HUMAN ANTIBODIES THAT BIND HUMAN TNFalpha

125697/2

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Human antibodies that bind human TNFa

Abbott Laboratories (Bermuda) Ltd. C.112430

ATTORNEYD

ΓΝΟ: BBI-043CPPC - 1 - HUMAN ANTIBODIES THAT BIND HUMAN TNFa

Background of the Invention

Tumor necrosis factor a (TNFa) is a cytokine produced by numerous cell types,including monocytes and macrophages, that was originally identified based on itscapacity to induce the necrosis of certain mouse tumors (see e.g., Old, L. (1985) Science230:630-632). Subsequently, a factor termed cachectin, associated with cachexia, wasshown to be the same molecule as TNFa. TNFa has been implicated in mediatingshock (see e.g., 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 diseasesand disorders, including sepsis, infections, autoimmune diseases, transplant rejectionand graft-versus-host disease (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169;U.S. Patent No. 5,231,024 to Moeller et al.·, European Patent Publication No. 260 610BI by Moeller, A., et α/.Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-5031.

Because of the harmful role of human TNFa (hTNFa) in a variety of humandisorders, therapeutic strategies have been designed to inhibit or counteract hTNFaactivity. In particular, antibodies that bind to, and neutralize, hTNFa have been soughtas a means to inhibit hTNFa activity. Some of the earliest of such antibodies weremouse monoclonal antibodies (mAbs), secreted by hybridomas prepared fromlymphocytes of mice immunized with hTNFa (see e.g., Hahn T; et al., (1985) Proc NatlAcad Sci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys. Res.Commun. 137:847-854: Hirai, M., et al. (1987) J. Immunol. Methods 96:57-62; Fendly,B.M., et al. (1987) Hybridoma 6:359-370; Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Patent No. 5,231,024 to Moeller et al.·, European Patent Publication No. 186833 BI by Wallach, D.; European Patent Application Publication No. 218 868 Al byOld et al.·, European Patent Publication No. 260 610 BI by Moeller, A., et al.). Whilethese mouse anti-hTNFa antibodies often displayed high affinity for hTNFa (e.g., Kd &lt;10"9M) and were able to neutralize hTNFa activity, their use in vivo may be limited byproblems associated with administration of mouse antibodies to humans, such as short* serum half life, an inability to trigger certain human effector functions and elicitation ofan unwanted immune response against the mouse antibody in a human (the "human anti-mouse antibody" (HAMA) reaction).

In an attempt to overcome the problems associated with use of fully-murine antibodies in humans, murine anti-hTNFa antibodies have been genetically engineered

ATTORNEYD' TNO: BBI-043CPPC -2- to be more "human-like." For example, chimeric antibodies, in which the variableregions of the antibody chains are murine-derived and the constant regions of theantibody 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, P.E., et al.).Additionally, humanized antibodies, in which the hypervariable domains of the antibodyvariable regions are murine-derived but the remainder of the variable regions and the.antibody constant regions are human-derived, have also been prepared (PCT PublicationNo. WO 92/11383 by Adair, J.R., et al.). However, because these chimeric andhumanized antibodies still retain some murine sequences, they still may elicit anunwanted immune reaction, the human anti-chimeric antibody (HACA) reaction,especially when administered for prolonged periods, e.g., for chronic indications, suchas rheumatoid arthritis (see e.g., Elliott, M.J., et al. (1994) Lancet 344:1125-1127; Elliot,M.J., et al. (1994) Lancet 344:1105-1110). A preferred hTNFa inhibitory agent to murine mAbs or derivatives thereof (e.g,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 prolongedperiods. Human monoclonal autoantibodies against hTNFa have been prepared usinghuman hybridoma techniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568;Boyle, P., et al. (1993) Cell. Immunol. 152:569-581; European Patent ApplicationPublication No. 614 984 A2 by Boyle, et al.). However, these hybridoma-derivedmonoclonal autoantibodies were reported to have an affinity for hTNFa that was toolow to calculate by conventional methods, were unable to bind soluble hTNFa and wereunable to neutralize hTNFa-induced cytotoxicity (see Boyle, et al.·, supra). Moreover,the success of the human hybridoma technique depends upon the natural presence inhuman peripheral blood of lymphocytes producing autoantibodies specific for hTNFa.Certain studies have detected serum autoantibodies against hTNFa in human subjects(Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog. Leukocyte Biol. 10B :447-452). whereas others have not (Leusch, H-G., etal. (1991) J. Immunol. Methods 139:145-1471.

Alternative to naturally-occurring human anti-hTNFa antibodies would be arecombinant hTNFa antibody. Recombinant human antibodies that bind hTNFa withrelatively low affinity (i.e., Kj ~10'7M) and a fast off rate (i.e., Koff ~ 10*2 sec1) havebeen described (Griffiths, A.D., et al. (1993) EMBO J. 12:725-734). However, becauseof their relatively fast dissociation kinetics, these antibodies may not be suitable fortherapeutic use. Additionally, a recombinant human anti-hTNFa has been describedthat does not neutralize hTNFa activity, but rather enhances binding of hTNFa to the 125697/2 -3- surface of cells and enhances internalization of hTNFa (Lidbury, A., et al. (1994)

Biotechnol. Ther. 5:27-45; PCT Publication No. WO 92/03145 by Aston, R. et al.)

Accordingly, human antibodies, such as recombinant human antibodies, that bindsoluble hTNFa with high affinity and slow dissociation kinetics and that have thecapacity to neutralize hTNFa activity, including hTNFa-induced cytotoxicity (in vitroand in vivo) and hTNFa-induced cell activation, are still needed.

Summary of the Invention

This invention provides human antibodies, preferably recombinant humanantibodies, that specifically bind to human TNFa.

