis or topical application to diabetic ulcers, alone or in combination with a :cyclohexane-ylidene derivative as described in PCT Publication No. WO 93/19751). An antibody, or antibody portion, of the invention also can be administered with one or more additional therapeutic agents useful in the treatment of autoimmune diseases, as discussed further in subsection III.

C. Infectious Diseases Tumor necrosis factor has been implicated in mediating biological effects observed in a variety of infectious diseases. For example, TNFc has been implicated in mediating brain inflammation and capillary thrombosis and infarction in malaria. TNFa also has been implicated in mediating brain inflammation, inducing breakdown of the blood-brain barrier, triggering septic shock syndrome and activating venous infarction in meningitis. TNFa also has been implicated in inducing cachexia, stimulating viral proliferation and mediating central nervous system injury in acquired 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 European Patent Application Publication No. EP 585 705), cerebral -38 malaria, AIDS and AIDS-related complex (ARC) (see European Patent Application Publication No. EP 230 574), as well as cytomegalovirus infection secondary to transplantation (see Fietze, et al. (1994) Transplantation 58:675-680). The antibodies, and antibody portions, of the invention, also can be used to alleviate symptoms associated with infectious diseases, including fever and myalgias due to infection (such as influenza) and cachexia secondary to infection secondary to AIDS or ARC).

D. Transplantation Tumor necrosis factor has been implicated as a key mediator of allograft rejection and graft versus host'disease (GVHD) and in mediating an adverse reaction that has been observed when the rat antibody OKT3, directed against the T cell receptor CD3 complex, is used to inhibit rejection of renal transplants (see Eason. et al.

(1995) Transplantation 59:300-305; Suthanthiran, M. and Strom, T.B. (1994) New Engl.

15 J. Med. 331:365-375). Accordingly, the antibodies, and antibody portions, of the invention, can be used to inhibit transplant rejection, including rejections of allografts and xenografts and to inhibit GVHD. Although the antibody or antibody portion may be used alone, more preferably it is used in combination with one or more other agents that inhibit the immune response against the allograft or inhibit GVHD. For example, in one 20 embodiment, an antibody or antibody portion of the invention is used in combination with OKT3 to inhibit OKT3-induced reactions. In another embodiment, an antibody or antibody portion of the invention is used in combination with one or more antibodies directed at other targets involved in regulating immune responses, such as the cell surface molecules CD25 (interleukin-2 receptor-a), CD1 la (LFA-I), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 In yet another embodiment, an antibody or antibody portion of the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.

E. Malignancy Tumor necrosis factor has been implicated in inducing cachexia, stimulating tumor growth, enhancing metastatic potential and mediating cytotoxicity in malignancies. Accordingly, the antibodies, and antibody portions, of the invention, can be used in the treatment of malignancies, to inhibit tumor growth or metastasis and/or to alleviate cachexia secondary to malignancy. The antibody, or antibody portion. may be administered systemically or locally to the tumor site.

-39- F. Pulmonary Disorders Tumor necrosis factor has been implicated in the pathophysiology of adult respiratory distress syndrome (ARDS), including stimulating leukocyte-endothelial activation, directing cytotoxicity to pneumocytes and inducing vascular leakage syndrome. Accordingly, the antibodies, and antibody portions, of the invention, can be used to treat various pulmonary disorders, including adult respiratory distress syndrome (see PCT Publication No. WO 91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The antibody, or antibody portion, may be administered systemically or locally to the lung surface, for example as an aerosol. An antibody, or antibody portion, of the invention also can be administered with one or more additional therapeutic agents useful in the treatment of pulmonary disorders. as discussed further in subsection III.

G. Intestinal Disorders 15 Tumor necrosis factor has been implicated in the pathophysiology of inflammatory bowel disorders (see Tracy, et al. (1986) Science 234:470-474; Sun, et al. (1988) J. Clin. Invest. 81:1328-1331; MacDonald, et al. (1990) Clin. Exp. Immunol. 81:301-305). Chimeric murine anti-hTNFa antibodies have undergone clinical testing for treatment of Crohn's disease (van Dullemen, et al.

20 (1995) Gastroenterology 109:129-135). The human antibodies, and antibody portions, of the invention, also can be used to treat intestinal disorders, such as idiopathic inflammatory bowel disease, which includes two syndromes. Crohn's disease and ulcerative colitis. An antibody, or antibody portion. of the invention also can 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, also can be used to treat various cardiac disorders, including ischemia of the heart (see European Patent Application Publication No. EP 453 898) and heart insufficiency (weakness of the heart muscle)(see PCT Publication No. WO 94/20139).

1. Others The antibodies, and antibody portions, of the invention, also can be used to treat various other disorders in which TNFo. activity is detrimental. Examples of other diseases and disorders in which TNFa activity has been implicated in the pathophysiology. and thus which can be treated using an antibody, or antibody portion, of the invention, include inflammatory bone disorders and bone resorption disease (see Berolini. et al. (1986) Nature 319:516-518; Konig, et al. (1988) J. Bone Miner. Res. 3:621-627; Lerer, U.H. and Ohlin, A. (1993) J. Bone Miner. Res. 8:147- 155; and Shankar. G. and Stem, P.H. (1993) Bone 14:871-876), hepatitis, including alcoholic hepatitis (see McClain, C.J. and Cohen, D.A. (1989) Hepatology 9:349.

351; Felver, et al. (1990) Alcohol. Clin. Exp. Res. 14:255-259; and Hansen, et al. (1994) Hepatology 20:461-474), viral hepatitis (Sheron, et al. (1991) J. Hepatol.

12:241-245; and Hussain, et al. (1994) J. Clin. Pathol. 47:1112-1115), and fulminant hepatitis: coagulation disturbances (see van der Poll, et al. (1990) N.

Engl. J. Med. 322:1622-1627; arid van der Poll, et al. (1991) Prog. Clin. Biol. Res.

367:55-60), burs (see Giroir, et al. (1994) Am. J. Physiol. 267:H118-124; and Liu. et al. (1994) Burns 20:40-44), reperfusion injury (see Scales. W.E., et al. (1994) Am. J. Physiol. 267:G1122-1127; Serrick, et al. (1994) Transplantation 15 58:1158-1162; and Yao, et al. (1995) Resuscitation 29:157-168), keloid formation (see 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 references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

EXAMPLE 1: Kinetic Analysis of Binding of Human Antibodies to hTNFa *see 25 Real-time binding interactions between ligand (biotinylated recombinant human TNFa (rhTNFa) immobilized on a biosensor matrix) and analyte (antibodies in solution) were measured by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor. Piscataway, NJ). The system utilizes the optical properties of SPR to detect alterations in protein concentrations within a dextran biosensor matrix.

Proteins are covalently bound to the dextran matrix at known concentrations.

Antibodies are injected through the dextran matrix and specific binding between injected antibodies and immobilized ligand results in an increased matrix protein concentration and resultant change in the SPR signal. These changes in SPR signal are recorded as resonance units (RU) and are displayed with respect to time along the y-axis of a sensorgram.

-41 To facilitate immobilization of biotinylated rhTNFa on the biosensor matrix, streptavidin is covalently linked via free amine groups to the dextran matrix by first activating carboxyl groups on the matrix with 100 mM N-hydroxysuccinimide (NHS) and 400 mM N-ethyl-N'-(3-diethylaminopropyl) carbodiimide hydrochloride (EDC).

Next, streptavidin is injected across the activated matrix. Thirty-five microliters of streptavidin (25 pg/ml), diluted in sodium acetate, pH 4.5, is injected across the activated biosensor and free amines on the protein are bound directly to the activated carboxyl groups. Unreacted matrix EDC-esters are deactivated by an injection of 1 M ethanolamine. Streptavidin-coupled biosensor chips also are commercially available 10 (Pharmacia BR-1000-16, Pharmacia Biosensor, Piscataway, NJ).

Biotinylated rhTNF was prepared by first dissolving 5.0 mg ofbiotin (Dbiotinyl-E-aminocaproic acid N-hydroxysuccinimide ester; Boehringer Mannheim Cat.

No. 1008 960) in 500 pl dimethylsulfoxide to make a 10 mg/ml solution. Ten microliters of biotin was added per ml of rhTNFa (at 2.65 mg/ml) for 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 PD-10 column, Sephadex G-25M (Pharmacia Catalog No. 17-0851-01) was equilibrated with 25 ml of cold PBS and loaded with 2 ml of rhTNFa-biotin per column. The column was eluted with 10 x 1 ml cold PBS. Fractions were collected and read at OD280 (1.0 OD 1.25 mg/ml). The appropriate fractions were pooled and stored at -80° C until use. Biotinylated rhTNFa also is commercially available (R D Systems Catalog No. FTAOO, Minneapolis, MN).

