AU2006241387A1 - Human antibodies that bind human TNFalpha - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant/s: Actual Inventor/s: Abbott Biotechnology Ltd 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 Tristan J. Vaughan and Michael White and Alison J. Wilton and David Schoenhaut Address for Service is: SHELSTON IP Margaret Street SYDNEY NSW 2000 CCN: 3710000352 Attorney Code: SW Telephone No: Facsimile No.

(02) 9777 1111 (02) 9241 4666 Invention Title: HUMAN ANTIBODIES THAT BIND HUMAN TNFalpha Details of Original Application No. 2004202769 dated 23 Jun 2004 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 29786AUP02 501029188 1.DOC/5844

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S- la- HUMAN ANTIBODIES THAT BIND HUMAN TNFca Z The present application relates to TNFa and more particularly to antibodies binding Sto human TNFa. This application isa divisional application of Australian Application No., 2004202769, which is incorporated in its entirety herein by reference.

Tumor necrosis factor a. (TNFa) is a cytokine produced by numerous cell types, M including monocytes and macrophages, that was originally identified based on its Scapacity to induce the necrosis of certain mouse tumors (see Old, L. (1985) Sciencei C" 230:630-632). Subsequently, a factor termed cachectin, associated with cachexia, was Sshown 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 arid graft-versus-host disease (see 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 B I 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 TNFcL (hTNFa) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract hTNFa activity. In particular, antibodies that bind to, and neutralize, hTNFca have been sought as a means to inhibit hTNFct activity. Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of niice immunized with hTNFa (see Hahn T; et al., (1985) Proc Natl AcadSci USA 82: 3814-3818: Liang, et al. (1986) Biochem. Biophys. Res.

S Commun. 137:847-854; Hirai, et al. (1987) Immunol. Methods 96:57-62; Fendly, et al. (1987) Hybridoma 6:359-370; Moeller, e al. (1990) Cytokine 2:162- 169; U.S. Patent No. 5,231,024 to Moeller et al.; European Patent Publication No. 186 833 B by Wallach. European Patent Application Publication No. 218 868 Al by Old et al.: European Patent Publication No. 260 610 B1 by Moeller, et While these mouse anti-hTNFa antibodies often displayed high affinity for hTNcFa Kd 9 M) and were able to neutralize hTNFa activity, their use in vivo may be limited by problems associated with administration of mouse antibodies to hu'mans, 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-hTNFac antibodies have been genetically engineered

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0 0 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 C1 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 00 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 CI No. WO 92/11383 by Adair, et However, because these chimeric and INO humanized antibodies still retain some murine sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, r C 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 hTNFoa inhibitory agent to murine mAbs or derivatives thereof 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 hTNFc and were unable to neutralize hTNFac-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 hTNFo.

Certain studies have detected serum autoantibodies against hTNFa 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 hTNFca antibody. Recombinant human antibodies that bind hTNFa with relatively low affinity Kd -10- 7 M) and a fast off rate Koff- 10- 2 sec- have been described (Griffiths, et al. (1993) EMBOJ. 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 O 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.) C' Accordingly, human antibodies, such as recombinant human antibodies, that bind soluble hTNFc with high affinity and slow dissociation kinetics and that have the capacity to neutralize hTNFa activity, including hTNFa-induced cytotoxicity (in vitro 0 0 and in vivo) and hTNFa-induced cell activation, are still needed.

C Summary of the Invention NO This invention piovides human antibodies, preferably recombinant human antibodies, that specifically bind to human TNFa. The antibodies of the invention are C characterized by binding to hTNFc 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 hTNFct but not hTNFP (lymphotoxin) and by having the 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 IgG1 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 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 TNFc with a Kd of l 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, 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 1 x 10- 4 s- 1 or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes -human TNFc cytotoxicity in a standard in vitro L929 assay with an IC 50 of 1 x 10- 8

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or less, even more preferably with an IC 50 of 1 x 10- 9 M or less and still more preferably with an ICs 0 of 5 x 10-10 M or less.

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Z In another embodiment, the invention provides a human antibody, or antigen- -binding portion thereof, with the following characteristics: a) dissociates from human TNFca with a K o ff 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 00 M 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; N 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 TNFc with a Koff of 5 x 10- 4 s-1 or less. Still more preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFcL 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

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SNO: 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 Ci 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, 00 SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: F" In yet another embodiment, the invention provides an isolated human antibody, O or antigen-binding portion thereof, that neutralizes the activity of human TNFc but not human TNFP (lymphotoxin). In a preferred embodiment, the human antibody, or C1 antigen-binding portion thereof,-neutralizes the activity of human TNFa, chimpanzee TNFc and at least one additional primate TNFa selected from the group consisting of baboon TNFct, marmoset TNFa, cynomolgus TNFc and rhesus TNFa. Preferably, the antibody also neutralizes the activity of at least one non-primate TNFa. For example, in one subembodiment, the isolated human antibody, or antigen-binding portion thereof, also neutralizes the activity of canine TNFc. 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.

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.

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- TNFcL 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 TNFc activity is detrimental such that human TNFa activity in the human subject is inhibited. The disorder can be, for example, sepsis, an autoimmune -6- 0 disease rheumatoid arthritis, allergy, multiple sclerosis, autoimmune diabetes.

Z autoimmune uveitis and nephrotic syndrome), an infectious disease, a malignancy, c=KI transplant rejection or graft-v'ersus-host disease, a pulmonary disorder, a bone disorder, an intestinal disorder or a cardiac disorder.

00 Brief Description of the Drawings Figures I A and IlB 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 IND 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 CK1 VL; also shown in SEQ ID NO:-9) and other D2E7-related light chain variable regions (EP B 12, \'LlIOE4, VL.1 00A9, VL IOOD2, VL1IOF4, LOES, VLLOF9, VLL0F VLLOG7, VLLOG9, VLLOHI, VLLOH 10, VLl B7, VLIC 1, VL1C7, VLO.I F4, VLO. 1H8, LOE7, LOE7.A and LOE7.T). Figure IlA shows the FRl1, CDRI1, FP.2 and CDR2 domains. Figure lB shows the FR.3, CDR3 and FR4 domains. The light chain CDR1 ("CDR CDR2 ("CDR L2") and CDR.3 ("CDR U3") domains are boxed.

Figures 2A and 2B show the amnino 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*.Al, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4, HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, IHD2E7*.A8 and HD2E7*.A9), the heavy chain variable region of the D2E7-related antibody 2SD4 (2SD4 VH; als6 shown in SEQ ID NO: and other D2E7-related heavy chain variable regions (VI-1lBI 11, VH1ID 8, VI IA 11, VH12, VHI-D2, VIE4, VH1F6, VHIG1, 3C-H2, VH1-D2.N and VHI-D2.Y).

Figure 2A shows the FRl, CDRI, FR2 and CDR,2 domains. Figure 2B shows the FR3, CDR3 and FR4 domains. The heavy chain CDR1 ("CDR Hl') CDR.2 ("CDR H2") and CDR3 ("CDR H3Y) domains are boxed.

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

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

Figure 5 is a graph depicting the inhibition of TNFci-induced ELAM- I expression on HUVEC by the human anti-hTNFcx antibody D2E7, as compared to the murine anti-hTNFcr. antibody MAK 195.

Figure 6 is a bar gr-aph depicting protection from TNFoa-induced lethality in

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S-7- 0 D-galactosamine-sensitized mice by administration of the human anti-hTNFa antibody D2E7 (black bars), as compared to the murine anti-hTNFa antibody MAK 195 (hatched Cbars).

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 00 CDR L1, CDR L2 and CDR L3 regions are underlined.

Figure 8 shows the nucleotide sequence of the heavy chain variable region of C" D2E7, with the predicted amino acid sequence below the nucleotide sequence. The \0 CDR HI, CDR H2 and CDR H3. regions are underlined.

Figure 9 is a graph depicting the effect of D2E7 antibody treatment on the mean C1 joint size of Tgl97 transgenic mice as a polyarthritis model.