The antibodies of the invention can be full-length (e.g., an IgGl or IgG4antibody) or can comprise only an antigen-binding portion (e.g., a Fab, F(ab')2 or scFvfragment). The most preferred recombinant antibody of the invention, termed D2E7, hasa light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and aheavy 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 theamino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID ΝΘ: 2.

In one embodiment, the invention provides an isolated human antibody, or anantigen-binding portion thereof, that dissociates from human TNFa with a K4 of 1 x10-8 M or less and a Karate constant of 1 x 10-3 s*1 or less, both determined by surfaceplasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitroL929 assay with an IC50 of 1 χ IO*7 M or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koffof 5 x IO-4 S'1 or less, or even more preferably, with a Koff of 1 x IO’4 s'1 or less. Morepreferably, the isolated human antibody, or antigen-binding portion thereof, neutralizeshuman TNFa cytotoxicity in a standard in vitro L929 assay with an IC50 of 1 χ 10"8 Mor less, even more preferably with an IC50 of 1 χ 10-9 M or less and still more preferablywith an IC50 of 5 χ IO'10 M or less. ATTORNEYD'

TNO: BBI-043CPPC -4-

In another embodiment, the invention provides a human antibody, or antigen-binding portion thereof, with the following characteristics: a) dissociates from human TNFa with a Koff of 1 x 10"3 s_l or less, asdetermined by surface plasmon resonance; b) has a light chain CDR3 domain comprising the amino acid sequence of SEQID 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 SEQID 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 atpositions 2,3,4, 5,6, 8,9,10,11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof, dissociatesfrom human TNFa with a of 5 χ 10*4 s'1 or less. Still more preferably, theantibody, or antigen-binding portion thereof, dissociates from human TNFa with a Kq^-of 1 χ 10-4 s*1 or less.

In yet another embodiment, the invention provides a human antibody, or anantigen-binding portion thereof, with an LCVR having CDR3 domain comprising theamino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1,4, 5,7 or 8, and with an HCVR having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQID NO: 4 by a single alanine substitution at position 2,3,4, 5,6, 8,9,10 or 11. Morepreferably, the LCVR further has a CDR2 domain comprising the amino acid sequenceof SEQ ID NO: 5 and the HCVR further has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 6. Still more preferably, the LCVR further has CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDR1domain 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 acidsequence of SEQ ID NO: 1 and an HCVR comprising the amino acid sequence of SEQID NO: 2. In certain embodiments, the antibody has an IgGl heavy chain constant t region or an IgG4 heavy chain constant region. In yet other embodiments, the antibodyis a Fab fragment, an F(ab')2 fragment or a single chain Fv fragment.

In still other embodiments, the invention provides antibodies, or antigen-bindingportions thereof, with an LCVR having CDR3 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID -5- 125697Z3 NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or with anHCVR having a CDR3 domain comprising an amino acid sequence selected from thegroup 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: 34and SEQ ID NO: 35.

In yet another embodiment, the invention provides an isolated human antibody,or antigen-binding portion thereof, that neutralizes the activity of human TNFa but nothuman TNFp (lymphotoxin). In a preferred embodiment, the human antibody, orantigen-binding portion thereof, neutralizes the activity of human TNFa, chimpanzeeTNFa and at least one additional primate TNFa selected from the group consisting ofbaboon TNFa, marmoset TNFa, cynomolgus TNFa and rhesus TNFa. Preferably, theantibody also neutralizes the activity of at least one non-primate TNFa. In anothersubembodiment, the isolated human antibody, or antigen-binding portion thereof, alsoneutralizes the activity of pig TNFa. In yet another subembodiment, the isolated humanantibody, or antigen-binding portion thereof, also neutralizes the activity of mouseTNFa.

All passages of the description which are not within the scope ofthe claims donot form part of the invention. -6- 125697/2

Brief Description of the Drawings

Figures 1A and IB show the amino acid sequences of the light chain variableregion of D2E7 (D2E7 VL; also shown in SEQ ID NO: 1), alanine-scan mutants ofD2E7 VL (LD2E7*.A1, LD2E7*.A3, LD2E7*.A4, LD2E7*.A5, LD2E7*.A7 andLD2E7*.A8), the light chain variable region of the D2E7-related antibody 2SD4 (2SD4VL; also shown in SEQ ID NO: 9) and other D2E7-related light chain variable regions(EP B12, VL10E4, VL100A9, VL100D2, VL10F4, LOE5, VLLOF9, VLL0F10,VLLOG7, VLLOG9, VLLOH1, VLLOHIO, VL1B7, VL1C1, VL1C7, VL0.1F4,VL0.1H8, LOE7, LOE7.A and LOE7.T). Figure 1A shows the FR1, CDR1, FR2 andCDR2 domains. Figure IB shows the FR3, CDR3 and FR4 domains. The light ChainCDR1 ("CDR Ll"), CDR2 ("CDR L2") and CDR3 ("CDR L3") domains are boxed.

Figures 2A and 2B show the amino acid sequences of the heavy chain variableregion of D2E7 (D2E7 VH; also shown in SEQ ID NO: 2), alanine-scan mutants ofD2E7 VH (HD2E7*.A1, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4, HD2E7*.A5,HD2E7*.A6, HD2E7*.A7, HD2E7*.A8 and HD2E7*.A9), the heavy chain variableregion of the D2E7-related antibody 2SD4 (2SD4 VH; also shown in SEQ ID NO: 10)and other D2E7-related heavy chain variable regions (VH1B11, VH1D8, VH1 Al 1,VH1B12, VH1-D2, VH1E4, VH1F6, VH1G1, 3C-H2, VH1-D2.N and VH1-D2.Y).Figure 2A shows the FRi, CDR1, FR2 and CDR2 domains. Figure 2B shows the FIG,CDR3 and FR4 domains. The heavy chain CDR1 ("CDR Hl"), CDR2 ("CDR H2") andCDR3 ("CDR H3") domains are boxed.