Biotinylated rhTNFa to be immobilized on the matrix via streptavidin was diluted in PBS running buffer (Gibco Cat. No. 14190-144, Gibco BRL, Grand Island, S. NY) supplemented with 0.05% (BIAcore) surfactant P20 (Pharmacia BR-1000-54, Pharmacia Biosensor, Piscataway, NJ). To determine the capacity ofrhTNFa-specific antibodies to bind immobilized rhTNFa, a binding assay was conducted as follows.

Aliquots of biotinylated rhTNFa (25 nM; 10 iil aliquots) were injected through the streptavidin-coupled dextran matrix at a flow rate of 5 pl/min. Before injection of the protein and immediately afterward, PBS buffer alone flowed through each flow cell.

The net difference in signal between baseline and approximately 30 sec. after completion of biotinylated rhTNFa injection was taken to represent the binding value (approximately 500 RU). Direct rhTNFa-specific antibody binding to immobilized biotinylated rhTNFa was measured. Antibodies (20 pg/ml) were diluted in PBS running buffer and 25 .pl aliquots were injected through the immobilized protein matrices at a flow rate of 5 il/min. Prior to injection of antibody, and immediately afterwards, PBS buffer alone flowed through each flow cell. The net difference in 42 baseline signal and signal after completion of antibody injection was taken to represent the binding value of the particular sample. Biosensor matrices were regenerated using 100 mM HCI before injection of the next sample. To determine the off rate (Koff), on rate (Kon), association rate (Ka) and dissociation rate (Kd) constants, BlAcore kinetic evaluation software (version 2. 1) was used.

Representative results of D2E7 (IgG4 full-length antibody) binding to biotinylated rhTNFct, as compared to the mouse inAb MAK 195 (F(ab')2 fragment), are shown below in Table 1.

10 Table 1: Bindin2 of D2E7 IeG4 or MAK 195 to Biotinvlated rhTNFa______ Anibdy [A],nM rhTNFa, Ab, bound, Koff, sec- 1 Atbd A, M bound. RUs RUs rhTNFa/Ab (Avg) D2E7 267 373 1215 1.14 8.45 x 10-5 133 420 1569 I 1.30 5.42 x 67 434 1633 1.31 4.75 x 10-5 33 450 1532 1.19 4.46 xI 17 460 1296 0.98 3.47 x 10-5 8 486 936 0.67 2.63 x 105 4 489 536 0.38 2.17x 1- 2 470 244 0.18 3.68 x10-' j(4.3 8 x MAKl195 400 375 881 1.20 5.3 8x 10- 1 200 400 1080 1.38 4.54 x 105 100 419 1141 1.39 3.54x 10-5 427 1106 1.32 1.67 x10-5 446 957 1.09 4.41 x10-5 13 464 708 0.78 3.66 x 10-5 6 474 433 0.47 7.37 x l0- 3 451 231 0.26 6.95 x I O0T (4.94 x 10-5) In a second series of experiments, the molecular kinetic interactions between an IgG I full-length form of D2E7 and biotinylated rhTNF was quantitatively analyzed using BlAcore technology, as described above, and kinetic rate constants were derived.

sumnmarized below in Tables 2. 3 and 4.

-43 Table 2: Apparent dissociation rate constants of the interaction between D2E7 and o* o o *o oo o Table 3: Apparent association rate constants of the interaction between D2E7 and biotinvlated rhTNF Experiment Ka s-1) 1 1.33 x 105 2 1.05 x 105 3 3.36 x 105 Average 1.91 1.26 x 105 Table 4: Apparent kinetic reate and affinity constants nof TP7 and ,A .I,'rn Experiment Ka (M1. s- 1 1 1.33 x 105 2 1.05x 10 5 3 3.36 x 10 5 Average 1.91 1.26 x 105

I

Kd Kd (M) 9.58 x 10-5 7.20 x 10-10 9.26 x 10-5 8.82 x 10-10 7.60 x 10-5 2.26 x 10-10 8.81 1.06 x 10- 5 6.09 3.42 x 10- 10 Dissociation and association rate constants were calculated by analyzing the dissociation and association regions of the sensorgrams by BIA analysis software. Conventional chemical reaction kinetics were assumed for the interaction between D2E7 and biotinylated rhTNF molecule: a zero order dissociation and first order association kinetics. For the sake of analysis, interaction only between one arm of the bivalent D2E7 antibody and one unit of the trimeric biotinylated rhTNF was considered in choosing molecular models for the analysis of the kinetic data. Three independent experiments were performed and the results were analyzed separately. The average apparent dissociation rate constant (kd) of the interaction between D2E7 and biotinvlated rhTNF was 8.81 1.06 x 10 5 s and the average apparent association rate constant, ka was 1.91 1.26 x 105 s The apparent intrinsic dissociation constant (Kd) was then calculated by the formula: Kd= kd ka. Thus, the mean Kd of D2E7 antibody for rhTNF -44 derived from kinetic parameters was 6.09 3.42 x 10- 1 0 M. Minor differences in the kinetic values for the IgGI form of D2E7 (presented in Tables 2, 3 and 4) and the IgG4 form of D2E7 (presented in Table I and in Examples 2 and 3) are not thought to be true differences resulting from the presence of either an IgGI or an IgG4 constant regions but rather are thought to be attributable to more accurate antibody concentration measurements used for the IgG1 kinetic analysis. Accoringly, the kinetic values for the IgGi form of D2E7 presented herein are thought to be the most accurate kinetic parameters for the D2E7 antibody.

10 EXAMPLE 2: Alanine Scanning Mutagenesis of D2E7 CDR3 Domains A series of single alanine mutations were introduced by standard methods along the CDR3 domain of the D2E7 VL and the D2E7 VH regions. The light chain mutations are illustrated in Figure IB (LD2E7*.A1, LD2E7*.A3, LD2E7*.A4, LD2E7*.A5, LD2E7*.A7 and LD2E7*.A8, having an alanine mutation at position 1,3, 4, 5, 7 or 8, respectively, of the D2E7 VL CDR3 domain). The heavy chain mutations are illustrated in Figure 2B (HD2E7*.A1, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4, HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8 and 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 ofrhTNF interaction with an antibody composed of wild-type D2E7 VL and VH was compared to that of antibodies composed of 1) a wildtype D2E7 VL paired with an alanine-substituted D2E7 VH; 2) a wild-type D2E7 VH paired with an alanine-substituted D2E7 VL; or 3) an alanine-substituted D2E7 VL paired with an alanine-substituted D2E7 VH. All antibodies were tested as full-length, IgG4 molecules.

Kinetics of interaction of antibodies with rhTNFa was determined by surface plasmon resonance as described in Example 1. The Koff rates for the different VH/VL pairs are summarized below in Table Table 5: Binding of D2E7 Alanine-Scan Mutants to Biotinvlated rhTNFa VH VL Koff(secl D2E7 VH D2E7 VL 9.65 x 10- HD2E7*.AI D2E7VL 1.4 x 10- 4 HD2E7*.A2 D2E7 VL 4.6 x 10- 4 HD2E7*.A3 D2E7 VL 8.15 x 10 4 HD2E7*.A4 D2E7 VL 1.8 x 10- 4 HD2E7*.A5 D2E7 VL 2.35 x 10- 4 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 D2E7 VH LD2E7*.A4 1.75 x D2E7 VH LD2E7*.A3 NOT DE T A-LE D2E7 VH LD2E7*.A4 1.75 x 10 4 D2E7 VH LD2E7*.A5 1.8 x D2E7 VH LD2E7*.A7 .4 x 10-4 D2E7 VH LD2E7.A3.65 x SHD2E7*.A9 ILD2E7*.A 1.05 x 0-4 These results demonstrate that the majority of positions of the CDR3 domains of the D2E7 VL region and VH region are amenable to substitution with a single alanine residue. Substitution of a single alanine at position 1, 4, 5, or 7 of the D2E7 VL CDR3 domain or at position 2, 5, 6, 8, 9 or 10 of the D2E7 VH CDR3 domain does not significantly affect the off rate of hTNFa binding as compared to the wild-type parental D2E7 antibody. Substitution of alanine at position 8 of the D2E7 VL CDR3 or at position 3 of the D2E7 VH CDR3 gives a 4-fold faster Koffand an alanine substitution at position 4 or 11 of D2E7 VH CDR3 gives an 8-fold faster Kof, indicating that these 10 positions are more critical for binding to hTNFa. However, a single alanine substitution at position 1, 4, 5, 7 or 8 of the D2E7 VL CDR3 domain or at 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 x 10- 3 sec-i or less.