Detailed Description of the Invention This invention pertains to isolated human antibodies, or antigen-binding portions 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 hTNFca, or simply hTNF), as used herein, is intended to refer to a human cytokine that exists as a 17 kD secreted form 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 TNFc is intended to include recombinant human TNFc (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

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-8- 0 Z region. The heavy chain constant region is comprised of three domains. CH1, 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 5 regions of hypervariability, termed complementarity determining regions (CDR), 00 C 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 aminoc N terminus to carboxy-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that 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 "antigenbinding 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 CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et 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, RJ., 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 0 0 -9- Z 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 5 polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et 00 eC 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 C as papain or pepsin digestion. respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard S 10 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 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.

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 (describedfurther 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.

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0 7Z An "isolated antibody", as used herein, is intended to refer to an antibody that is j substantially free of other antibodies having different antigenic specificities an isolated antibody that specifically binds hTNFa is substantially free of antibodies that specifically_bind antigens other than hTNFa). An isolated antibody that specifically b 5 binds hTNFL may, however, have cross-reactivity to other antigens, such as TNFa 00 M molecules from other species (discussed in further detail below). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

C( A "neutralizing antibody", as used herein (or an "antibody that neutralized O hTNFa activity"), is intended to refer to an antibody whose binding to hTNFa results in inhibition of the biological activity of hTNFa. This inhibition of the biological activity of hTNFa can be assessed by measuring one or more indicators of hTNFca biological activity, such as hTNFac-induced cytotoxicity (either in vitro or in vivo), hTNFa-induced cellular activation and hTNFa binding to hTNFa receptors. These indicators of hTNFc biological activity can be assessed by one or more of several standard in vitro or in vivo 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 hTNFca-induced expression of ELAM-1 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 using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Example I and Jbnsson, U, et al. (1993) Ann. Biol.

Clin. 51:19-26; J6nsson, 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 "Kof", 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.

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-11 Z is intended to refer to a nucleic acid molecule in which the nucleotide sequences 4 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 5 example, an isolated nucleic acid of the invention encoding a VH region of an anti- 00 C TNFa antibody contains no other sequences encoding other VH regions that bind antigens other than TNFca.

C 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 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 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.

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-12- 0 1 I. Human Antibodies that Bind Human TNFa This invention provides isolated human antibodies, or antigen-binding portions

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1 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-hTNFc antibodies. The most preferred recombinant, 00 00 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 N sequence of the D2E7 VH region is also shown in SEQ ID NO: The binding 010 properties of D2E7, as compared to the murine anti-hTNFc 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-

1 sec- 1 M metry D2E7 IgGI 8.81 x 10-5 1.91 x 105 6.09 x 10-10 .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 10 5 4.60 x 10-10 1.4 The D2E7 antibody, and related antibodies, also exhibit a strong capacity to neutralize hTNFc activity, as assessed by several in vitro and in vivo assays (see Example For example, these antibodies neutralize hTNFa-induced cytotoxicity of L929 cells with IC 50 values in the range of about 10- 7 M to about 10- 1 0 M. D2E7, when expressed as a full-length IgGl antibody, neutralizes hTNFa-nduced cytotoxicity of L929 cells with IC 5 o 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 TNFac-induced cellular activation, as measured by hTNFa-induced ELAM-1 expression on HUVEC (IC 50 about 1.85 x 10- 1 0 and binding of hTNFa to hTNFc receptors on U-937 cells (IC56 about 1.56 x 10- 1 0 Regarding the latter, D2E7 inhibits the binding of hTNFac 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 TNF in various forms, including soluble hTNFa, transmembrane hTNFc and hTNFa. bound to cellular receptors. D2E7 does not specifically bind to other cytokines, such as lymphotoxin (TNFP), IL-la, IL-1p, IL-2, IL-4, IL-6, IL-8, IFN-y and TGFp. However,

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-13- 0 Z 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 IC50 values as for neutralization of hTNFa (see Example 4, subsection E).

5 D2E7 also neutralizes the activity of mouse TNFa, although approximately 1000-fold 00 C less well than human TNFa (see Example 4, subsection D2E7 also binds to canine and porcine TNFa.

C In one aspect, the invention pertains to D2E7 antibodies and antibody portions, D2E7-related antibodies and antibody portions, and other human antibodies and 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, and neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an 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 Kff of 5 x 10- 4 s- 1 or less, or even more preferably, with a Koff of 1 x 10- 4 s I or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes human TNFc cytotoxicity in a standard in vitro L929 assay with an IC50 of I x 10-8 M or less, even more preferably with an 1050 of 1 x 10- 9 M or less and still more preferably with an IC50 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 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 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, 1B, 2A and 2B (see also Examples 2, 3 and 4 for kinetic and neutralization analyses): [D2E7 VH/D2E7 VL]; [HD2E7*.A/lD2E7 VL], [HD2E7'.A2/D2E7 VL], [HD2E7*.A3/D2E7 VL], 14- Z [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 VHILD2E7*.A8], [HD2E7*.A9/LD2E7*.A1], [VH1- 00 5 D2/LOE7], [VH1-D2.N/LOE7.T], [VH1-D2.Y/LOE7.A], [VHI-D2.N/LOE7.A], [VH1- 00 C D2/EP B12] and [3C-H2/LOE7]..

It is well known in the art that antibody heavy and light chain CDR3 domains 1 play an important role in the binding specificity/affinity of an antibody for an antigen.

0 Accordingly, in another aspect, the invention pertains to human antibodies that have 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 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 Sdomains 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 0 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 hTNFct.

As shown in Example 3, positions 2 and 5 of the D2E7 VL CDR3 and positions I and 7 00" of the D2E7 VH CDR3 appear to be critical for interaction with hTNFa and thus, 00 M 5 conservative amino acid substitutions preferably are not made at these positions (although an alanine substitution at position S of the D2E7 VL CDR3 is acceptable. as rI described above).

Accordingly, in another embodiment, the invention provides an isolated human antibody, or antigen-binding portion thereof, with the following characteristics: a) dissociates from human TNFc. 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 II 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 TNFct with a Koff of 5 x 10 4 s-I 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

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-16- 0 are from the V.I human germline family, more preferably from the A20 human CK1 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 0 0 5 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) ^O 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 the D2E7 VH). In certain embodiments. the antibody comprises a heavy chain constant region. such as an IgG, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgGI 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 TNFx but not human TNF(3. Preferably, antibody, or antigen-binding portion thereof.

also neutralizes the activity of chimpanzee TNFa and at least one additional primate TNFca selected from the group consisting of baboon TNFa, marmoset TNFca, cynomolgus TNFa and rhesus TNFa. Preferably, the antibody, or antigen-binding portion thereof, neutralizes the human, chimpanzee and/or additional primate TNFc in a

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-17- 0 Z standard 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 TNFta, preferably in a standard in vitro L929 assay with an IC 50 of 1 x 10 7 M or less, more preferably lx 10- 8 M or less and even more preferably 5 x 10- 9 M or less. In another subembodiment, the antibody also C neutralizes the activity of pig TNFa, preferably with an IC 50 of I 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 N embodiment, the antibody also neutralizes the activity of mouse TNFa, preferably with 0 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, 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 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

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s- 0 Z 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.

00 II. Expression of Antibodies M 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 O 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 and heavy chain variable regions are first obtained. These DNAs can be obtained by 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 germline sequence database; see also Kabat, et al. (199 )-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 V, 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 VH 3 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 VKI family of human germline VL genes is amplified by standard PCR. Most preferably, the A20 VL germline sequence is

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C> -19- Z amplified. 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 00 5 mutated to encode the D2E7 or D2E7-related amino acid sequences disclosed herein.

C

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 cN amino acid residues in the D2E7 or D2E7-related sequence that differ from germline.

O Then. the appropriate nucleotides of the germline DNA sequences are mutated such that C1 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 "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-encodiig 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 (CH1, 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 lmmunological Interest. Fifth Edition. U.S.

Department of Health and Human Services, NIH Publication No. 91-3242) and DNA

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0 fragments encompassing these regions can be obtained by standard PCR amplification.

C, The heavy chain constant region can be an IgG 1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGI or IgG4 constant region. For a Fab .fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another 00 5 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 s 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., C1 10 Kabat, 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 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. Nail.