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

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

Figure 5 is a graph depicting the inhibition of TNFa-induced ELAM-1expression on HUVEC by the human anti-hTNFa antibody D2E7, as compared to themurine anti-hTNFa antibody MAK 195.

Figure 6 is a bar graph depicting protection from TNFa-induced lethality in 01124304X33-01

ATTORNEYDi ΓΝΟ: BBI-043CPPC Ί- .· ** 10 10.02.1997 D-galactosamine-sensitized mice by administration of the human anti-hTNFa antibodyD2E7 (black bars), as compared to the murine anti-hTNFa antibody MAK 195 (hatchedbars).

Figure 7 shows the nucleotide sequence of the light chain variable region ofD2E7, with the predicted amino acid sequence below the nucleotide sequence. TheCDR LI, CDR L2 and CDR L3 regions are underlined.

Figure 8 shows the nucleotide sequence of the heavy chain variable region ofD2E7, with the predicted amino acid sequence below the nucleotide sequence. TheCDR Hl, CDR H2 and CDR H3 regions are underlined.

Figure 9 is a graph depicting the effect of D2E7 antibody treatment on the meanjoint 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 15 thereof, that bind to human TNFa with high affinity, a low off rate and high neutralizingcapacity. Various aspects of the invention relate to antibodies and antibody fragments,and pharmaceutical compositions thereof, as well as nucleic acids, recombinantexpression vectors and host cells for making such antibodies and fragments. Methods ofusing the antibodies of the invention to detect human TNFa or to inhibit human TNFa 20 activity, either in vitro or in vivo, are also encompassed by the invention.

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

The term "human TNFa" (abbreviated herein as hTNFa, or simply hTNF), asused herein, is intended to refer to a human cytokine that exists as a 17 kD secreted form 25 and a 26 kD membrane associated form, the biologically active form of which iscomposed of a trimer of noncovalently bound 17 kD molecules. The structure ofhTNFa is described further in, for example, Pennica, D., et al. (1984) Nature 312:724-729; Davis, J.M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E.Y., et al. (1989) Nature 338:225-228. The term human TNFa is intended to include recombinant 30 human TNFa (rhTNFa), which can be prepared by standard recombinant expressionmethods or purchased commercially (R &amp;amp; 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 (H) chains and two light (L) 35 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

ATTORNEYD' ~~ 'TNO: BBI-043CPPC -8- region. The heavy chain constant region is comprised of three domains, CHI, CH2 andCH3. Each light chain is comprised of a light chain variable region (abbreviated hereinas LCVR or VL) and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed framework regions (FR).

Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term "antigen-binding portion" of an antibody (or simply "antibodyportion"), as used herein, refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., hTNFa). It has been shown that theantigen-binding function of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalentfragment 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 consistingof the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward etal., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the two domains ofthe Fv fragment, VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalent molecules(known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426: andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term "antigen-bindingportion" of an antibody. Other forms of single chain antibodies, such as diabodies arealso encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VLdomains are expressed on a single polypeptide chain, but using a linker that is too shortto 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 antigenbinding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may be part of alarger immunoadhesion molecules, formed by covalent or noncovalent association of the

ATTORNEYDC

ΓΝΟ: BBI-043CPPC -9- antibody or antibody portion with one or more other proteins or peptides. Examples ofsuch immunoadhesion molecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S.M., et al. (1995) Human Antibodies andHybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminalpolyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., etal. (1994) Mol. Immunol. 31.:1047-1058). Antibody portions, such as Fab and F(ab')2fragments, can be prepared from whole antibodies using conventional techniques, suchas papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies,antibody portions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein.

The term "human antibody", as used herein, is intended to include antibodieshaving variable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g, mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term "human antibody", asused herein, is not intended to include antibodies in which CDR sequences derived fromthe germline of another mammalian species, such as a mouse, have been grafted ontohuman framework sequences.

The term "recombinant human antibody", as used herein, is intended to includeall human antibodies that are prepared, expressed, created or isolated by recombinantmeans, such as antibodies expressed using a recombinant expression vector transfectedinto a host cell (described further in Section II, below), antibodies isolated from arecombinant, combinatorial human antibody library (described further in Section III,below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means thatinvolves splicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable and constant regions derived fromhuman germline immunoglobulin sequences. In certain embodiments, however, suchrecombinant 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 theamino acid sequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH and VLsequences, may not naturally exist within the human antibody germline repertoire invivo.

ATTORNEYDf TNO: BBI-043CPPC C - 10-

An "isolated antibody", as used herein, is intended to refer to an antibody that issubstantially free of other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hTNFa is substantially free of antibodies thatspecificallybind antigens other than hTNFa). An isolated antibody that specificallybinds hTNFa may, however, have cross-reactivity to other antigens, such as TNFamolecules from other species (discussed in further detail below). Moreover, an isolatedantibody may be substantially free of other cellular material and/or chemicals. A "neutralizing antibody", as used herein (or an "antibody that neutralizedhTNFa activity"), is intended to refer to an antibody whose binding to hTNFa results ininhibition of the biological activity of hTNFa. This inhibition of the biological activityof hTNFa can be assessed by measuring one or more indicators of hTNFa biologicalactivity, such as hTNFa-induced cytotoxicity (either in vitro or in vivo), hTNFa-inducedcellular activation and hTNFa binding to hTNFa receptors. These indicators of hTNFabiological activity can be assessed by one or more of several standard in vitro or in vivoassays known in the art (see Example 4). Preferably, the ability of an antibody to'neutralize hTNFa activity is assessed by inhibition of hTNFa-induced cytotoxicity ofL929 cells. As an additional or alternative parameter of hTNFa activity, the ability ofan antibody to inhibit hTNFa-induced expression of ELAM-1 on HUVEC, as a measureof hTNFa-induced cellular activation, can be assessed.