EXA PLE 3: Binding Analysis of D2E7-Related Antibodies A series of antibodies related in sequence to D2E7 were analyzed for their binding to rhTNFa, as compared to D2E7, by surface plasmon resonance as described in Example 1. The amino acid sequences of the VL regions tested are shown in Figures IA and lB. The amino acid sequences of the VH regions tested are shown in Figures 2A and 2B. The Koff rates for various VH/VL pairs (in the indicated format, either as a fulllength IgG or IgG4 antibody or as a scFv) are summarized below in Table 6: 46 VHJ-LDl LOE7 I LOEI VH I-D2 LOE7 lgGl/lgG4 7.7 x 103 scFv I 4.6 X 10-4 LOE7 v r I -UL.

LOE7.T LOE7. fci4 II LA A VHI-D2.Y J LOE7.A IgG4 2.7 x VH1-D2.N LOE7.A I IgG4 I 3.2 x 10-5 V riII EP ES ,Z(%PV Iil IrF vtll- Z 2SD4 L LSD4 VLsc Fv 8.0 x -10-4 1.94 x103 15 0j-3 3 L~tih LUh Iii LZWJQ v iI LOE7 LUE] scFv 2SD4 VT-I 2SD4 VL Qr-v I 1 '7 VHII 2D4 L c~ 1.34 x 10-2 VHIB12 2SD4 VL scFv I1.01 x j- VHIB1 1 -2SD4 VL scFv 1 9.8 x IO- VH1E4 I 2SD4 VL I cv 1 1.59 x 10-2 VH1F6 2SD4 VLC scFv' I 22 10-2 VHID8 j 2SD4 VL scFv 9.5 x I0- VHIGI -2SD4 VL scFv I 2.14 x 10-2 2SD4 VH EP B12 scFv 6.7 x 10-3 -2SD4 VH VL1OE4 scFv 1 9.6 x 10-3 2SD4 VT- VL1I00A9 scFv I 1. 33 x 1'-2 2SD4 VH VL100D2 scFv 1. 41 X 10-2 -2SD4 VH VL1OF4 sc v 1.11 X 1- 2SD4 VH VLLOES scFv I 1.16 x 10-2 2SD4 VH VLLOF9 scFv 6.09 x 10-3 2SD4 VH I VLLOF 10 scFv j 1.34 x 10-2 2SD4 VH I VLLOG7 scFv 1 1.56 x 10-2 2SD4 VH VLLOG9 r scFv I 1.46 x 10-2 2SD4 VH VLLOH1I scFv 1.17 x 1- 2SD4 R VLLOHI10 scFv I 1.12 x 10-2 2SD4 VH VL1B7 scFv 1.3 x 10-2 2SD4 VH VLICI scFv 1.36 x 10-2 2SD4 VH VL1C7 scFv 2.0Ox 1-2 2SD4 VH I VLO.1IF4 cv176 x 10-2 V.Lty- V LU. 1171 scF%! sc'1.14 x 1- The slow off rates Koff:5 1 x 10-4 sec-1) for ftill-length antibodies IgG format) having a VL selected from D2E7, LOE7, LOE7.T and LOE7.A, which have either a threonine or an alanine at position 9, indicate that position 9 of the D2E7 VL CDR.3 can be occupied by either of these two residues without substantially affecting the

K

0 Accordingly, a consensus motif for the D2E7 VL CDR3 comprises the amino acid sequence: (SEQ ID NO: Furthermore, the slow off rates Kof: I x 10-4 sc- 1 for antibodies having a VH selected from D2E7, VH I -D2.N -47 and VHI-D2.Y, which have either a tyrosine or an asparagine at position 12, indicate that position 12 of the D2E7 VH CDR3 can be occupied by either of these two residues without substantially affecting the Koff. Accordingly, a consensus motif for the D2E7 VH CDR3 comprises the amino acid sequence: (SEQ ID NO: 4).

The results shown in Table 6 demonstrate that, in scFv format, antibodies containing the 2SD4 VL or VH CDR3 region exhibit a faster Koff Koff 1 x 10- 3 sec- 1 as compared to antibodies containing the D2E7 VL or VH CDR3 region. Within the VL CDR3, 2SD4 differs from D2E7 at positions 2, 5 and 9. As discussed above, 10 however, position 9 may be occupied by Ala (as in 2SD4) or Thr (as in D2E7) without substantially affecting the Koff. Thus, by comparison of 2SD4 and D2E7, positions 2 and 5 of the 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 in the antibody binding site or could contribute critically to maintaining the scaffolding architecture of the antibody molecule in this region. Regarding the importance of position 2, replacement of Arg (in LOE7, which has the same VL CDR3 as D2E7) with Lys (in EP B12) accelerates the off rate by a factor of two. Regarding the importance of position 5, replacement of Arg (in D2E7) with Ala (in LD2E7*.A5), as described in Example 2, also accelerates the off rate two-fold. Furthermore, without either Arg at positions 2 and 5 (in 2SD4), the off rate is five-fold faster. However, it should be noted that although position 5 is important for improved binding to hTNFca, a change at this position can be negated by changes at other positions, as seen in VLLOE4, VLLOHI or VL0.1H8.

Within the VH CDR3, 2SD4 differs from D2E7 at positions 1, 7 and 12. As discussed above, however, position 12 may be occupied by Asn (as in 2SD4) or Tyr (as in D2E7) without substantially affecting the Koff. Thus, by comparison of 2SD4 and D2E7, positions 1 and 7 of the 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 in the antibody binding site or could contribute critically to maintaining the scaffolding architecture of the antibody molecule in this region. Both positions are important for binding to hTNFa since when the 3C-H2 VH CDR3 (which has a valine to alanine change at position 1 with respect to the D2E7 VH CDR3) is used, the scFv has a 3-fold faster off rate than when the D2E7 VH CDR3 is used but this off rate is still four times slower than when the 2SD4 VH CDR3 is used (which has changes at both positions 1 and 7 with respect to the D2E7 VH CDR3).

-48 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 hTNFa activity, either in vitro Sor in vivo.

A. Neutralization of TNFa-Induced Cvtotoxicitv in L929 Cells Human recombinant TNFa (rhTNFa) causes cell cytotoxicity to murine L929 10 cells after an incubation period of 18-24 hours. Human anti-hTNFa antibodies were evaluated in L929 assays by coincubation of antibodies with rhTNFa and the cells as follows. A 96-well microtiter plate containing 100 pl of anti-hTNFa Abs was serially diluted 1/3 down the plate in duplicates using RPMI medium containing 10% fetal bovine serum (FBS). Fifty microliters of rhTNFa was added for a final concentration of 15 500 pg/ml in each sample well. The plates were then incubated for 30 minutes at room temperature. Next, 50 l of TNFa-sensitive L929 mouse fibroblasts cells were added for a final concentration of 5 x 104 cells per well, including 1 pg/ml Actinomycin-D.

Controls included medium plus cells and rhTNFa plus cells. These controls, and a TNFa standard curve, ranging from 2 ng/ml to 8.2 pg/ml, were used to determine the quality of the assay and provide a window of neutralization. The plates were then incubated overnight (18-24 hours) at 370 C in 5% CO 2 One hundred microliters of medium was removed from each well and 50 tl of -5 mg/ml 3 4 4 -dimethylthiazol-2-yl)2.5-diphenyl-tetrazolium bromide (MTT; .commercially available from Sigma Chemical Co., St. Louis,'MO) in PBS was added.

.The plates were then incubated for 4 hours at 370 C. Fifty microliters of 20% sodium dodecyl sulfate (SDS) was then added to each well and the plates were incubated overnight at 370 C. The optical density at 570/630 nm was measured, curves were plotted for each sample and IC 5 os were determined by standard methods.

Representative results for human antibodies having various VL and VH pairs, as compared to the murine MAK 195 mAb, are shown in Figure 3 and in Table 7 below.

Table 7: Neutralization of TNFa-Induced L929 Cvtotoxicitv VH VL Structure D2E7 D2E7 scFv 1.1 x 10-10 D2E7 D2E7 IgG4 4.7 x 10-11 2SD4 2SD4 scFv/fgGl/IgG4 3.0 x 10-7 -49 2SD4 LOE7 ]scFv 4.3 x 10-8 VH I-D2 I 2SD4 IscFv 1.0OX 10-8 VHI-D2 LOE7 I scFv/IgGI/lgG4 3.4 x VHlI.D2.Y LOE7.T IgG4 8.1 x 10-11 VI-D2.N I LOE7.T IgG4 1.3 x 10-10 VHl-D2.Y LOE7.A IgG4 2.8 x 10-11 VHI-D2.N LOE7.A IgG4 6.2 x 10-11 MAK 195 MAK 195 scFv 1.9 x 10.8 MAK 195 1MAK195 F(ab')2 6.2 x101 The results in Figure 3 and Table 7 demonstrate that the D2E7 human anti-hTNFct antibody, and various D2E7-related antibodies, neutralize TNFcz-induced L929 cytotoxicity with a capacity approximately equivalent to that of the murine anti-hTNFa mAb MAK 195.