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

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-21- Z 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 alternatively, 0 5 the recombinant expression vector can encode a signal peptide that facilitates secretion c 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 Santibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide a signal peptide from a non-immunoglobulin protein).

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.

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

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-22- Z include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of 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 00 5 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 i eukaryotic host cell, electroporation. calcium-phosphate precipitation, DEAE- O dextran transfection and the like. Although it is theoretically possible to express the Santibodies of the invention in either prokaryotic or eukaryotic host cells, expression of K 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. Nail. 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 iO 0 Z 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 0 5 the 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 Sthe 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 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 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 CDR1 domain of the LCVR encompasses nucleotides 70-102, the CDR2 domain encompasses nucleotides 148-168 and the CDR3 domain encompasses nucleotides 265- 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 O IND -24- Z or, 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 Camino 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 00 M 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 N substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino NO 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 CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 the D2E7 VH CDR1).

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, SSEQ 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 still another embodiment, the invention provides an isolated nucleic acid encoding an antibody light chain variable region comprising-the 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 nucleotide sequence of SEQ ID NO: 37, although the skilled artisan will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences can encode the amino acid sequence of SEQ ID NO: 2. The nucleic acid can encode only the HCVR or

O

0 z can also encode a heavy chain constant region. operatively linked to the HCVR. For C example, the nucleic acid can comprise an IgG1 or IgG4 constant region. In one embodiment, this nucleic acid is in a recombinant expression vector.

The invention also provides recombinant expression vectors encodingboth an 00 5 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 \0 sequence of SEQ ID NO: 1 the D2E7 LCVR); and b) an antibody heavy chain having a variable region comprising the amino acid 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 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.

Ill. 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 SurJZAPTM 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 91117271; 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; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) lio/Technology 9:1370-1372; Hay et al. (1992)

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-26- 0 Z Hum Antibod Hybridomas 3:81-85; Huse er al. (1989) Science 246:1275-128i; McCafferty et al., Nature (1990) 48:552-554; 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 Acid Res 19:4133-4137; 00 and Barbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with high affinity and a C low off rate constant for hTNFa, a murine anti-hTNFa antibody having high affinity O aind a low off rate constant for hTNFar MAK 195,the hybridoma for which has 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 p_repared-and-screened-as-described-inMcCaffert.y-et-aL -ECE-ublication-NoW-- 92/01047, McCafferty et al., Nature (1990) 348:552-554; and Griffiths el al., (1993) EMBO J 12:725-734. The scFv antibody libraries-preferably are screened using recombinant human TNFct 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 screened for hTNFac binding, are performed to select preferred VL/VH pair combinations. Additionally, to further improve the affinity and/or lower the off rate constant for hTNFc 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 hTNFa 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 (N 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 0 0 5 "germlining") of framework residues can be accomplished by standard molecular Sbiology methods for introducing specific mutations site-directed mutagenesis; PCR-mediated mutagenesis. and the like).

O Following screening and isolation of an anti-hTNFot antibody of the invention Sfrom a recombinant immunoglobulin display library, nucleic acid encoding the selected 1 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 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,

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K .1 -28 0 Z liposomes 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 o0 5 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 I\D intramuscular or subcutaneous injection.

STherapeutic compositions typically must be sterile and stable under the conditions C 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 preparation 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 ofsurfactants. 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 an. See, e.g..

0 (O 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 S" orally administered, for example, with an inert diluent or an assimilable edible carrier.

M 5 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 1 diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, 0elixirs, suspensions, syrups, wqfers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the 4 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 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 TNFc 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 theinvention may be used in combination with two or more of the foregoing therapeutic agerts. 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; CelltechlBayer); 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. 4, 235A); 55 kdTNFR- IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB S210396 (non-depleting.primatized anti-CD4 antibody; IDEC/SmithKline; see e.g.,

(O

0 Z Arthritis Rheumatism (1995) Vol. S185); DAB 486-IL-2 and/or DAB 389-1L-2 c, (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, anti- 00 5 inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists agonist

C

antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF binding protein; see Arthritis Rheumatism (1996) Vol. 39, No. 9 S, (supplement), S284; Amer. J. Physiol. Heart and Circulatory Physiology (1995) Vol.

O 268 pp. 37-42); R973401 (phosphodiesterase Type IV inhibitor; see Arthritis c-,1 10 Rheumatism (1996) Vol. 9, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g.,Arthritis Rheumatism (1996) Vol. 3, No. 9 (supplement), S81); Iloprost (see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S82); methotrexate: thalidomide (see Arthritis Rheumatism (1996) Vol. 39, No. 9 (supplement), S282) and thalidomide-related drugs Celgen); leflunomide (anti-inflammatory and 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. 29, No. 9 (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-1 P converting enzyme); zap-70 and/or Ick inhibitor (inhibitor of the tyrosine kinase zap-7 0 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); corticosteroid anti-inflammatory drugs SB203580);

TNF-

convertase inhibitors; anti-IL-12 antibodies; interleukin- 1 (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-17 inhibitors (see Arthritis Rheumatism (1996) Vol. 39. No. 9 (supplement), S120); Z gold: penici Ilamine; chloroquine: hNvdroxvchloroquine: chlorambucil; cyclophospharnide. cyclosporine; total lymphoid irradiation; anti-thvmocyte globulin; anti-CD4 antibodies; CD5-toxins;, oral ly-admini stered peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and H-P466 (1-oughten 00 5 Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor I (TP 10; T Cell Sciences. Inc.); prednisone: orgotein; glycosaminoglycan polysulphate; minocvcline; 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; meclofenamnic acid; flufenamnic acid; intravenous immune globulin;, zileuton; mycophenolic acid (RS 6 1443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxvadenosine); 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 following: budenoside; epidermal growth factor; corticosteroids; cyclosporin, sulfasaazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalainine; olsalazine; balsalazide; antioxidants; thrornboxane inhibitors;, IL- I receptor antagonists; anti-IL- I P3 monoclonal antibodies; anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; CDP-57lIBAY-10-3356 (humanized anti-TNFa antibody; Celltech/B aver); 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. 23 5A); 55 kdTNFR-IgG (55 kD TNF receptor-I gG fusion protein; Hoffmann-LaRoche); interleukin- 10 (SCH 52000; Schering Plough); IL-4; IL.-10 and/or ILA4 agonists agonist antibodies); interleukin-l 11; glucuronide- or dextran-conjugated prodrugs of prednisolone, dexaniethasone or budesonide; ICAM-l antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor I (TP 10; T Cell Sciences, Inc.); slow-release mesalazine; methotrexate; antagonists of Platelet Activating Factor (PAF); 30 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-arninopyridine; tizanidine; interferon-P3 I a (AvoneX

T

M;

Biogen); interferon-jllb (BetaseronTM; Chiron!Berlex); Copolymer I (Cop-I; CopaxoneTM; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous

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C> 32- Z immunoglobulin; clabribine; CDP-571/BAY-10-3356 (humanized anti-TNFc 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 00 5 receptor-IgG 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 ND antibody portion, of the invention can be combined include the following: hypertonic O saline solutions; antibiotics; intravenous gamma globulin; continuous hemofiltration; C1 10 carbapenems meropenem); antagonists of cytokines such as TNFa, IL- 13, IL-6 and/or IL-8; CDP-571/BAY-10-3356 (humanized anti-TNFc antibody; Celltech/Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-lgG (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; Hoffinann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular peptide; SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (Picower Institute); Tissue Factor Pathway Inhibitor (TFPI; Chiron); PHP (chemically modified hemoglobin; APEX Bioscience); iron chelators and chelates, including diethylenetriamine pentaacetic acid iron (III) complex (DTPA iron (III); Molichem Medicines); lisofylline (synthetic small molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan (aqeuous soluble 31,3glucan; Alpha-Beta Technology); apolipoprotein A- reconstituted with lipids; chiral hydroxamic acids (synthetic antibacterials that inhibit lipid A biosynthesis); antiendotoxin 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-TNFac antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-lgG 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).