The term "surface plasmon resonance", as used herein, refers to an opticalphenomenon that allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensor matrix, for exampleusing the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,NJ). For further descriptions, see Example 1 and JonsS'on, U., et al. (1993) Ann. Biol.Clin. 51:19-26: Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al.(1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.198:268-277.

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

The term "Kj", as used herein, is intended to refer to the dissociation constant ofa 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 ordouble-stranded, but preferably is double-stranded DNA.

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

ATTORNEYD' ’ TNO: BBI-043CPPC - 11 - is intended to refer to a nucleic acid molecule in which the nucleotide sequencesencoding the antibody or antibody portion are free of other nucleotide sequencesencoding antibodies or antibody portions that bind antigens other than hTNFa, whichother sequences may naturally flank the nucleic acid in human genomic DNA. Thus, forexample, an isolated nucleic acid of the invention encoding a VH region of an anti-TNFa antibody contains no other sequences encoding other VH regions that bindantigens other than TNFa.

The term "vector", as used herein, is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has been linked. One type ofvector is a "plasmid", which refers to a circular double stranded DNA loop into whichadditional 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 vectorsare capable of autonomous replication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectors are referred toherein as "recombinant expression vectors" (or simply, "expression vectors"). Ingeneral, expression vectors of utility in recombinant DNA techniques are often in theform of plasmids. In the present specification, "plasmid" and "vector" may be usedinterchangeably 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 viralvectors (e.g., replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term "recombinant host cell" (or simply "host cell"), as used herein, isintended to refer to a cell into which a recombinant expression vector has beenintroduced. It should be understood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Because certain modificationsmay 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 i the scope of the term "host cell" as used herein.

Various aspects of the invention are described in further detail in the followingsubsections. 72 5 ATTORNEY!)

TNO: BBI-043CPPC - 12- I. Human Antibodies that Bind Human TNFa

This invention provides isolated human antibodies, or antigen-binding portions thereof, that bind to human TNFa with high affinity, a low off rate and high neutralizingcapacity. Preferably, the human antibodies of the invention are recombinant, 5 neutralizing human anti-hTNFa antibodies. The most preferred recombinant, neutralizing antibody of the invention is referred to herein as D2E7 and has VL and VHsequences as shown in Figure 1 A, IB and Figure 2A, 2B, respectively (the amino acidsequence of the D2E7 VL region is also shown in SEQ ID NO: 1; the amino acidsequence of the D2E7 VH region is also shown in SEQ ID NO: 2). The binding 10 properties of D2E7, as compared to the murine anti-hTNFa MAK 195 mAb thatexhibits high affinity and slow dissociation kinetics and another human anti-hTNFaantibody related in sequence to D2E7,2SD4, are summarized below: 10.02.1997

Antibody Koff sec*1 k-on M'1 sec’1 Kd M Stoichio- metry D2E7 IgGl 8.81 x IO"5 1.91 x 10s 6.09 x IO"10 1.2 2SD4 lgG4 8.4 x 10-3 4.20 x 105 2.00 x 10-8 0.8 MAK 195 F(ab')2 8.70 x IO'5 1.90 x 105 4.60 x 10-10 1.4 15 The D2E7 antibody, and related antibodies, also exhibit a strong capacity to neutralize hTNFa activity, as assessed by several in vitro and in vivo assays (seeExample 4). For example, these antibodies neutralize hTNFa-induced cytotoxicity ofL929 cells with ICsq values in the range of about 10'7 M to about IO*10 M. D2E7, whenexpressed as a full-length IgGl antibody, neutralizes hTNFa-induced cytotoxicity of 20 L929 cells with IC50 of about 1.25 x 10-10 M. Moreover, the neutralizing capacity of D2E7 is maintained when the antibody is expressed as a Fab, F(ab')2 or scFv fragment.D2E7 also inhibits TNFa-induced cellular activation, as measured by hTNFa-inducedELAM-1 expression on HUVEC (IC50 = about 1.85 χ IO*10 M), and binding of hTNFato hTNFa receptors on U-937 cells (IC50 = about 1.56 χ IO*10 M). Regarding the latter, 25 D2E7 inhibits the binding of hTNFa to both the p55 and p75 hTNFa receptors.

Furthermore, the antibody inhibits hTNFa-induced lethality in vivo in mice (ED50 =1-2.5 pg/mouse).

Regarding the binding specificity of D2E7, this antibody binds to human TNFa in various forms, including soluble hTNFa, transmembrane hTNFa and hTNFa bound 30 to cellular receptors. D2E7 does not specifically bind to other cytokines, such as lymphotoxin (TNFP), IL-Ια, IL-1 β, IL-2, IL-4, IL-6, IL-8, IFNy and TGFp. However,

ATTORNEY!) 'NO: BBI-043CPPC - 13- D2E7 does exhibit crossreactivity to tumor necrosis factors from other species. Forexample, the antibody neutralizes the activity of at least five primate TNFas(chimpanzee, baboon, marmoset, cynomolgus and rhesus) with approximatelyequivalent IC5Q values as for neutralization of hTNFa (see Example 4, subsection E).D2E7 also neutralizes the activity of mouse TNFa, although approximately 1000-foldless well than human TNFa (see Example 4, subsection E). D2E7 also binds to canineand porcine TNFa.

In one aspect, the invention pertains to D2E7 antibodies and antibody portions,D2E7-related antibodies and antibody portions, and other human antibodies andantibody portions with equivalent properties to D2E7, such as high affinity binding tohTNFa with low dissociation kinetics and high neutralizing capacity. In oneembodiment, the invention provides an isolated human antibody, or an antigen-bindingportion thereof, that dissociates from human TNFa with a IQ of 1 x 10‘8 M or less and aKoff rate constant of 1 x IO*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 IC5Qof 1 x 10*7 M or less. More preferably, the isolated human antibody, or antigen-bindingportion thereof, dissociates from human TNFa with a IQffOf 5 x 10*4 s*1 or less, or evenmore preferably, with a Koff of 1 x 10‘4 s’1 or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFa cytotoxicity in astandard in vitro L929 assay with an IC50 of 1 x 10‘8 M or less, even more preferablywith an IC50 of 1 x IO*9 M or less and still more preferably with an IC5Q of 5 x IO*10 Mor less. In a preferred embodiment, the antibody is an isolated human recombinantantibody, or an antigen-binding portion thereof. In another preferred embodiment, theantibody also neutralizes TNFa-induced cellular activation, as assessed using a standardin vitro assay for TNFa-induced ELAM-1 expression on humah umbilical veinendothelial cells (HUVEC).