In another series of experiments, the ability of the IgG I formn of D2E7 to neutralize T-NFca-induced L929 cvtotoxicitv was examined as described above. The results from three independent experiments, and the average thereof, are summarized 10 below in Table 8: Table 8: Neutralization of TNFcz-lnduced L929 Cvtotoxicitv by D2E7 IkGI Experiment 1050 [M) 1.26sx 10-10 2 1.33 x 10-10 1.15 x10- 1 0 Average 1.25 ±0.01 x 10- This series of experiments confirmed that D2E7, in the full-length IgG I form, neutralizes TNFcz-induced L929 cvtotoxicity with an average 1C50 of 1.25 0.01 x 10-10.

B Inhibition of TNKFa Bindine to TNFx Receptors on U-937 Cells The ability of human anti-hTNFx antibodies to inhibit the binding of hTNFc to hT'NFcx receptors on the surface of cells was examined using the U-937 cell line (ATCC No. CRL 1593), a human histiocytic cell line that expresses hTNFa receptors. U-937 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone A- I11l. Hyclone Laboratories. Logan, UT), L-glutamine (4 nM), HEPES buffer solution (10 mM), penicillin (100 jg/ml) and streptomycin (100 pg/ml). To examine the activity of full-length IgG antibodies, U-937 cells were preincubated with PBS supplemented with I mg/ml of human IgG (Sigma 1-4506, Sigma Chemical Co.. St.

Louis, MO) for 45 minutes on ice and then cells were washed three times with binding buffer. For the receptor binding assay, U-937 cells (5 x 106 cells/well) were incubated in a binding buffer (PBS supplemented with 0.2% bovine serum albumin) in 9 6-well microtiter plates (Costar 3799, Costar Corp., Cambridge, MA) together with 125 1-labeled rhTNFa (3 x 10-10 M; 25 pCi/ml; obtained from NEN Research Products, Wilmington, DE), with or without anti-hTNFa antibodies, in a total volume of 0.2 ml. The plates were incubated on ice for 1.5 hours. Then, 75 pl of each sample was transferred to ml test tubes (Sarstedt 72.700, Sarstedt Corp., Princeton. NJ) containing dibutylphthalate (Sigma D-2270, Sigma Chemical Co., St. Louis, MO) and dinonylphthalate

(ICN

15 210733, ICN, Irvine, CA). The test tubes contained a 300 pl mixture of dibutylphthalate and dinonylphthalate, 2:1 volume ratio, respectively. Free unbound) 1 25 1-labeled rhTNFa was removed by microcentrifugation for five minutes. Then, each test tube end containing a cell pellet was cut with the aid of a microtube scissor (Bel-Art 210180001, Bel-Art Products, Pequannock, NJ). The cell pellet contains 125 1-labeled rhTNFa bound *20 to the p60 or p80 TNFa receptor, whereas the aqueous phase above the oil mixture contains excess free 1 2 5I-labeled rhTNFa. All cell pellets were collected in a counting tube (Falcon 2052, Becton Dickinson Labware, Lincoln Park, NJ) and counted in a scintillation counter.

Representative results are shown in Figure 4. The IC 50 value for D2E7 inhibition of hTNFa binding to hTNFa receptors on U-937 cells is approximately 3 x 10-10 M in these experiments. These results demonstrate that the D2E7 human antihTNFa antibody inhibits rhTNFa binding to hTNFa receptors on U-937 cells at concentrations approximately equivalent to that of the murine anti-hTNFa mAb MAK 195.

In another series of experiments, the ability of the IgGI form of D2E7 to inhibit rhTNFa binding to hTNFa receptors on U-937 cells was examined as described above.

The results from three independent experiments, and the average thereof, are summarized below in Table 9: -51 Table 9: Inhibition of TNF Receptor Binding on U-937 Cells by D2E7 leG1 Experiment

IC

50

(M]

1 1.70 x 10-10 2 1.49x 10-10 3 1.50 x 10-10 Average 1.56 0.12 x This series of experiments confirmed that D2E7, in the full-length IgGI form, inhibits TNF receptor binding on U-937 cells with an average IC 50 of 1.56 0.12 5 x 10-10.

To investigate the inhibitory potency of D2E7 in the binding of 125 1-rhTNF binding to individual p55 and p75 receptors. a solid phase radioimmunoassay was performed. To measure the IC 50 values of D2E7 for separate TNF receptors, varying concentrations of the antibody were incubated with 3 x 10-10 concentration of 125I.

rhTNF. The mixture was then tested on separate plates containing either the p55 or the p75 TNF receptors in a dose dependent manner. The results are summarized below in Table 1 15 Table 10: Inhibition of TNF Receptor Binding to p55 and p75 TNFR by D2E7 IeG1 I ICn [Ml 9.9.

9 9 .9* Reagent p55 TNFR D2E7 1.47 x 10-9 1.26 x 10-9 rhTNF 2.31 x 10-9 2 .70 x 10- 9 Inhibition of 21I-rhTNF binding to the p55 and p75 TNF receptors on 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, ICso values for inhibition of 125I-rhTNF binding to the p55 and the receptors by D2E7 were calculated as 1.47 x 10-9 and 1.26 x 10-9 M, respectively. The decrease in ICo 5 values in the solid phase was probably due to higher density of receptors in the RIA format. as unlabeled rhTNF also inhibited with similar ICso values. The IC 5 o values for inhibition of 25 I-rhTNF binding to the p55 and the p75 receptors by unlabeled rhTNF were 2.31 x 10-9 and 2.70 x 10-9 M, respectively 52 C. Inhibition of ELAM-1 Expression on HUVEC Human umbilical vein endothelial cells (HUVEC) can be induced to express endothelial cell leukocyte adhesion molecule 1 (ELAM-1) on their cell-surface by treatment with rhTNFa, which can be detected by reacting rhTNFa-treated

HUVEC

with an mouse anti-human ELAM-1 antibody. The ability of human anti-hTNFa antibodies to inhibit this TNFa-induced expression of ELAM-I on HUVEC was examined as follows: HUVEC (ATCC No. CRL 1730) were plated in 9 6-well plates x 104 cells/well) and incubated overnight at 37 OC. The following day, serial dilutions of human anti-hTNFa antibody (1:10) were prepared in a microtiter plate, starting with 0: 20-100 tg/ml ofantibody. A stock solution of rhTNFa was prepared at 4.5 ne/ml aliquots of rhTNFa were added to each antibody-containing well and the contents were mixed well. Controls included medium alone, medium plus anti-hTNFa antibody and medium plus rhTNFa. The HUVEC plates were removed from their overnight incubation at 370 C and the medium gently aspirated from each well. Two hundred microliters of the antibody-rhTNFa mixture were transferred to each well of the HUVEC plates. The HUVEC plates were then further incubated at 370 C for 4 hours.

Next, a murine anti-ELAM-1 antibody stock was diluted 1:1000 in RPMI. The medium in each well of the HUVEC plate was gently aspirated, 50 pl/well of the anti-ELAM-1 antibody solution was added and the HUVEC plates were incubated 60 minutes at room i temperature. An 1 2 5 1-labeled anti-mouse Ig antibody solution was prepared in RPMI (approximately 50.000 cpm in 50 pl). The medium in each well of the HUVEC plates was gently aspirated, the wells were washed twice with RPMI and 50 pl of the 1251.

labeled anti-mouse Ig solution 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 eighty microliters of 5% SDS was 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.

Representative results are shown in Figure 5. The IC 50 value for D2E7 inhibition ofhTNFa-induced expression of ELAM-1 on HUVEC is approximately 6 x 1M in these experiments. These results demonstrate that the D2E7 human antihTNFa antibody inhibits the hTNFa-induced expression of ELAM-1 on HUVEC at concentrations approximately equivalent to that of the murine anti-hTNFa mAb MAK 195.

-53 In another series of experiments, the ability of the IgGi form of D2E7 to inhibit hTNFa-induced expression of ELAM-I on HUVEC was examined as described above.

The results from three independent experiments, and the average thereof, are summarized below in Table 11: Table 11: Inhibition ofTNFa-Induced ELAM- Expression bV D2E7 leG Receptor cExperiment IC50 [M] 1 -1.95 x 10-10o 2 1.69 x 10-10 3 1.90 x 10-10o Average 1.85 0.14 x 10-10 This series of experiments confirmed that D2E7, in the full-length IgGI form.

inhibits TNFa-induced ELAM-I expression on HUVEC with an average IC50 of 10 1.85 0.14 x 10-10.