0

INO

Z 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 5 portion of the invention. A "therapeutically effective amount" refers to an amount 00 0 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 Saccording to factors such as the disease state, age, sex, and weight of the individual, and O the ability of the antibody or antibody portion to elicit a desired response in the S 10 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 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 asunitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosageunit 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

ID

0 -34- Z supervising the administration of the compositions, and that dosage ranges set forth 4j herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

5 IV. Uses of the Antibodies of the Invention 00 M Given their ability to bind to hTNFa, the anti-hTNFa antibodies, or portions thereof, of the invention can be used to detect hTNFo in a biological sample, such C1 as serum or plasma), using a conventional immunoassay, such as an enzyme linked I 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 hTNFac in the biological sample. The antibody is directly or indirectly labeled with a detectable 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, 13. 35S or 3

H.

S Alternative to labeling the antibody, hTNFc can be assayed in biological fluids by a competition immunoassay utilizing rhTNFca standards labeled with a detectable substance and an unlabeled anti-hTNFa antibody. In this assay, the biological sample, the labeled rhTNFra 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 hTNFc 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, Smarmoset, cynomolgus and rhesus), pig and mouse, since D2E7 can bind to each of these TNFcs (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

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0 the antibodies of the invention. such as D2E7. can neutralize TNFat activity from other C, species. _Accordingly, the antibodies and antibody portions of the invention can be used to inhibit TNFa activity, in a cell culture containing hTNFca. in human subjects or in other mammalian subjects having TNFas with which an antibody of the invention 0 0 5 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 \0 invention such that TNFt activity is inhibited. Preferably, the TNFoa is human TNFca.

For example, in a cell culture containing, or suspected of containing hTNFa, an 10 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 TNFao activity in a subject suffering from a disorder in which TNFca activity is detrimental.

TNFa has been implicated in the pathophysiology of a wide variety of disorders (see Moeller, el 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, the TNFa is human TNFc 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 hTNFa by administration of hTNFc or by expression of an hTNFt 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 0

ON

-36- 0 Z symptoms 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 5 anti-TNFca antibody as described above. There are numerous examples of disorders in 00 CM which TNFa 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 clotting cascade (see 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, 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 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 ofplatelet activating factor (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 pg/ml. 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, TNFca has been implicated in activating tissue inflammation and causing joint destruction in rheumatoid

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-37- 0 arthritis (see Moeller, et al. (1990) Cvtokine 2:162-169; U.S. Patent No.

'1 5,231,024 to Moeller et al.; European Patent Publication No. 260 610 BI by Moeller, Tracey 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 00 5 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 O induction of inflammatory plaques in multiple sclerosis (see Tracey and Cerami, 0 supra). Chimeric and humanized murine anti-hTNFc antibodies have undergone C"1 10 clinical testing for treatment of rheumatoid arthritis (see Elliott, et al. (1994) Lancet 344:1125-1127; Elliot, Mi.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 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 adrhinistered 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 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, TNFa 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

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O -38malaria, AIDS and AIDS-related complex (ARC) (see European Patent Application l 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 0 0 5 symptoms associated with infectious diseases, including fever and myalgias due to' infection (such as influenza) and cachexia secondary to infection secondary to C AIDS or ARC).

D. Transplantation 1 10 Tumor necrosis factor has been implicated as a key mediator of allograft rejection and graft versus hostdisease (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.

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 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-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 In yet another embodiment, an antibody or antibody portion of the invetion 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.

0 0

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-39- SF. Pulmonary Disorders C1 Tumor necrosis factor has been implicated in the pathophysiology of adult respiratory distress syndrome (ARDS), including stimulating leukocyte-endothelial I> activation, directing cytotoxicity to pneumocytes and inducing vascular leakage 0 0 5 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 O 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 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.

(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).

I. Others The antibodies, and antibody portions, of the invention, also can be used to treat various other disorders in which TNFcL activity is detrimental. Examples of other diseases and disorders in which TNFa activity has been implicated in the

CO

0-40- 0 Z 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 Bertolini. et al. (1986) Nature 319:516-518; Konig, et al. (1988) J. Bone Miner. Res. 3:621-627; Lerner, U.H. and Ohlin, A. (1993)J. Bone Miner. Res. 8:147- 0 5 155; and Shankar. G. and Stem, P.H. (1993) Bone 14:871-876), hepatitis, including S. 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.

O 12:241-245; and Hussain, et al. (1994) J. Clin. Pathol. 47:1112-1115), and 10 fulminant hepatitis: coagulation disturbances (see van der Poll, et al. (1990) N.

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

367:55-60), bums (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 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 exanples 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 hTNFc Real-time binding interactions between ligand (biotinylated recombinant human TNFct (rhTNFa) immobilized on a biosensor matrixJ 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.

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0 -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) 00 and 400 mM N-ethyl-N'-(3-diethylaminopropyl) carbodiimide hydrochloride (EDC).

C 5 Next, streptavidin is injected across the activated matrix. Thirty-five microliters of streptavidin (25 lg/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 Sethanolamine. Streptavidin-coupled biosensor chips also are commercially available (Pharmacia BR-1000-16, Pharmacia Biosensor, Piscataway, NJ).

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

No. 1008 960) in 500 lil 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 rhTNF-biotin per column. The column was eluted with 10 x I 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, NY) supplemented with 0.05% (BIAcore) surfactant P20 (Pharmacia BR-1000-54, Pharmacia Biosensor, Piscataway, NJ). To determine the capacity of rhTNFa-specific antibodies to bind immobilized rhTNFa, a binding assay was conducted as follows.

Aliquots of biotinylated rhTNFc (25 nM; 10 1l aliquots) were injected through the streptavidin-coupled dextran matrix at a flow rate of 5 Rl/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 yalue (approximately 500 RU). Direct rhTNFa-specific antibody binding to immobilized biotinylated rhTNFa was measured. Antibodies (20 pg/m) were diluted in PBS running buffer and 25 p.i aliquots were injected through the immobilized protein matrices at a flow rate of 5 l.l/min. Prior to injection of antibody, and immediately afterwards, PBS buffer alone flowed through each flow cell. The net difference in -42baseline 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, BIAcore kinetic evaluation software (version 2.1) was used.

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

in T,1 1.I. R;n;ngo f D2E7 loG4 or MAK 195 to Biotinvlated rhTNFc 1 U rhTNFca, Ab, bound, Koff, sec- 1 Antibody nM bound. RUs RUs rhTNFca/Ab (Avg) D2E7 267 373 1215 1.14 8.45 x 10- 133 420 1569 1.30 5.42 x 10- 67 434 1633 1.31 4.75 x 33 450 1532 1.19 4.46 x 17 460 1296 0.98 3.47 x 10 8 486 936 0.67 2.63 x 10- 4 489 536 0.38 2.17 x 10- 2 470 244 0.18 3.68 x (4.38 x 10-5) MAK 195 400 375 881 1.20 5.38 x 10- 200 400 1080 1.38 4.54 x 10- 100 419 1141 1.39 3.54x 10- 427 1106 1.32 3.67 x 10- 3 446 957 1.09 4.41 x 10- 13 464 708 0.78 3.66 x 10- 6 474 433 0.47 7.37 x 10 3 451 231 0.26 6.95 x (4.94 x 10- 5 In a second series of experiments, the molecular kinetic interactions between an IgGI full-length form of D2E7 and biotinylated rhTNF was quantitatively analyzed using BlAcore technology, as described above, and kinetic rate constants were derived.

summarized below in Tables 2. 3 and 4.