Surface plasmon resonance analysis for determining IQ and IQff can beperformed as described in Example 1. A standard in vitro L929 assay for determiningIC50 values is described in Example 4, subsection A. A standard in vitro assay forTNFa-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 andneutralization criteria include antibodies having the following [VH/VL] pairs, thesequences of which are shown in Figures 1 A, IB, 2A and 2B (see also Examples 2, 3and 4 for kinetic and neutralization analyses): [D2E7 VH/D2E7 VL]; [HD2E7*.A1/D2E7 VL], [HD2E7*.A2/D2E7 VL], [HD2E7*.A3/D2E7 VL],

ATTORNEYD( TNO: BBI-043CPPC - 14- [HD2E7*.A4/D2E7 VL], [HD2E7*.A5/D2E7 VL], [HD2E7*.A6/D2E7 VL],[HD2E7*.A7/D2E7 VL], [HD2E7*.A8/D2E7 VL], [HD2E7*.A9/D2E7 VL], [D2E7VH/LD2E7*.A1], [D2E7 VH/LD2E7*.A4], [D2E7 VH/LD2E7*.A5], [D2E7VH/LD2E7*.A7], [D2E7 VH/LD2E7*.A8], [HD2E7*.A9/LD2E7*.A1], [VH1-D2/LOE7], [VH1-D2.N/LOE7.T], [VH1-D2.Y/LOE7.A], [VH1-D2.N/LOE7.A], [VH1-D2/EP B12] and [3C-H2/LOE7].

It is well known in the art that antibody heavy and light chain CDR3 domainsplay an important role in the binding specificity/affinity of an antibody for an antigen.Accordingly, in another aspect, the invention pertains to human antibodies that haveslow dissociation kinetics for association with hTNFa and that have light and heavychain CDR3 domains that structurally are identical to or related to those of D2E7. Asdemonstrated in Example 3, position 9 of the D2E7 VL CDR3 can be occupied by Alaor Thr without substantially affecting the Koff. Accordingly, a consensus motif for theD2E7 VL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQID NO: 3). Additionally, position 12 of the D2E7 VH CDR3 can be occupied by Tyr orAsn, without substantially affecting the Koff. Accordingly, a consensus motif for theD2E7 VH CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N)(SEQ ID NO: 4). Moreover, as demonstrated in Example 2, the CDR3 domain of theD2E7 heavy and light chains is amenable to substitution with a single alanine residue (atposition 1,4, 5,7 or 8 within the VL CDR3 or at position 2, 3,4,5,6, 8,9,10 or 11within the VH CDR3) without substantially affecting the Koff- Still further, the skilledartisan will appreciate that, given the amenability of the D2E7 VL and VH CDR3domains to substitutions by alanine, substitution of other amino acids within the CDR3domains may be possible while still retaining the low off rate constant of the antibody,in particular substitutions with conservative amino acids. A "conservative amino acidsubstitution", as used herein, is one in which one amino acid residue is replaced withanother amino acid residue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, ' proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Preferably, no more than one to five conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains. Morepreferably, no more than one to three conservative amino acid substitutions are made

ATTORNEYS

ΓΝΟ: BBI-043CPPC - 15- within the D2E7 VL and/or VH CDR3 domains. Additionally, conservative amino acidsubstitutions should not be made at amino acid positions critical for binding to hTNFa.As shown in Example 3, positions 2 and 5 of the D2E7 VL CDR3 and positions 1 and 7of the D2E7 VH CDR3 appear to be critical for interaction with hTNFa and thus,conservative amino acid substitutions preferably are not made at these positions(although an alanine substitution at position 5 of the D2E7 VL CDR3 is acceptable, asdescribed above).

Accordingly, in another embodiment, the invention provides an isolated humanantibody, or antigen-binding portion thereof, with the following characteristics: a) dissociates from human TNFa with a rate constant of 1 χ IO'3 s*1 or less,as determined by surface plasmon resonance; b) has a light chain CDR3 domain comprising the amino acid sequence of SEQID 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 SEQID 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 atpositions 2,3,4, 5,6, 8,9,10,11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof, dissociatesfrom human TNFa with a IQff of 5 χ 10*4 s'1 or less. Even more preferably, theantibody, or antigen-binding portion thereof, dissociates from human TNFa with aof 1 χ 10*4 s'1 or less.

In yet another embodiment, the invention provides an isolated human antibody,or an antigen-binding portion thereof, with a light chain variabfe region (LCVR) havinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1,4, 5, 7 or 8, and with aheavy chain variable region (HCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution 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 (i.e., the D2E7 VL‘ CDR2) and the HCVR further has a CDR2 domain comprising the amino acid sequenceof SEQ ID NO: 6 (i.e., the D2E7 VH CDR2). Even more preferably, the LCVR furtherhas CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e., the D2E7VL CDR1) and the HCVR has a CDR1 domain comprising the amino acid sequence ofSEQ ID NO: 8 (i.e., the D2E7 VH CDR1). The framework regions for VL preferably

ATTORNEYD

:ΤΝΟ: BBI-043CPPC - 16- are from the VKI human germline family, more preferably from the A20 human germline Vk gene and most preferably from the D2E7 VL framework sequences shown in Figures 1A and IB. The framework regions for VH preferably are from the Vjj3 human germline family, more preferably from the DP-31 human germline VH gene and most preferably from the D2E7 VH framework sequences shown in Figures 2A and 2B.