The neutralization potency of D2E7 IgGi was also examined for the rhTNF induced expression of two other adhesion molecules, ICAM-1 and VCAM-1. Since the rhTNF titration curve for ICAM-1 expression at 16 hours was very similar to the curve of ELAM-1 expression, the same concentration of rhTNF was used in the antibody neutralization experiments. The HUVEC were incubated with rhTNF in the presence of varying concentrations of D2E7 in a 37"C CO, incubator for 16 hours, and the ICAM-1 expression was measured by mouse anti-ICAM-1 antibody followed by 12sI-labeled sheep anti-mouse antibody. Two independent experiments were performed and the ICso 20 values were calculated. An unrelated human IgGI antibody did not inhibit the ICAM-1 expression.

The experimental procedure to test inhibition of VCAM-1 expression was the same as the procedure for ELAM-1 expression, except anti-VCAM-1 MAb was used instead of anti-ELAM-1 MAb. Three independent experiments were performed and the ICo 5 values were calculated. An unrelated human IgGI antibody did not inhibit VCAM- 1 expression.

The results are summarized below in Table 12: -54- Table 12: Inhibition of ICAM-l and VCAM-I Expression bvD2E7 IIGI ICAM-1 Inhibition

IC

50

[M)

Experiment ico 5 Experiment

IC

5 1_ 1.84 x 10-10 I 1.03 2 2 .49x 10-10 2 9.26 0oI M] x 10-10 Average 2.17 0.46 x 10-10 3 1.06 x 10-10 Average 1.01i 0.01 x 10-10 These experiments demonstrate that treatment of primary human umbilical vein endothelial cells with rhTNF led to optimum expression of adhesion molecules: 5 ELAM-1 and VCAM-1 at four hours, and the maximum up-regulated expression of ICAM-1 at 16 hours. D2E7 was able to inhibit the expression of the three adhesion molecules in a dose dependent manner. The ICo 5 values for the inhibition of ELAM-1, ICAM-1 and VCAM-1 were 1.85 x 10-10. 2.17 x 10-10 and 1.01 x 10-10 M, respectively.

These values are very similar, indicating similar requirements for the dose of rhTNF 10 activation signal to induce ELAM-1, ICAM-1 and VCAM-1 expression. Interestingly, D2E7 was similarly effective in the longer inhibition assay of the the 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 the ELAM- and the VCAM-I inhibition assays. Since D2E7 has a slow off-rate for rhTNF, it is conceivable that during the 16 hour co-incubation period there was no significant competition by the TNF receptors on the HUVEC.

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

I. Inhibition of TNF-lnduced Lethality in D-Galactosamine-Sensitized Mice Injection of recombinant human TNF c (rhTNFa) to D-galactosamine sensitized mice causes lethality within a 24 hour time period. TNFa neutralizing agents have been shown to prevent lethality in this model. To examine the ability of human anti-hTNFa antibodies to neutralize hTNFa in vivo in this model. C57B1/6 mice were injected with varying concentrations of D2E7-IgGl. or a control protein, in PBS intraperitoneally Mice were challenged 30 minutes later with 1 gg of rhTNFa and 20 mg of Dgalactosamine in PBS and observed 24 hours later. These amount of rhTNFa and D-galactosamine were previously determined to achieve 80-90% lethality in these mice.

Representative results, depicted as a bar graph of% survival versus antibody concentration, are shown in Figure 6. The black bars represent D2E7, whereas the hatched bars represent MAK 195. Injection of 2.5-25 pg of D2E7 antibody per mouse protected the animals from TNFa-induced lethality. The ED 50 value is approximately 1-2.5 pg/mouse. The positive control antibody, MAK 195, was similar in its protective ability. Injection of D2E7 in the absence of rhTNFa did not have any detrimental effect on the mice. Injection of a non-specific human IgGI antibody did not offer any protection from TNFa-induced lethality.

In a second experiment, forty-nine mice were divided into 7 equal groups. Each :i group received varying doses of D2E7 thirty minutes prior to receiving an LD 80 dose of rhTNF/D-galactosamine mixture (1.0 p.g rhTNF and 20 mg D-galactosamine per 15 mouse). Control group 7 received normal human IgGI kappa antibody at 25 pg/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 Group Survival (alive/total) Survival 1 (no antibody) 0/7 0 2 1 pg) 1/7 14 3 (2.6 4g) 5/7 71 4 (5.2 Ag) 6/7 86 5 (26 pg) 6/7 86 6 (26 pg; no rhTNF) 7/7 100 7 (25 Pg Hu IgGl) 1/7 14 II. Inhibition of TNF-Induced Rabbit Pvrexia The efficacy of D2E7 in inhibiting rhTNF-induced pyrexia response in rabbits was examined. Groups of three NZW female rabbits weighing approximately 2.5 kg each were injected intravenously with D2E7, rhTNF, and immune complexes of D2E7 and rhTNF. Rectal temperatures were measured by thermistor probes on a Kaye thermal recorder every minute for approximately 4 hours. Recombinant human TNF in saline, injected at 5 pg/kg, elicted a rise in temperature greater than 0.4 0 C at approximately 56minutes after injection. The antibody preparation by itself, in saline at a dose of 138 Ug/kg, did not elicit a rise in temperature in the rabbits up to 140 minutes after administration. In all further experiments, D2E7 or control reagents (human IgGI or a saline vehicle) were injected i.v. into rabbits followed 15 minutes later by an injection of rhTNF in saline at 5 pg/kg i.v. Representative results of several experiments are summarized below in Table 14: Table 14: Inhibition of rhTNF-induced Pvrexia with D2E7 in Rabbits Temp. rise*, °C Molar Ratio Peak Temp.

D2E7 dose rhTNF D2E7: minutes (pg/kg) rhTNF D2E7 Inhib.** rhTNF post rhTNF 14 0.53 0.25 53 1 24 0.43 0.13 70 1.6 48 0.53 0.03 94 3.3 137 0.53 0.00 100 9.5 792 0.80 0.00 100 55 *=Peak temperature inhibition=(l-{temperature rise with rhTNF D2E7/temperature rise with rhTNF alone}) x 100.

Intravenous pretreatment with D2E7 at a dose of 14 pg/kg partially inhibited the pyrogenic response, compared to rabbits pre-treated with saline alone. D2E7 administered at 137 pg/kg totally suppressed the pyrogenic response of rhTNF in the same experiment. In a second experiment, D2E7 administered at 24 pg/kg also partially suppressed the pyrogenic response, compared to rabbits pretreated with saline alone.

The molar ratio of D2E7 to rhTNF was 1/6:1 in this experiment. In a third experiment, D2E7 injected i.v. at 48 pg/kg (molar ratio D2E7:rhTNF 3.3:1) totally suppressed the pyrogenic response, compared to rabbits pretreated With the control human IgGI in saline at 30 pg/kg. In the final experiment, rabbits pretreated with D2E7 (792 pg/kg) at a very high molar ratio to rhTNF (55:1) did not develop any rise in temperature at any time up to 4 hours of observation. Treatment of rabbits with immune complexes generated from a mixture of D2E7 and rhTNF incubated at 37 0 C for 1 hour at a molar ratio of 55:1, without subsequent rhTNF administration, also did not elicit any rise in temperature in the same experiment.

57 III. Prevention of Polvarthritis in Tel 97 Transeenic Mice The effect of D2E7 on disease development was investigated in a transgenic murine model of arthritis. Transgenic mice (Tg197) have been generated that express human wild type TNF (modified in the 3' region beyond the coding sequences) and these mice develop chronic polyarthritis with 100% incidence at 4-7 weeks of age (see EMBO J (1991) 10:4025-4031 for further description of the Tgi 97 model of polyarthritis).

Transgenic animals were identified by PCR at 3 days of age Litters of transgenic mice were divided into six groups. Transgenic mice were verified by slot-blot 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 Group 4=D2E7 at 15 g/g; Group 5=D2E7 at 30 pg/g; and Group 6=IgGl isotype control at 30 p.g/g. A litter with non transgenic mice was also included in the study to 15 serve as a control (Group 7 nontransgenic; no treatment). Each group received three i.p. injections per week of the indicated treatments. Injections continued for 10 weeks.

Each week, macroscopic changes in joint morphology were recorded for each animal.

At 10 weeks, all mice were sacrificed and mouse tissue was collected in formalin.

Microscopic examination of the tissue was performed.

Animal weight in grams was taken for each mouse at the start of each week. At the same time measurements of joint size (in mm) were also taken, as a measurement of disease severity. Joint size was established as an average of three measurements on the hind right ankle using a micrometer device. Arthritic scores were recorded weekly as Sfollows: 0 No arthritis, (normal appearence and flexion); =mild arthritis (joint 25 distortion); moderate arthritis (swelling, joint deformation) and heavy arthritis (ankylosis detected on flexion and severely impaired movement).