-43 Table 2: Apparent dissociation rate constants of the interaction between D2E7 and biotinvlated rhTNF Experiment Kd (s' 1 1 9.58 x 10- 2 9.26 x 10-5 3 7.60 x 10-5 Average 8.81 1.06 x 10- Table 3: Apparent association rate constants of the interaction between D2E7 and biotinvlated rhTNF Experiment I Ka s- 1 1 1.33 x 105 2 1.05 x 3 3.36 x 105 Average 1.91 1.26 x 105 Table 4: Apparent kinetic reate and affinity constants of D2E7 and biotinvlated rhTNF Experiment Ka K

K

d

(M)

1 1.33 x 105 9.58 x 10- 5 7.20 x 10-10 2 1.05 x 105 9.26 x 10-5 8.82 x 10-10 3 3.36 x 105 7.60 x 10-5 2.26 x 10-10 Average 1.91 1.26 x 105 8.81 1.06 x 10- 5 6.09 t 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 biotinylated rhTNF was 8.81 1.06 x 10- 5 and the average apparent association rate constant, k, was 1.91 1.26 x 105 M' The apparent intrinsic dissociation constant (Kd) was then calculated by the formula: Kd= kd/ Thus, the mean Kd of D2E7 antibody for rhTNF

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-44- 0 derived from kinetic parameters was 6.09 3.42 x 10-10 M. Minor differences in the C. kinetic values for the IgGI form of D2E7 (presented in Tables 2, 3 and 4) and the IgG4 form of D2E7 (presented in Table 1 and in Examples 2 and 3) are not thought to be true differences resulting from the presence of either an IgG or an IgG4 constant regions but 0 0 5 rather are thought to be attributable to more accurate antibody concentration Smeasurements used for the IgG1 kinetic analysis. Accoringly, the kinetic values for the IgG1 form of D2E7 presented herein are thought to be the most accurate kinetic INO parameters for the D2E7 antibody.

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C 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 of rhTNFc 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 rhTNFc was determined by surface plasmon resonance as described in Example 1. The Koff rates for the different VHIVL pairs are summarized below in Table Table 5: Binding of D2E7 Alanine-Scan Mutants to Biotinviated rhTNFcx VH VL Koff(sec') D2E7 VH D2E7 VL 9.65 x 10- HD2E7*.A1 D2E7 VL 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- A 27VL29 HD2E7*.A6 D2E7 VL 2.9 x 16- 4 I n 117- HD2E7*.A7 D2E7 VL HD2E7*.A8 D2E7 VL 3.1 x 10-4 HD2E7*.A9 D2E7 VL 8.1 x 10-4 D2E7 VH LD2E7*.A1 6.6 x 10-3 D2E7 VH LD2E7*.A3 NOT DETECTABLE D2E7 VH I LD2E7*.A4 1.75 x 10-4 D2E7 VH LD2E7*.A5 1.8 x 10-4 D2E7 VH LD2E7*.A7 1.4 x 10-4 D2E7 VH LD2E7*.A8 3.65 x 10-4 HD2E7*.A9 LD2E7*.A1 1.05 x 10-4 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 Koff and an alanine substitution at position 4 or 11 of D2E7 VH CDR3 gives an 8-fold faster Koff, indicating that these 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, or 11 of the D2E7 VH CDR3 domain still results in an anti-hTNFa antibody having a Koffof 1 x 10 3 sec- 1 or less.

EXAMPLE 3: Binding Analysis of D2E7-Related Antibodies A series of antibodies related in sequence to D2E7 were analyzed for their binding to rhTNFca, 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 1A and IB. 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 IgGI or IgG4 antibody or as a scFv) are summarized below in Table 6: Table 6: Binding of D2E7-Related Antibodies to Biotinvlated rhTNFct VH .VL Format Koff(sec D2E7 VH D2E7 VL IgGl/IgG4 9.65 x 10- -46- VHI-D2 LOE7 I gG/IgG4 7.7 x VH I-D2 LQE7 F scFv 4.6 x 104 VH I-D2.N LOE7.T IgG4 2.1 x VH1-D2.Y LOE7.A IgG4 2.7 x VH1-D2.N LOE7.A IgG4 3.2x 10-5 VH1-D2 EP B12 scFv 8.0 x 10-4 VH1-D2 2SD4 VL scFv 1.94 x 10- 3 3C-H2 LOE7 scFv 1.5 x 10-3 2SD4 VH LOE7 scFv. 6.07 x 10-3 2SD4 VH 2SD4 VL scFv 1.37 x 10-2 VHIAII 2SD4 VL scFv 1.34 x 10-2 VHI1B12 2SD4 VL scFv 1.01 x 10-2 VHIBI 1 2SD4 VL scFv 9.8 x 10-3 VH1E4 2SD4 VL scFv 1.59 x 10-2 VH1F6 2SD4 VL scFv 2.29 x 10-2 VHID8 2SD4 VL scFv 9.5 x 10-3 VH1GI 2SD4 VL scFv 2.14 x 10-2 2SD4 V- EP B12 scFv 6.7 x 10-3 2SD4 VH VLIOE4 scFv 9.6 x 10-3 2SD4 VH VLIOOA9 scFv 1.33 x 10-2 2SD4 VH VLI10OD2 scFv 1.41x 10-2 2SD4 VH VLIOF4 scFv 1.11x 10-2 2SD4 VH VLLOE5 scFv 1.16 x 10-2 2SD4 VH VLLOF9 scFv 6.09,x 103 2SD4VH VLLOF1O scFv 1.34 x 10-2 2SD4 VH VLLOG7 scFv 1.56 x 10-2 2SD4 VH VLLOG9 scFv 1.46 x 10-2 2SD4 VH VLLOHI scFv 1.17 x 10-2 2SD4 VH VLLOH10 scFv 1.12 x 10-2 2SD4 VH VLIB7 scFv 1.3 x 10-2 2SD4 VH VLIC7 scFv 1.36 x 10-2 2SD4 V j VLIC7 scFv .0 x 10-2 2SD4 VH VLO.1F4 scFv 1.76x 10-2 2SD4 VH VLO.1HS scFv 1.14 x 10-2 The slow off rates Koff 1 x 10- sec- 1 for ,11-length antibodies lgG 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 CDR3 can be occupied by either of these two residues without substantially affecting the Kof. Accordingly, a consensus motif for the D2E7 VL CDR3 comprises the amino acid sequence: (SEQ ID NO: Furthermore, the slow off rates KffrS I x 10-4 sec-1) for antibodies having a VH selected from D2E7. VHI-D2.N

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-47z and VH -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

00 5 IDNO:4).

SThe 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 ^0 sec- 1 as compared to antibodies containing the D2E7 VL or VH CDR3 region. Within Sthe 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 hTNFac. 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 hTNFat, a change at this position can be negated by changes at other positions, as seen in VLLOE4, VLLOH1 or VLO.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 hTNFca. 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).

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-48- 0 Z 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 00 5 or in vivo.

A. Neutralization of TNFa-Induced Cvtotoxicitv in L929 Cells 0 Human recombinant TNFa (rhTNFa) causes cell cytotoxicity to murine L929 Ci 10 tells after an incubation period of 18-24 hours. Human anti-hTNFa antibodies were evaluated in L929 assays by coincubation of antibodies with rhTNF and the cells as follows. A 96-well microtiter plate containing 100 il 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 500 pg/ml in each sample well. The plates were then incubated for 30 minutes at room temperature. Next, 50 pl ofTNFa-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-..

One hundred microliters of medium was removed from each well and 50 p1 of mg/ml 3,(4,4-dimethylthtazol-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 37* C. The optical density at 570/630 nm was measured, curves were plotted for each sample and IC 50 s 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/IgG1/IgG4 3.0 x 10-7 49 2SD4 -LOE7 IscFv 4.3 x 10-9 VHl*-D2 I 2SD4 IscFv 1.0Ox l0-8 VHI2LE scFvfIgGI/IgG4 3.4 x 10-10 VHI.D2.Y LOE7.T IgG4 8.1 x 10-11 VH I-D2.N LOE7.T IgG4 1.3 x 10-10 VI-D2.Y LOE7.A IgG4 2.8 x 10-11 VI-D2.N LOE7.A IgG4 6.2 x 10-11 MAK 195 MAK 195 scFv 1.9 x I o- MAK 195 MAK195 IF(ab')2 6.2 x 10I1 The results in Figure 3 and Table 7 demonstrate that the D2E7 human anti-hTNFax antibody, and various D2E7-related antibodies, neutralize ThiFa-induced L929 cytotoxicity with a capacity approximately equivalent to that of the murine anti-hTNFct mAb MAK 195.