In still another embodiment, the invention provides an isolated human antibody,or an antigen binding portion thereof, with a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 (i.e., the D2E7 VL) and a heavychain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2(i.e., the D2E7 VH). In certain embodiments, the antibody comprises a heavy chainconstant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgGl heavy chain constantregion or an IgG4 heavy chain constant region. Furthermore, the antibody can comprisea light chain constant region, either a kappa light chain constant region or a lambda lightchain constant region. Preferably, the antibody comprises a kappa light chain constantregion. Alternatively, the antibody portion can be, for example, a Fab fragment or asingle chain Fv fragment.

In still other embodiments, the invention provides an isolated human antibody, oran antigen-binding portions thereof, having D2E7-related VL and VH CDR3 domains,for example, antibodies, or antigen-binding portions thereof, with a light chain variableregion (LCVR) having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or with a heavy chainvariable region (HCVR) having a CDR3 domain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33,SEQ ID NO: 34 and SEQ ID NO: 35.

In yet another embodiment, the invention provides a recombinant humanantibody, or antigen-binding portion thereof, that neutralizes the activity of human ‘ TNFa but not human TNFp. Preferably, antibody, or antigen-binding portion thereof,also neutralizes the activity of chimpanzee TNFa and at least one additional primateTNFa selected from the group consisting of baboon TNFa, marmoset TNFa,cynomolgus TNFa and rhesus TNFa. Preferably, the antibody, or antigen-bindingportion thereof, neutralizes the human, chimpanzee and/or additional primate TNFa in a

ATTORNEYDi ΓΝΟ: BBI-043CPPC - 17- standard in vitro L929 assay with an IC5Q of 1 χ 10*8 M or less, more preferably lx 10*9M or less, and even more preferably 5 x 10'10 M or less. In one subembodiment, theantibody also neutralizes the activity of canine TNFa, preferably in a standard in vitroL929 assay with an IC50 of 1 x 10‘7 M or less, more preferably lx IO"8 M or less andeven more preferably 5 x 10‘9 M or less. In another subembodiment, the antibody alsoneutralizes the activity of pig TNFa, preferably with an IC50 of 1 χ IO*5 M or less, morepreferably lx 10'6 M or less and even more preferably 5 x 10‘7 M or less. In yet anotherembodiment, the antibody also neutralizes the activity of mouse TNFa, preferably withan IC50 of 1 χ 10"4 M or less, more preferably lx 1 O'5 M or less and even morepreferably 5 χ IO*6 M or less.

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

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to create bispecific antibodies).Suitable crosslinkers include those that are heterobifunctional, having two distinctlyreactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company, Rockford, IL.

Useful detectable agents with which an antibody or antibody portion of theinvention may be derivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like. An antibodymay also be derivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatizedwith a detectable enzyme, it is detected by adding additional reagents that the enzymeuses to produce a detectable reaction product. For example, when the detectable agenthorseradish peroxidase is present, the addition of hydrogen peroxide and ATTORNEYD' ~ ΎγΝΟ: BBI-043CPPC ί &lt; Ί - 18- diaminobenzidine leads to a colored reaction product, which is detectable. An antibodymay also be derivatized with biotin, and detected through indirect measurement ofavidin or streptavidin binding. II. Expression of Antibodies

An antibody, or antibody portion, of the invention can be prepared byrecombinant 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 morerecombinant expression vectors carrying DNA fragments encoding the immunoglobulinlight and heavy chains of the antibody such that the light and heavy chains are expressedin the host cell and, preferably, secreted into the medium in which the host cells arecultured, from which medium the antibodies can be recovered. Standard recombinantDNA methodologies are used obtain antibody heavy and light chain genes, incorporatethese genes into recombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular-Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),Ausubel, F.M. et al. (eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (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 lightand heavy chain variable regions are first obtained. These DNAs can be obtained byamplification and modification of germline light and heavy chain variable sequencesusing the polymerase chain reaction (PCR). Germline DNA sequences for human heavyand light chain variable region genes are known in the art (see e.g., the "Vbase" humangermline sequencedatabase; see also Rabat, E.A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) "The Repertoire of HumanGermline VH Sequences Reveals about Fifty Groups of Vpj Segments with DifferentHypervariable Loops" J. Mol. Biol. 227:776-798: and Cox, J.P.L. et al. (1994) "ADirectory of Human Germ-line VK Segments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated hereinby reference). To obtain a DNA fragment encoding the heavy chain variable region of ί * D2E7, or a D2E7-related antibody, a member of the V^3 family of human germline VHgenes is amplified by standard PCR. Most preferably, the DP-31 VH germline sequenceis amplified. To obtain a DNA fragment encoding the light chain variable region ofD2E7, or a D2E7-related antibody, a member of the VKI family of human germline VLgenes is amplified by standard PCR. Most preferably, the A20 VL germline sequence is

<img img-format="tif" img-content="drawing" file="IL125697AD00022.tif" id="idf0002" />

ATTORNEYD-^ 'NO: BBI-043CPPC - 19- 12 10.02.1997 10 15 20 25 30 « amplified. PCR primers suitable for use in amplifying the DP-31 germline VH and A20germline VL sequences can be designed based on the nucleotide sequences disclosed inthe references cited supra, using standard methods.

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

The amino acid sequences encoded by the germline VH and VL DNA sequences are firstcompared to the D2E7 or D2E7-related VH and VL amino acid sequences to identifyamino acid residues in the D2E7 or D2E7-related sequence that differ from germline.Then, the appropriate nucleotides of the germline DNA sequences are mutated such thatthe 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 PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCRprimers such that the PCR product contains the mutations) or site-directed mutagenesis.