Histopathological scoring based on haematoxylin/eosin staining of joint sections 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 mean joint size of the Tg197 transgenic arthritic mice is shown in the graph of Figure 9. The histopathological and arthritic scores of the Tg197 transgenic mice, at 11 weeks of age, are summarized below in Table -58 Table 15: ffect of D2E7 on Hisoatholov and Arthritic Score in Tel 97 Mice Group Treatment I Histopathological Score Arthritic Score I none 3 (7/70 (7/7) 2 saline 3 i (8/8) 6 IgGI control 3 8(7/9) 3 D2E7 at 1.5 gg/g 0 0(8/8) 4 D2E7 at 15 pg/g 0 0 (8/8) D2E7 at 30 gg/g j0 0(8/8) 0 (8/8) This experiment demonstrated that the D2E7 antibody has a definite beneficial effect on transgenic mice expressing the wild-type human TNF (Tg197) with no arthritis 5 evident after the study period.

SE. D2E7 Neutralization of TNFas from Other Species The binding specificity of D2E7 was examined by measuring its ability to neutralize tumor necrosis factors from various primate species and from mouse, using an L929 cytotoxicity assay (as described in Example 4, subsection A, above). The results are summarized in Table 16 below: Table 16: Ability of D2E7 to Neutralize TNF from Different Species in the L929 Assa o:o• IC50 for D2E7 *TNFa* -Fa- S o u r c e Neutralization H um a n Recombinant 7.8 x 10-11 Chimpanzee LPS-stimulated PBMC 5.5 x 10-11 baboon Recombinant 6.0 x 10-11 marmoset LPS-stimulated PBMC 4.0 x 10-10 cynomolgus LPS-stimulated PBMC 8.0 x 10-1 I rhesus LPS-stimulated PBMC 3.0 x 10-11 canine LPS-stimulated WBC 2.2 x 10-

I

o porcine Recombinant 1.0 x 10-7 murine Recombinant >1.0 x 10-7 The results in Table 16 demonstrate that D2E7 can neutralize the activity of five primate TNFas approximately equivalently to human TNFa and, moreover, can -59neutralize the activity of canine TNFa (about ten-fold less well than human TNFa) and porcine and mouse TNFa (about -1000-fold less well than human TNFa). Moreover, the binding of D2E7 to solution phase rhTNFa was not inhibited by other cytokines, such as lymphotoxin (TNFP), IL-la, IL-lp, IL-2, IL-4, IL-6, IL-8, IFNy and TGFp, indicating that D2E7 is very specific for its ligand TNFa.

F. Lack of Cvtokine Release by Human Whole Blood Incubated with D2E7 In this example, the ability of D2E7 to induce, by itself, normal human blood cells to secrete cytokines or shed cell surface molecules was examined. D2E7 was incubated with diluted whole blood from three different normal donors at varying concentrations for 24 hours. An LPS positive control was run at the same time, at a concentration previously determined to stimulate immunocompetent blood cells to secrete cytokines. The supematants were harvested and tested in a panel often soluble 15 cytokine, receptor and adhesion molecule ELISA kits: IL-la, IL-0, IL-1 receptor antagonist, IL-6, IL-8, TNFa, soluble TNF receptor I, soluble TNF receptor II, soluble ICAM-1 and soluble E-selectin. No significant amounts of cytokines or shed cell surface molecules were measured as a result of D2E7 antibody co-incubation, at concentrations up to 343 p.g/ml. Control cultures without the addition of the antibody also did not yield any measurable amounts of cytokines, whereas the LPS co-culture control yielded elevated values in the high picogram to low nanogram range. These results indicate that D2E7 did not induce whole blood cells to secrete cytokines or shed cell surface proteins above normal levels in ex vivo cultures.

60 Forming part of the present disclosure is the appended Sequence Listing, the contents of which are summarized in the table below:

ANTIBODY

SEQ ID NO: CHAIN J REGION SEQUENCE TYPE 1 D2E7 {VL amino acid 2 D2E7 VH amino acid 3 D2E7 VL CDR3 E 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 amino acid 9 2SD4 VL amino acid 10 2SD4 VH amino acid 11 2SD4 VL CD)R3 amino acid 12 EP B12 VL CDR3 amino acid 13 VL1OE4 VL CDR3 Jamino acid 14 VL100A9 VL CDR3 amino acid 15 VLL100D2 VL CDR3 amino acid 1.6 VLLOF4 VL CDR3 amino acid 17 L OEB VL CDR3 amino acid 18 VLLOG7 VL CDR3 amino acid 19 VLLOG9 VL CDR3 amino acid VLLOH. .VL CDR3 amino acid 21 VLLOH1O f VL CDR3 amino acid 22 VL1B7 VL CD.R3 amino acid 23 VL1C1 VL CDR3 -amino acid 24 VLO.1F4 VL CDR3 amino acid VLO.1H8 VL CDR3 amino acid 26 LOE7.A VL CDR3 amino acid 27 2SD4 yR CDR3 amino acid 28 VH1B11 VH CDR3 amino acid 29 VI-11D8 VH- CDR3 amino acid VH1A11 VH CDR3 T amino acid 31 VH1B12 yR CDR3 amino acid 32 VH1E4 yR CDR3 amino acid 33V HlF6 '11- CDR3 amino acid 34 3C-H-2 J VH CDR3 amino acid VH1-D2.N VH CDR3 [amino acid 36 I D2E7 I VL Inucleic acid 37 j D2E7 Inucleic acid" -61

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Page(s)]7 87 are claims pages they appear after the sequence listing -62- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: BASF Aktiengesellschaft STREET: Carl-Bosch Str. 38 CITY: 67056 Ludwigshafen STATE: Rheinland-Pfalz COUNTRY: Federal Republic of Germany (ii) TITLE OF INVENTION: Human Antibodies that Bind Human TNFa (iii) NUMBER OF SEQUENCES: 37 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston 20 STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/599,226 FILING DATE: 09-FEB-1996

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/031,476 FILING DATE: 25-NOV-1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: DeConti, Giulio Jr.

REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043CPPC (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (617)227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO:1: 63 SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:1: Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly C C C. C

C

e.g.

C.

C

C.

C C

C.

C C

CCC.

C

CC..

4 C

C

CC C C C

CC

Asp Arg Val Thr 20 Leu Ala Trp Tyr 35 Tyr Ala Ala Ser 5o Ser Gly Ser Giy 65 Giu Asp Val Ala Thr Phe Gly Gin 100 Ile Thr Cys Arg Ala 25 Ser Gin Gly Ile Gln Gin Lys Pro Gly Lys Ala Pro 40 Thr Leu Gin Ser Gly Val Pro Ser 55 Thr Asp Phe Thr Leu Thr Ile Ser 70 Lys Arg Arg Asn Tyr Leu Leu Ile Phe Ser Gly Ser Leu Gin Thr Tyr Tyr Cys Gin Giy Thr Lys Val Giu 105 Arg Tyr Asn Arg Ala Ile Lys 35 INFORMATION FOR SEQ ID NO:2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 121 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRALGMENTI TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Giu Val Gin Leu Val Giu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 1 5 10 1 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 25 Ala Met His Trp Val Arg Gin. Ala Pro Gly Lys Gly Leu Giu Trp Val.

35 40 .64 Ser Ala 11e Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val s0 55 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 70 75 Leu Gin Met Asri Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 90 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu A-sp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 15115 120 06.9 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 0600 FRAGMENT TYPE: internal *Too (ix) FEATURE: NAME/KEY: modified-site 006(B) LOCATION: 9 /oe OTHER INFORMATION: /nt="Xaa is Thr or Ala" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: *000 Gin Arg Tyr Asn Arg Ala Pro Tyr Xaa 1. INFORMATION FOR SEQ ID NO:4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide Cv) FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 122 CD) OTHER INFORMAXTION: /note= "Xaa is Tyr or Asn,, (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID 20 Ala Ala Ser Thr Leu Gin Ser 1 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal 35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu 1 5 10 Gly INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 66 Arg Ala Ser Gin Gly Ile Arg Asn Tyr Leu Ala 1 5 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: S amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Asp Tyr Ala Met His INFORMATION FOR SEQ ID NO: 9: Wi SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear 0 (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ::00.35 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Serr Ala Ser Ile Gly 1 10i is Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly Ile Arg Asn Tyr 20 25 Leu Ala Tr-p Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 40 Tyr Ala Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 55 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 70 75 Giu Asp Val Ala Thr Tyr Tyr Cys Gin Lys Tyr Asn Ser Ala Pro Tyr 90 Ala Phe Gly Gin Gly Thr Lys Val Giu Ile Lys 100 105 67 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 122. amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide Cv) FRAGMENT TYPE: internal Cxi) SEQUENCE DESCRIPTION: SEQ ID Gln Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Leu Arg Leu Ser 20 Met His Trp Val Cys Ala Ala Ser Gly Ph-e Thr Gin Pro Gly Arg Phe Asp Asp Tyr Leu Asp Trp Val Ala Asp Ser Val Ala.