In another series of experiments, the ability of the IgG I form of D2E7 to neutralize TN~a-induced L929 cvtotoxicity was examined as described above. -The results from three independent experiments, and the average thereof, are summnarized below in TableS8: Table 8: Neutralization of TNFa-lnduced L929 Cvtotoxicitv by D2E7 12GI Experiment IC 50

[M]

1 1l.26x 10-10 2 1.33.x 10-10 3 1.15 x10- 10 Average I1.25 iz0.01 x 10-10 This series of experiments confirmed that D2E7, in the full-length IgGI form, neutralizes TNFa-induced L929 cytotoxicity with an average IC 50 of 1.25 0.01 x 10-10.

B. Inhibition of fl'JFc Bindine to TNFa Recentors on U-937 Cells The ability of human anti-hTNFct antibodies to inhibit the binding of hTNFc to hTNFc receptors on the surface of cells was examined using the U-937 cell line (ATCC No. CRL 1 593), a human histiocviic cell line that expresses hTNFcx receptors. U-937

(O

5-50- Scells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone A-1111, Hyclone Laboratories, Logan, UT), L-glutamine (4 nM), HEPES buffer solution (10 mM), penicillin (100 Vg/ml) and streptomycin (100 pg/ml). To examine the activity of full-length IgG antibodies, U-937 cells were preincubated with 00 5 PBS supplemented with 1 mg/ml of human IgG (Sigma 1-4506, Sigma Chemical Co.. St.

Cc Louis, MO) for 45 minutes on ice arid then cells were washed three times with binding buffer. For the receptor binding assay, U-937 cells (5 x 106 cells/well) were incubated C in-a binding buffer (PBS supplemented with 0.2% bovine serum albumin) in 96-well microtiter plates (Costar 3799, Costar Corp., Cambridge, MA) together with 125 1-labeled rhTNFa (3 x 10- 1 0 M; 25 .iCilml; 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 210733, ICN, Irvine, CA). The test tubes contained a 300 ul mixture of dibutylphthalate and dinonylphthalate, 2:1 volume ratio, respectively. Free unbound) 125 1-labeled rhTNFc 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 1 25 -labeled rhTNFa bound 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 5 'value for D2E7 inhibition of hTNFa binding to hTNFa receptors on U-937 cells is approximately 3 x 10 M in these experiments. These results demonstrate that the D2E7 human antihTNFc antibody inhibits rhTNFa binding to hTNFa receptors on U-937 cells at concentrations approximately equivalent to that of the murine anti-hTNFc 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: 0

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-51- Table 9: Inhibition of TNF Receotor Bindine on U-937 Cells by D2E7 I eGl Extoeriment I5 M Average 0.12____x__10-10 This series of experiments confirmed that D2E7, in the full-length IgG I form, inhibits TNF receptor binding on U-937 cells with an average IC 50 of 1.56 0.12 10 -1o.

To investig-ate the inhibitokry potency of D2E7 in the binding of 125 1-rhTNF binding to individual p55 and p75 receptors. a solid phase radio immunoassay was performed. To measure the ICS 0 values of D2E7 for separate TNF receptors, varying concentrations of the antibody were incubated with 3 x 10 -10 concentration of 12S1rhTNF. The mixture was then tested on separate plates containing either the p55 or the TNF receptors in a dose dependent manner. The results are summarized below in Table Table 10: Inhibition of TNF Receptor Bindine to r)55 'and P75 TNFR by D2E7 IqGl

IC

50

[M]

Reaeent I p55 TNFR D2E7 1.47 x 10-9 J .6x 16-9 rhTNF 2.31 x 10-9 21.70 x 10-9 Inhibition of I25-rhTNF binding to the p55 and p75 TNF receptors on U937 cells by D2E7 followed a simple sigmoidal* curve, indicating similar IC 50 values for each receptor. In the solid phase radioimrnunoassay (RIA) experiments with recombinant TNF receptors, IC 50 values for inhibition of 25 1I-rhTNF binding to the p55 and the receptors. by D2E7 were calculated as 1.47 x 10Q- and 1.26 x 10o-9 M, respectively. The decrease in IC 50 values in the solid phase was probably due to higher density of receptors in the RIA format, as unlabeled rhTNF also inhibited with similar IC 5 0 values. The IC 50 values for inhibition of 1 25 I-rhTNF binding to the p5 *5 and the p75 receptors by unlabeled rhTNF were 2.31 x 10-9 and 2.70 x 10-9 M. respectively

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0 >-52- Z C. Inhibition of ELAM-1 Exoression 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 C with an mouse anti-human ELAM-1 antibody. The ability of human anti-hTNFa antibodies to inhibit this TNFa-induced expression of ELAM-1 on HUVEC was examined as follows: HUVEC (ATCC No. CRL 1730) were plated in 96-well plates x 10 4 cells/well) and incubated overnight at 37 The following day, serial dilutions of human anti-hTNFa antibody (1:10) were prepared in a microtiter plate, starting with 20-100 lg/ml of antibody. A stock solution of rhTNFa. was prepared at 4.5 ng/ml, aliquots of rhTNFca 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 37* 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- 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 temperature. An 1 25 1-labeled anti-mouse Ig antibody solution was prepared in RPMI (approximately 50.000 cpm in 50 1l). The medium in each well of the HUVEC plates was gently aspirated, the wells were washed twice with RPMI and 50 pl of the 1251labeled 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 5 0 value for D2E7 inhibition of hTNFa-induced expression of ELAM-1 on HUVEC is approximately 6 x 10-11 M 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.

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-53 SIn another series of experiments, the ability of the IgG1 form of D2E7 to inhibit hTNFa-induced expression of ELAM-1 on HUVEC was examined as described above.

The results from three independent experiments, and the average thereof, are summarized below in Table 11: 00 Cc Table 11: Inhibition of TNFa-Induced ELAM-1 Expression by D2E7 IeGI Recepnr

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Exoeriment ICso [M] 1 1.95 x 10- 1 0 2 1.69 x 10o 3 _1.90 x 10-1o Average 1.85 0.14 x 10-10 This series of experiments confirmed that D2E7, in the full-length IgG 1 form, inhibits TNFa-induced ELAM-1 expression on HUVEC with an average IC 50 of 1.85 ±0.14 x 10-0.

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 2 incubator for 16 hours, and the ICAM-1 expression was measured by mouse anti-ICAM-1 antibody followed by 12 5 I-labeled

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sheep anti-mouse antibody. Two independent experiments were performed and the ICSo 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 ICso values were calculated. An unrelated human IgGI antibody did not inhibit VCAM- 1 expression.

The results are summarized below in Table 12:

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-54- Table 12: Inhibition of ICAM-1 and VCAM-I Expression bvD2E7 leGI ICAM-1 Inhibition

IC

50

[M]

Experiment f IC50 Experiment

IC

50

[M]

1 1.84 x 10-10 1 I1.03 x 10- 10 2 2.49 x 10-10 2 9 2 6x 10-11 3 1.06x 10-10 Average 2.17 0.46 x 10-10 Average 1.01 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: 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 ICso 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-1 and the VCAM-1 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 hTNFo activity in vivo.

I. Inhibition of TNF-Induced Lethality in D-Galactosamine-Sensitized Mice Injection of recombinant human TNFa (rhTNFa) to D-galactosamine sensitized mice causes lethality within a 24 hour time period. TNF 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-IgG or a control protein, in PBS intraperitoneally Mice were challenged 30 minutes later with 1 pg of rhTNFa and 20 mg of D-

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Z galactosamine 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 5 hatched bars represent MAK 195. Injection of 2.5-25 pg of D2E7 antibody per mouse 00 0 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 CI ability. Injection of D2E7 in the absence of rhTNFa did not have any detrimental effect N on the mice. Injection of a non-specific human IgG1 antibody did not offer any protection from TNFa-induced lethality.