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

Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments areobtained (by amplification and mutagenesis of germline VH and VL genes, as describedabove), these DNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes to full-length antibodychain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- orVH-encoding DNA fragment is operatively linked to another DNA fragment encodinganother 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 DNAfragments are joined such that the amino acid sequences encoded by the two DNAfragments remain in-frame.

The isolated DNA encoding the VH region can be converted to a full-lengthheavy chain gene by operatively linking the VH-encoding DNA to another DNAmolecule encoding heavy chain constant regions (CHI, CH2 and CH3). The sequencesof human heavy chain constant region genes are known in the art (see e.g., Kabat, E.A.,et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242) and DNA 35

ATTORNEY!} TNO: BBI-043CPPC -20- fragments encompassing these regions can be obtained by standard PCR amplification.

The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or

IgD constant region, but most preferably is an IgGl or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.

The isolated DNA encoding the VL region can be converted to a full-length lightchain gene (as well as a Fab light chain gene) by operatively linking the VL-encodingDNA to another DNA molecule encoding the light chain constant region, CL. Thesequences of human light chain constant region genes are known in the art (see e.g.,Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standard PCRamplification. 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 operativelylinked to another fragment encoding a flexible linker, e.g, encoding the amino acidsequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as acontiguous single-chain protein, with the VL and VH regions joined by the flexiblelinker (see e.g, Bird et al. (1988) Science 242:423-426: Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions of the invention, DNAs encodingpartial or full-length light and heavy chains, obtained as described above, are insertedinto expression vectors such that the genes are operatively linked to transcriptional andtranslational control sequences. In this context, the term "operatively linked" is intendedto mean that an antibody gene is ligated into a vector such thaftranscriptional andtranslational control sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. The expression vectorand expression control sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chain gene can beinserted into separate vector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expression vector by( standard methods (e.g., ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites are present). Prior toinsertion of the D2E7 or D2E7-related light or heavy chain sequences, the expressionvector may already carry antibody constant region sequences. For example, oneapproach to converting the D2E7 or D2E7-related VH and VL sequences to full-length ATTORNEY!)

ΓΝΟ: BBI-043CPPC -21 - antibody genes is to insert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that the VH segment isoperatively linked to the CH segment(s) within the vector and the VL segment isoperatively linked to the CL segment within the vector. Additionally or alternatively,the recombinant expression vector can encode a signal peptide that facilitates secretionof the antibody chain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors ofthe invention carry regulatory sequences that control the expression of the antibodychain genes in a host cell. The term "regulatory sequence" is intended to includespromoters, enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibody chain genes. Suchregulatory sequences are described, for example, in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Itwill 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 choiceof the host cell to be transformed, the level of expression of protein desired, etc.

Preferred regulatory sequences for mammalian host cell expression include viralelements that direct high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMVpromoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer),adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For —further description of viral regulatory elements, and sequences thereof, see e.g., 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 Schafiner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g., origins of replication)and selectable marker genes. The selectable marker gene facilitates selection of host‘cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216,4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, ona host cell into which the vector has been introduced. Preferred selectable marker genes &lt; ATTORNEYD'

TNO: BBI-043CPPC -22- include the dihydrofolate reductase (DHFR) gene (for use in dhfi- host cells withmethotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vectors) encodingthe heavy and light chains is transfected into a host cell by standard techniques. Thevarious forms of the term "transfection" are intended to encompass a wide variety oftechniques commonly used for the introduction of exogenous DNA into a prokaryotic oreukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express theantibodies of the invention in either prokaryotic or eukaryotic host cells, expression ofantibodies in eukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammalian cells, are morelikely than prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibody genes has beenreported 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 theinvention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells,described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, usedwith a DHFR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp(1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introduced into mammalianhost cells, the antibodies are produced by culturing the host cells for a period of timesufficient 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 proteinpurification methods.

Host cells can also be used to produce portions of intact antibodies, such as Fabfragments or scFv molecules. It will be understood that variations on the aboveprocedure are within the scope of the present invention. For example, it may bedesirable to transfect a host cell with DNA encoding either the light chain or the heavychain (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 lightand heavy chains that is not necessary for binding to hTNFa. The molecules expressedfrom such truncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy and light chain are

<img img-format="tif" img-content="drawing" file="IL125697AD00023.tif" id="idf0003" />

ATTORNEYD TNO: BBI-043CPPC -23- specific for an antigen other than hTNFa by crosslinking an antibody of the invention toa second antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding boththe antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cellsby 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 (e.g., derived from SV40, CMV, adenovirus andthe like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40enhancer/AdMLP promoter regulatory element) to drive high levels of transcription ofthe genes. The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells are culture to allow forexpression of the antibody heavy and light chains and intact antibody is recovered fromthe culture medium. Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select for transformants, culturethe 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 theantibodies and antibody portions of the invention. The nucleotide sequence encodingthe D2E7 light chain variable region is shown in Figure 7 and SEQ ID NO: 36. TheCDR1 domain of the LCVR encompasses nucleotides 70-102, the CDR2 domainencompasses nucleotides 148-168 and the CDR3 domain encompasses nucleotides 265-291. The nucleotide sequence encoding the D2E7 heavy chain variable region is shownin Figure 8 and SEQ ID NO: 37. The CDR1 domain of the HCVR encompassesnucleotides 91-105, the CDR2 domain encompasses nucleotides 148-198 and the CDR3domain encompasses nucleotides 295-330. It will be appreciated by the skilled artisanthat nucleotide sequences encoding D2E7-related antibodies, or portions thereof (e.g., aCDR domain, such as a CDR3 domain), can be derived from the nucleotide sequencesencoding the D2E7 LCVR and HCVR using the genetic code and standard molecularbiology techniques.