Ser Ala Ile Arg Gin Ala 40 Asn Ser Gly Pro Gly Lys Gly Thr Trp His Ile Asp 50 Glu Gly Arg Phe Ala Leu Val Ser 70 Leu Arg Arg Asp Asn Pro Glu Asp 90 Ala Lys Asn Ala Leu Tyr 75 so Thr Ala Val Tyr Tyr Cys Gin Met Asn Ser Thr Lys Ala Ser Tyr 100 Leu Ser Thr Ser Ser Leu Asp Asn Trp Gly 110 Gin Gly Thr Leu Val Thr Val 115 INFORMATION FOR SEQ ID NO:ll: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRI PTION: SEQ ID NO:2.1: Gin Lys Tyr Asn Ser Ala Pro Tyr Ala 1 -68- INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Gin Lys Tyr Asn Arg Ala Pro Tyr Ala 1 20 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal SEQUENCE DESCRIPTION: SEQ ID NO:13: Gln Lys Tyr Gln Arg Ala Pro Tyr Thr 1 INFORMATION FOR SEQ ID N0:14: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Gin Lys Tyr Ser Ser Ala Pro Tyr Thr 1 INFORMATION FOR SEQ ID -69- SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Lys Tyr Asn Ser Ala Pro Tyr Thr 1 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Gin Lys Tyr Asn Arg Ala Pro Tyr Thr 1 s INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Gin Lys Tyr Asn Ser Ala Pro Tyr Tyr 1 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Gin Lys Tyr Asn Ser Ala Pro Tyr Asn 1 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: 15 LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

S

Gin Lys Tyr Thr Ser Ala Pro Tyr Thr .1 INFORMATION FOR SEQ ID 3 SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Lys Tyr Asn Arg Ala Pro Tyr Asn 1 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal -71- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Gin Lys Tyr Asn Ser Ala Ala Tyr Ser 1 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear 15 (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 20 Gln Gln Tyr Asn Ser Ala Pro Asp Thr 1 25 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid 30 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Gin Lys Tyr Asn Ser Asp Pro Tyr Thr 1 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 72- Gln Lys Tyr Ile Ser Ala Pro Tyr Thr 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Gln Lys Tyr Asn Arg Pro Pro Tyr Thr 1 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: 2 5 LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 35 Gln Arg Tyr Asn Arg Ala Pro Tyr Ala 1 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 1 5 -73- INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys S: 15 1 s INFORMATION FOR SEQ ID NO:29: 20 SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear 25 (ii) MOLECULE TYPE: peptide *o FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: SAla Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr 1 5 35 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp 1 5 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: (A).LENGTH: 12 amino acids -74- TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Ala Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr 1 5 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear .0 (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr 1 5 1* 2) INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: se. LENGTH: 12 amino acids 35 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr 1 s INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 75 0 0 6S 0 0.00 0..

FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: A-la Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr 1 5 INFORMATION FOR SEQ ID Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Asn 1 5 INF ORMATION FOR SEQ ID NO:36:.

Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 321 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:36: TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGGGA CAGAGTCACC ATCACTTGTC GGGCAAGTCA GGGCATCAGA AATTACTTAG CCTGGTATCA

GCAAAAACCA

GGGAAAGCCC CTAAGCTCCT GATCTATGCT GCATCCACTT TGCAATCAGG

GGTCCCATCT,

CGGTTCAGTG GCAGTGGATC TGGGACAGAT TTCACTCTCA CCATCAGCAG

CCTACAGCCT

GAAGATGTTG CA.ACTTATTA CTGTCAAAGG TATAACCGTG CACCGTATAC

TTTTGGCCAG

GGGACCAAGG TGGAATCAA

A

120 180 240 300 321 INFORMATION FOR SEQ ID NO 37: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 363 base pairs 76 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GAGGTGCAGC TGGTGGAGTC TGGGGGAGGC TTGGTACAGC TCCTGTGCGG CCTCTGGATT CACCTTTGAT GATTATGCCA CCAGGGAAGG GCCTGGAATG GGTCTCAGCT ATCACTTGGA GCGGACTCTG TGGAGGGCCG ATTCACCATC TCCAGAGACA CTGCAAATGA ACAGTCTGAG AGCTGAGGAT ACGGCCGTAT TACCTTAGCA CCGCGTCCTC CCTTGACTAT TGGGGCCAAG

AGT

*o

CCGGCAGGTC

TGCACTGGGT

ATAGTGGTCA

ACGCCAAGAA

ATTACTGTGC

GTACCCTGGT

CCTGAGACTC

CCGGCAAGCT

CATAGACTAT

CTCCCTGTAT

GAAAGTCTCG

CACCGTCTCG

120 180 240 300 360 363

Claims (58)

1. An isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFa with a Kd of 1 x 10- 8 M or less and a Koff rate constant of I x 10- 3 or less, both determined by surface plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC 50 of I x 10- 7 M or less.

2. The isolated human antibody, or antigen-binding portion thereof, of claim 10 1, which dissociates from human TNFa with a Koff rate constant of5 x 10- 4 s- 1 or less,

3. The isolated human antibody, or antigen-binding portion thereof. of claim 1, which dissociates from human TNFa with a Koff rate constant of 1 x 10- 4 s- 1 or less.

4. The isolated human antibody, or antigen-binding portion thereof, of claim 1, which neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC 50 of I x 10-8 M or less. The isolated human antibody, or antigen-binding portion thereof, of claim 1, which neutralizes human TNFac cytotoxicity in a standard in vitro L929 assay with an IC 50 of x 10 9 M or less.

6. The isolated human antibody, or antigen-binding portion thereof. of claim 1, which neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an ICso of 5 x 010 M or less.

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

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

9. An isolated human antibody, or antigen-binding portion thereof. with the following characteristics: -78 a) dissociates from human TNFa with a Koff rate constant of 1 x 10- 3 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 alanine substitution 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 alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at 10 positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

10. The isolated human antibody of claim 9, or an antigen-binding portion thereof, which dissociates from human TNFa with a Koff rate constant of 5 x 10- 4 s-I or less.

11. The isolated human antibody of claim 9, or an antigen-binding portion thereof, which dissociates from human TNFa with a Koff rate constant of I x 10 4 s'l 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 alanine substitution 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 of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.

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

14. The isolated human antibody, or an antigen-binding portion thereof, of claim 13, wherein the LCVR further has CDRI domain comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDRI domain comprising the amino acid sequence of SEQ ID NO: 8. -79- 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, which has an IgG1 heavy chain constant region. 10 17. The isolated human antibody of claim 15, which has an IgG4 heavy chain constant region.

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

19. The isolated human antibody of claim 15, which is a single chain Fv fragment. An isolated human antibody, or an antigen-binding portions thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid 20 sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, 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 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 and SEQ ID NO: 34.

21. A recombinant human antibody, or antigen-binding portion thereof, that neutralizes the activity of human TNFa but not human TNF.

22. The recombinant human antibody, or antigen-binding portion thereof. of claim 21, which also neutralizes the activity of chimpanzee TNFa and at least one additional primate TNFa selected from the group consisting of baboon TNFa, marmoset TNFa, cynomolgus TNFa and rhesus TNFa.

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

24. The recombinant human antibody, or an antigen-binding portion thereof, of claim 22, which also neutralizes the activity of pig TNFa. An isolated nucleic acid encoding 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 alanine substitution at position 1, 4, 5, 7 or 8, or by one to five conservative amino acid 10 substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9.

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

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

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

29. An 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 alanine substitution 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, 9, 10, 11 and/or 12. The isolated nucleic acid 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 antibody HCVR comprises the amino acid sequence of SEQ ID NO: 6.

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

33. An isolated nucleic acid encoding a CDR3 domain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO 4, -81 SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: SEQ ID NO: 16. SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.

34. An isolated nucleic acid encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 1. 10 35. The isolated nucleic acid of claim 34, which encodes the antibody light chain.variable region and an antibody light chain constant region.

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

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

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

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

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

41. The isolated nucleic acid of claim 38, which is in 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. -82-

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

44. A method of synthesizing a human antibody that binds human TNFa, comprising culturing the host cell of claim 43 in a culture medium until a human antibody that binds human TNFa is synthesized by the cell. A pharmaceutical composition comprising the antibody, or antigen- binding portion thereof, of any of claims 1-24, and a pharmaceutically acceptable carrier. o. 46. The pharmaceutical composition of claim 45, which further comprises at least one additional therapeutic agent for treating a disorder in which TNFa activity is detrimental.