In a second experiment, forty-nine mice were divided into 7 equal groups. Each group received varying doses of D2E7 thirty minutes prior to receiving an LD80 dose of rhTNF/D-galactosamine mixture (1.0 pg rhTNF and 20 mg D-galactosamine per mouse). Control group 7 received normal human IgGI kappa antibody at 25 g/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) /7 14 3 (2.6 Vg) 5/7 71 4 (5.2 Vg) 6/7 86 (26 Vg) 6/7 86 6 (26 Vg; 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 lg/kg, elicted a rise in temperature greater than 0.4 0 C at approximately

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-56- Z minutes after injection.. The antibody preparation by itself, in saline at a dose of 138 lg/kg, did not elicit a rise in temperature in the rabbits up to 140 minutes after

C

1 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 ilg/kg i.v. Representative results of several experiments are 00 Mc, summarized below in Table 14: N Table 14: Inhibition of rhTNF-induced Pvrexia with D2E7 in Rabbits

IND

Temp. rise*, OC Molar Ratio Peak Temp.

D2E7 dose rhTNF D2E7: minutes (lg/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 ag/kg partially inhibited the pyrogenic response, compared to rabbits pre-treated with saline alone. D2E7 administered at 137 rg/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 plg/kg (molar ratio D2E7:rhTNF 3.3:1) totally suppressed the pyrogenic response, compared to rabbits pretreated with the control human IgG1 in saline at 30 gg/kg. In the final experiment, rabbits pretreated with D2E7 (792 lg/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.

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0 .57- III. Prevention of Polvarthritis in Te197 Transeenic Mice The effect of D2E7 on disease development was investigated in a transgenic murine model of arthritis. Transgenic mice (Tgl97) have been generated that express 00 5 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 Tg197 model of O polyarthritis).

2 Transgenic animals were identified by PCR at 3 days of age Litters of transgenic 10 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 jlg/g; Group 4=D2E7 at 15 Ig/g; Group 5=D2E7 at 30 ig/g; and Group 6=gG 1 isotype control at 30 .tg/g. A litter with non transgenic mice was also included in the study to 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 follows: 0 No arthritis, (normal appearence and flexion); =mild arthritis (joint 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: Effect of D2E7 on Histopatholoev and Arthritic Score in Tel197 Mice Group Treatment Histopatho logical Score Arthritic Score none 13(7/70 2 J saline 13(8/8) 6 J IgGi control 13(9/9) (7/9) 3 ID2E7 at 1.5 pg/g ]0 0 (8/8) 4 1 2E7 at 15 jig/g 0(7/8) 0(8/8) 1D2E7 at 30 .ggg 0 0(88 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 evident after the study period.

E. D2E7 Neutralization of TNFas from Other Secies 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 Tiffe.rent 1 .k T OQfl I j-5 0 for D2E7 TNFar Source jNeutralization

(M)I*

Human Recombinant7.x101 Chimpanzee LPS-stimulated PBMC 5iS x 1011 baboon Recombinant j 6.0 x101 ssay marmoset LPS-stimulated PBMC marmsetLPSstiulaed B C4.0 x10-10 7 cynomolgus LPS-stimulated PBMC cynomolgus~~~ L0-tmuae xBM 10-1 rhesus LPS-stimulated PBMC rhesus~~~~ L0-tmuae xB 10-11 canine canin LPS-stimulated

WBC

porcine murine Recombinant Recombinant 2.2 x 10-10 1.0X 10-7 1.0 X 10-7 The results in Table 16 demonstrate that D2E7 can neutralize the activity of five primate TNFcas approximately equivalently to human TNFcz and, moreover, can I-D 0O -59- Z neutralize the activity of canine TNFc (about ten-fold less well than human TNFa) and porcine and mouse TNFc (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-lI, IL-2, IL-4, IL-6, IL-8, IFNy and TGF3, C 5 indicating that D2E7 is very specific for its ligand TNFa.

00 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 of ten soluble cytokine, receptor and adhesion molecule ELISA kits: IL-la, IL-13, IL-1 receptor antagonist, IL-6, IL-8, TNFc, soluble TNF receptor 1, soluble TNF receptor II, soluble ICAM-1 and soluble E-selectin. No significant amounts ofcytokines or shed cell surface molecules were measured as a result of D2E7 antibody co-incubation, at concentrations up to 343 lig/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.

IN

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 REGIO SEQUENCE TYPE 1 D2E7 ON amino acid 2 D2E7 VII amino acid 3 D2E7 VL CDR3 amino acid 4 D2E7 VII CDR3 amino acid D2E7 VL CDR2 amino acid 6 D2E7 VII CDR2 aminoai 7 D2E7 VL CDR1 amino acid 8 D2E7 VII CDR1- amino acid 9 2SD4 VL amino acid 2SD4 VH amino acid 11 2SD4 VL CDR3 (amino acid 12 E.P B 12__ VL CDR3 amino acid 13 VL10E4 VL CDR3 amino acid 14 VL100A9 VL CDR3 amino acid VLL100D2 VL CDR3 amino acid 16 VLLOF4 VL CDR3 amino acid 17 LOE5 VL CDR3 amino acid i8 VLLOG7 VL CDR3 amino acid 19 VLLOG9 VL CDR3 amino acid' VLLOI1 VL CDR3 amino acid 21 VLLOI10 VL CDR3 amino acid 22 VL1B7 VL CDR3 amino acid 23 VL1C1 VL CDR3 -amino acid 24 VLO.1F4 VL CDR3 amino acid VLO. 1148 VL CDR3 amino acid 26 LOE7.A VL CDR3 amino acid 27 2SD4 VII CDR3 amino acid 28 VH1B11 VII CDR amino acid 29 VH1D8 VII CDR3 amino acid VIHlA11 VII CDR3 amino acid 31 VHIB12 VII CDR3 amino acid 32 VH1lE4 VII CDR3 amino acid 33 VII1F6 VII CDR3 amino acid 34 3C-H2 VII CDR3 amino acid 35VH1-D2.N VII CDR3 amino acid 36 02E7 VL nucleic acid 37 D2E7 VII nucleic acid

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0 0 6 1

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EQUIVALENTS

Those skilled in the art will recognize. or be able to ascertain using no more than 0 0 5 routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following C claims.

0 (Nq

Claims (57)

1. An isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFc with a K d of 1 x 10-8 M or less and a Kff rate.constant of 1 x 10-3 s- 1 or less, both determined by surface plismon resonance, and neutralizes -Q human TNFc cytotoxicity in a standard in vitro L929 assay with an ICso of 1 x 10- 7 M Sor less. S2. The isolated human antibody, or antigen-binding pdrtion thereof, of claim 0 10 1, which dissociates from human TNFc with a Koff rate constant of 5 x 10-4 s-1 or less,.

3. The isolated htiman antibody, or antigen-binding portion thereof, of claim 1, which dissociates from human TNFa with a Koff rate constant of I x i0- 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 of 1 x 0 8 M or less. 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 of i x 10-9 M or less.

6. The isolated human antibody, or antigen-binding portiori thereof, of claim 1, which neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an ICo of 5 x 0-10 M orless.

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-bindihg potion 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: M -78- O a) dissociates from human TNFc with a Koff rate constant of 1 x 10-3 s- 1 or less, as determined by surface plasmon resonance; b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, S 5 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 00 7, 8 and/or 9; Sc) 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, \O 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. The isolated human antibody of claim 9, or an antigen-binding portion thereof, which dissociates from human TNFa with a Kof 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 1 x 10- 4 s- 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 CDR1 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. -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 00 5 SEQ ID NO: 2.

16. The isolated human antibody of claim 15, which has an IgG heavy chain constant region.

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 sequence selected from the group consisting of SEQ IDNO: 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 IDNO: 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 TNFc but not human TNFP.

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. (N Z 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, 5 of claim 22, which also neutralizes the activity of pig TNFa. 00 An isolated nucleic acid encoding a light chain CDR3 domain comprising C' the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single O alanine substitution at position 1, 4, 5, 7 or 8, or by one to five conservative amino acid 0 10 substitutions at positions 1, 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, 6,8, 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 CDRI 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, C -81- >SEQIDNO:11 SEQID NO: 12,SEQ IDNO:13,SEQ IDNO: 14,SEQ 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: 5 SEQ1DNO: 3 1,SEQIDNO:32,SEQIDNO:33andSEQ IDNO: 34 00

34. An isolated nucleic acid encoding an antibody light chain variable region j comprising the amino acid sequence of SEQ ID NO: 1.