47. A method for inhibiting human TNFct activity comprising contacting human TNFa with the antibody, or antigen-binding portion thereof, of any of claims 1-24 such that human TNFc activity is inhibited. 20 48. A method for inhibiting human TNFa activity in a human subject suffering from a disorder in which TNFa activity is detrimental, comprising administering to the human subject the antibody, or antigen-binding portion thereof, of any of claims 1-24 such that human TNFa activity in the human subject is inhibited.

49. The method of claim 48, wherein the disorder is sepsis. The method of claim 49, wherein the antibody is administered to the human subject together with the cytokine interleukin-6 (IL-6) or is administered to a human subject with a serum or plasma concentration of IL-6 above 500 pg/ml.

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 spondylitis. osteoarthritis and gouty arthritis. -83

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. The method of claim 48, wherein the disorder is transplant rejection or graft-versus-host disease. 10 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. S 58. The method of claim 48, wherein the disorder is an intestinal disorder.

59. The method of claim 48, wherein the disorder is a cardiac disorder. 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, fulminant hepatitis, coagulation disturbances, burns, reperfusion injury, keloid formation, scar tissue formation, pyrexia, periodontal disease, obesity and radiation toxicity.

61. Use of the antibody, or antigen-binding portion thereof, of any of claims 1-24 in the manufacture of a medicament for the treatment of a a disorder in which TNFo activity is detrimental.

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

63. The use of claim 62, wherein the antibody is administered to the human subject together with the cytokine interleukin-6 (IL-6) or is administered to a human subject with a serum or plasma concentration of IL-6 above 500 pg/ml.

64. The use of claim 61, wherein the disorder is an autoimmune disease. -84- The use of claim 64, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and Poutv 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 transplant rejection or graft- versus-host disease.

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

72. The use of claim 61, wherein the disorder is a 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 hepatitis, coagulation disturbances, bums. reperfusion injury keloid formation, scar tissue formation, pyrexia, periodontal disease, obesity and radiation toxicity.

74. The pharmaceutical composition of claim 46, wherein the additional therapeutic agent is selected from the group consisting of non-steroidal anti- inflammatory drugs, cytokine suppressive anti-inflammatory drugs, CDP-571/BAY-10- 3356, cA2, 75 kdTNFR-IgG 55 kdTNFR-IgG, IDEC-CE9.1/SB 210396, DAB 486-IL- 2, DAB 389-IL-2, Anti-Tac, IL-4, IL-O10, IL-4 agonists, IL-O10 agonists, IL-IRA, TNF- bp/s-TNFR, S284, R973401, MK-966, Iloprost, methotrexate, thalidomide, thalidomide- related drugs, leflunomide, tranexamic acid, T-614, prostaglandin El, Tenidap, Naproxen, Meloxicam, Piroxicam, Diclofenac, Indomethacin, Sulfasalazine, Azathioprine, ICE inhibitors, zap-70 inhibitors, Ick inhibitors, VEGF inhibitors, VEGF- 10 R inhibitors, corticosteroids, TNF-convertase inhibitors, anti-IL-12 antibodies, interleukin-11, interleukin-l1, interleukin-17 inhibitors, gold, penicillamine, chloroquine, hydroxychloroquine, chlorambucil, cyclophosphamide, cyclosporin, anti- .thymocyte globulin, anti-CD4 antibodies, CDS-toxins, orally-administered peptides, .collagen, lobenzarit disodium, Cytokine Regulating Agents HP228 and HP466, ICAM-I antisense phosphorothioate oligodeoxynucleotides, soluble complement receptor 1, prednisone, orgotein, glycosaminoglycan polysulphate, minocycline, anti-IL2R antibodies, marine lipids, botanical lipids, auranofin, phenylbutazone, meclofenamnic acid,. flufenamic acid, intravenous immune globulin, zileuton, mycophenolic acid, tacrolimus, sirolimus, amiprilose, cladribine, azaribine, budenoside, epidermal growth 20 factor, aminosalicylates, 6-mercaptopurine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL- 1 receptor antagonists, anti-IL-10 monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, glucuronide-conjugated prodrugs of prednisolone, dexamethasone or budesonide, dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide, soluble complement receptor 1, slow- release mesalazine, antagonists of Platelet Activating Factor (PAF), ciprofloxacin, lignocaine, prednisolone, methylprednisolone, cyclophosphamide, 4-aminopyridine, tizanidine, interferon-pla, interferon-pl b, Copolymer 1, hyperbaric oxygen, intravenous immunoglobulin, clabribine, hypertonic saline solutions, antibiotics, continuous hemofiltration, carbapenems, antagonists of cytokines such as TNFa, IL-I, IL-6 and/or IL-8, SK&F 107647, tetrava'ent guanylhydrazone CNI-1493, Tissue Factor Pathway Inhibitor, PHP, iron chelators and chelates, including diethylenetriamine pentaacetic acid iron (III) complex, lisofylline, PGG-Glucan, apolipoprotein A-I reconstituted with lipids, chiral hydroxamic acids, anti-endotoxin antibodies, E5531, rBPI 2 1 Synthetic Anti-Endotoxin Peptides, surfactant replacement therapy and anti-IL-8 antibodies. -86- Use of the antibody, or antigen-binding portion thereof, of any of claims 1-24 in therapy.

76. Use of the antibody, or antigen-binding portion thereof, of any of claims 1-24 in therapy in combination with at least additional therapeutic agent for treating a disorder in which TNFa activity is detrimental.

77. The use of claim 76, wherein the additional therapeutic agent is selected from the group consisting of non-steroidal anti-inflammatory drugs, cytokine suppressive anti- inflammatory drugs, CDP-571/BAY-10-3356, cA2, 75 kdTNFR-IgG, 55 kdTNFR-IgG, IDEC-CE9.1/SB 210396, DAB 486-IL-2, DAB 389-IL-2, Anti-Tac, IL-4, IL-10, IL-4 agonists, IL-10 agonists, IL-1RA, TNF-bp/s-TNFR, S284, R973401, MK-966, Iloprost, methotrexate, thalidomide, thalidomide-related drugs, leflunomide, tranexamic acid, T- 614, prostaglandin El, Tenidap, Naproxen, Meloxicam, Piroxicam, Diclofenac, Indomethacin, Sulfasalazine, Azathioprine, ICE inhibitors, zap-70 inhibitors, Ick inhibitors, VEGF inhibitors, VEGF-R inhibitors, corticosteroids, TNF-convertase inhibitors, anti-IL-12 antibddies, interleukin- 11, interleukin-13, interleukin-17 inhibitors, gold, penicillamine, chloroquine, hydroxychloroquine, chlorambucil, cyclophosphamide, cyclosporin, anti-thymocyte globulin, anti-CD4 antibodies, 20 toxins, orally-administered peptides, collagen, lobenzarit disodium, Cytokine Regulating Agents HP228 and HP466, ICAM-I antisense phosphorothioate oligodeoxynucleotides, soluble complement receptor 1, prednisone, orgotein, glycosaminoglycan polysulphate, minocycline, anti-IL2R antibodies, marine lipids, botanical lipids, auranofin, phenylbutazone, meclofenamic acid, flufenamic acid, intravenous immune globulin, zileuton, mycophenolic acid, tacrolimus, sirolimus, amiprilose, cladribine, azaribine, budenoside, epidermal growth factor, aminosalicylates, 6-mercaptopurine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-I receptor antagonists, anti-IL- 10 monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, glucuronide-conjugated prodrugs of prednisolone, dexamethasone or budesonide, dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide, soluble complement receptor 1, slow-release mesalazine, antagonists of Platelet Activating Factor (PAF), ciprofloxacin, lignocaine, prednisolone, methylprednisolone, cyclophosphamide, 4 -aminopyridine, tizanidine, interferon- I a, interferon-p Ilb, Copolymer 1, hyperbaric oxygen, intravenous immunoglobulin, clabribine, hypertonic saline solutions, antibiotics, continuous hemofiltration, -87- carbapenems, antagonists of cytokines such as TNFa, IL-11P, IL-6 and/or IL-8, SK&F 107647, tetravalent guanyihydrazone CNI- 1493, Tissue Factor Pathway Inhibitor, PHP, iron chelators and chelates, including diethylenetiarine pentaacetic acid iron (III) complex, lisofylline, PGG-Glucan, apolipoprotein A- I reconstituted with lipids, chiral hydroxamic acids, anti-endotoxin antibodies, E553 1, rBP1 2 1 Synthetic Anti-Endotoxin Peptides, sur-factant replacement therapy and anti-IL-S antibodies. DATED this 20th Day of October 2000 BASF AKTIENGESELLSCHAFT Attorney: IVAN A. RAJKOVIC Fellow Institute of Patent and Trade Mark Attorneys of Australia of BALDWIN SHELSTON WATERS