35. The isolated nucleic acid of claim 34, which encodes the antibody light N 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 IgG 4 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. O S-82- 0

43. A host cell into which the recombinant expression vector of claim 42 has Z 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 0 antibody that binds human TNFa is synthesized by the cell. A pharmaceutical composition comprising the antibody, or antigen- IS binding portion thereof, of any of claims 1-24, and a pharmaceutically acceptable carrier.

46. The pharmaceutical composition of claim 45, which further comprises at least one additional therapeutic agent for treating a disorder in which TNFc activity is detrimental.

47. A method for inhibiting human TNFa activity comprising contacting human TNFa with the antibody, or antigen-binding portion thereof, of any of claims 1-24 such that human TNFca activity is inhibited. :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 TNFc 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. O 00> 0 -83 Z

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. 5 54. The method of claim 48, wherein the disorder is an infectious disease. 00 The method of claim 48, wherein the disorder is transplant rejection or 1 graft-versus-host disease.

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

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

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

59. The method of claim 48, wherein 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, bums, 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 TNFa 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. i" C' -84- The use of claim 64, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty C arthritis. S 5 66. The use of claim 64, wherein the autoimmune disease is selected from the M¢3 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.

71. 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, burns, reperfusion injury, keloid formation, scar tissue formation, pyrexia, periodontal disease, obesity and radiation toxicity. Z74. The pharmaceutical comnposition of claim,46, wherein the additional therapeutic Agent is selected from the group consisting of wa-steroida1l anti- CIinflammatory drugs, cytokine suppressive anti-inflammratory druigs CDP-57l/13AY-.0- 3 3 56, rA2, 75 kdTNR-JgG, 55 kdTNFR-IgG, WDEC-CE9.l/SB 71036, DAB 486-IL- DAI 389-IL, Anti-Tac, IL-A, IL-10, IL-4 agoists, IL-10 agonist, I]L-IRA, TNF- 00bpls"TNFR, &284,9f973401, MK-966, Iloprost, methotrexate, thalidomide,-thaldomide- related ditgt,Ielflunoxnide, tranexamic acid, T-614, prostaglandin El, Tenidap, Naproxen, Meloxicam, Pir~xicam, Diclofenac, Indometha~in, Sufaalazine, NO Azathioprine, ICE: inhibitors, zap-70 inhbitors, Ick inhibitors, VEGF inhibitors, VEGF- R inhibitors, corticosteroids, TNF-convertase inhibitors, at--12 antibodies, interleukin-1 1, initerleukin-13, interleukin-17 inhibitors, gold, penicillamnine, cllooquine, hydroxychloroquine, chiorainbucil, cyclophosphamide, cyclosporin, anti- thym.Qcyte globulin, anti-CD4 antibodies, *CD5-toxins, orally-adruinistered peptides, collagen, lobenzarit disodium, Cytokine Regulating Agents 1W228 and HP466', ICAM-l anti~ense phosjphorothioate oligodeoxynucleotides, soluble complement receptor 1, prednisone, orgotein, glycosamninoglycan polysulphate, minocyc-line, aniti-IL2R antibodies, marine lipids, botanical 1ipids, auranofin, phenylbutazne, mneclofenarni- acid, flufetiarnlc acid, intravenous immune globulin, zileuton, mycophenolic acid, tacrolimus, sirolimus, amiprilose, cladribine, azaribine, budenoside, epidermal growth factor, atainosalicylates, 6-mercaptopurine, metronidazole, lipoxygenose'inhibitors, mesalamine, olsaazine, balsalazide, antioxidants, thromboxane inhibitors, IL-A receptqr antagonists, qnti-IL- I monoclonal antibodies, anti-IL-6 uionoclonal antibodies, growth factors, elasta~e inhibitors, pyridinyl-imiclazole compounds, glucuironide-conjugAted prodrugs of prednisolone, dexamethasone or budegonide, dextran-~conjugated prodrugs of prednisolo'ne, dexamethasone or budesonide, soluble complement receptor 1, slow- release mesalazine, Antagonists. of Platelet Activating Factor (PAP), ciprofloxacin, lignocame, prednisolone, methylprednisolone, cyclophosphamide, 4-aininopyridine, tizanidine, interferon-p 1 a, interferon-1b, Copolymer 1, hyperbaric. oxygen, intravenous immunoglobulin, clabribine, hypertonic saline solutions, antibiotics, continuous hemnofiltration, carbapenerns, antagonists of cytokines such as TNFca, IL- 1 P, IL-6 and/or -IL-8, SK&F 107647, tetrr'va'.eit guanyihydrazone CNI-14.93, Tissue Factor Pathway Inhibitor, PIP, iron chelators and chelates,- including diethylenetriamine pentaacetlc acid iron (III) complex, lisofline, PG(3-Glucd, apolipop rotein A;-i reconstituted with lipids, chiral hydroxamic acids, anti-eridotoxin antibodies, E553 1, rBPI 21 Synthetic Aniti-Endotoxin Peptides, surfactant replacement therapy and anti-IL-S antibodies. -86- 0 75. *Use of the antibody, or antigen-binding portion thereof, of any of claims 1-24 in Z therapy.

76. Use of the antibody, or antigen-binding portion thereof, of any of claims 1-24 ir) therapy in~ combination with at least additional therapeutic agent for treating a disorder 00 in which TNFa activity is detrimental. c-i77. 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-57lIBAY-10-3356, cA2, 75 kdTNFR-IgG 55 kdTNFR-IgG, IDEC-CE9.1/SB 210396, DAB 486-1t,-2, DAB 389-IL-2, Anti-Tac, IL-A, IL-lO, IL-4 agonists, IL-10 agonists, IL-IRA, TNF-bp/s-TNFR, S284, R973401, MK-966, Iloprost, methotrexate, thalidomide, thalidomide-related drugs, leflunonide, tranexamic acid, T- 6 14, prostaglandin El, Teni dap, Naproxen, Meloxicarn, Piroxicam, Diclofenac, Indomethacin, Sulfasalazine, Azathioprine, ICE inhibitors, zap-70 inhibitors, Ick inhibitors, VEGE inhibitors, VEGF-R inhibitors, corticosteroids, TNF-convertase inhibitors., anti-IL, 12 antibo'dies, interleukin- 11, interleukin- 13, interleukin-17 inhibitors, gold, penicillamine, chloroquine,,hydroxychloroqine, chlorambucil, cyclophosphamide, eyclosporin, anti-thymocyte globulin, anti-CD)4 antibodies, toxins, orally-administered peptides, collagen, lobenzarit disodium, Cytokine Regulating Agents HP228 and HP466, ICAM-l antisense phosphorotbioAto Qligodeoxynucleotides, soluble complement receptor 1, prednisone, orgotein, glycosaninoglycan polysulphate, minocycline, anti-IL2R antibodies, marine lipids, botanical lipids, auranof in, phenylbutazone, meclofenamic acid,* flufenamic acid, intravenous immune globulin, zileuton, mycophenolic acid, tacrolimus, sirolimus, amiprilose, cladribine, azaribine, budenoside, epidermal growth factor, amninosalicylates, 6-mercaptopurine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-l1 receptor antagonists, anti-IL- I P monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-irnidazole compounds, glucuronide-conjugated prodiugs 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, intoerferon-P 1 a, interferon-p3lb. Copolymer 1, hyperbaric oxygen, intravenous immunoglobulin, clabribine, hypertonic saline solutions, antibiotics, continuous hemofiltration, -87- 0 carbaPtnemn tagonjtsof cytokies such as TNFQ, IL-.1 P, IL-6 and/or tL-8, SK&F Z107647, tetravalent p8m-ny~lh.drazoneCNI.1493, Tissue Factor Pathway Inhibitor, PIP, -iroft rftektors adceasincluding diethylete~ianie pe'ntaacetic- acid ]"iTon(1) CoQraplex, lisof iline, PGG-Glic-An, apoliploprbt~m A- I reconsfituted with lpd,~ia laydroxarnie acids, iiati-et~dotoxin antibodies, E553 1, .rBPI 2 1 Synthetic Anti- End tx 00 Peptides, turfactant replacemnent therapy and wni-IL-;8 anilbodies.