In one embodiment, the invention provides an isolated nucleic acid encoding alight chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 (i.e.,the D2E7 VL CDR3), or modified from SEQ ID NO: 3 by a single alanine substitutionat position 1,4, 5, 7 or 8 or by one to five conservative amino acid substitutions atpositions 1,3,4, 6, 7, 8 and/or 9. This nucleic acid can encode only the CDR3 region ATTORNEYD;

TNO: BBI-043CPPC -24- or, more preferably, encodes an entire antibody light chain variable region (LCVR). Forexample, the nucleic acid can encode an LCVR having a CDR2 domain comprising theamino acid sequence of SEQ ID NO: 5 (i.e., the D2E7 VL CDR2) and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 7 (i.e., the D2E7 VL CDR1).

In another embodiment, the invention provides an isolated nucleic acid encodinga heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4(z.e., the D2E7 VH CDR3), or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2,3,4, 5,6, 8, 9,10 or 11 or by one to five conservative aminoacid substitutions at positions 2, 3,4, 5,6, 8, 9, 10,11 and/or 12. This nucleic acid canencode only the CDR3 region or, more preferably, encodes an entire antibody heavychain variable region (HCVR). For example, the nucleic acid can encode a HCVRhaving a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 (i.e., theD2E7 VH CDR2) and a CDR1 domain comprising the amino acid sequence of SEQ IDNO: 8 (/.e., the D2E7 VH CDR1).

In yet another embodiment, the invention provides isolated nucleic acidsencoding a D2E7-related CDR3 domain, e.g., comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO: 3, SEQ ID NO 4, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.

In still another embodiment, the invention provides an isolated nucleic acidencoding an antibody light chain variable region comprising the amino acid sequence ofSEQ ID NO: 1 (i.e., the D2E7 LCVR). Preferably this nucleic acid comprises thenucleotide sequence of SEQ ID NO: 36, although the skilled artisan will appreciate thatdue to the degeneracy of the genetic code, other nucleotide sequences can encode theamino acid sequence of SEQ ID NO: 1. The nucleic acid can encode only the LCVR orcan 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 sequenceof SEQ ID NO: 2 (i.e., the D2E7 HCVR). Preferably this nucleic acid comprises thenucleotide sequence of SEQ ID NO: 37, although the skilled artisan will appreciate thatdue to the degeneracy of the genetic code, other nucleotide sequences can encode theamino acid sequence of SEQ ID NO: 2. The nucleic acid can encode only the HCVR or

ATTORNEYD' NO: BBI-043CPPC -25- can also encode a heavy chain constant region, operatively linked to the HCVR. Forexample, the nucleic acid can comprise an IgGl or IgG4 constant region. In oneembodiment, this nucleic acid is in a recombinant expression vector.

The invention also provides recombinant expression vectors encoding both anantibody heavy chain and an antibody light chain. For example, in one embodiment, theinvention provides a recombinant expression vector encoding: a) an antibody light chain having a variable region comprising the amino acidsequence of SEQ ID NO: 1 (i.e., the D2E7 LCVR); and b) an antibody heavy chain having a variable region comprising the amino acidsequence of SEQ ID NO: 2 (i.e., the D2E7 HCVR).

The invention also provides host cells into which one or more of the recombinantexpression vectors of the invention have been introduced. Preferably, the host cell is amammalian host cell, more preferably the host cell is a CHO cell, an NSO cell or a COScell.

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

The method can further comprise isolating the recombinant human antibody from theculture medium. III. Selection of Recombinant Human Antibodies

Recombinant human antibodies of the invention in addition to the D2E7 orD2E7-related antibodies disclosed herein can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library, prepared usinghuman VL and VH cDNAs prepared from mRNA derived from’human lymphocytes.Methodologies for preparing and screening such libraries are known in the art. Inaddition to commercially available kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP'™. phage display kit, catalog no. 240612), examples of methodsand reagents particularly amenable for use in generating and screening antibody displaylibraries 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. WO91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCTPublication 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 PublicationNo. WO 92/09690; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992)

ATTORNEY!)' &gt;NO: BBI-043CPPC 10 15 20 25 30 -26- 10.02.1997

Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281:McCafferty et al., Nature (1990) 348:552-554: Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad etal. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137:and Barbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with high affinity and alow off rate constant for hTNFa, a murine anti-hTNFa antibody having high affinityand a low off rate constant for hTNFa (e.g., MAK 195, the hybridoma for which hasdeposit number ECACC 87 050801) is first used to select human heavy and light chainsequences having similar binding activity toward hTNFa, using the epitope imprinting,or guided selection, methods described in Hoogenboom et al., PCT Publication No. WO93/06213. The antibody libraries used in this method are preferably scFv librariesprepared and screened as described in McCafferty et al., PCT Publication No. WO92/01047, McCafferty et al., Nature (1990) 348:552-554; and Griffiths et al., (1993)EMBO J 12:725-734. The scFv antibody libraries preferably are screened usingrecombinant human TNFa as the antigen.

Once initial human VL and VH segments are selected, "mix and match"experiments, in which different pairs of the initially selected VL and VH segments arescreened for hTNFa binding, are performed to select preferred VL/VH paircombinations. Additionally, to further improve the affinity and/or lower the off rateconstant for hTNFa binding, the VL and VH segments of the preferred VL/VH pair(s)can be randomly mutated, preferably within the CDR3 region of VH and/or VL, in aprocess analogous to the in vivo somatic mutation process responsible for affinitymaturation of antibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying VH and VL regions using PCR primerscomplimentary 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 suchthat the resultant PCR products encode VH and VL segments into which randommutations have been introduced into the VH and/or VL CDR3 regions. These randomlymutated VH and VL segments can be rescreened for binding to hTNFa and sequencesthat 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 becompared to germline heavy and light chain amino acid sequences. In cases wherecertain framework residues of the selected VL and/or VH chains differ from thegermline configuration (e.g., as a result of somatic mutation of the immunoglobulin 35

ATTORNEYD' '" TNO: BBI-043CPPC 12 -27 - 10.02.1997 10 15 genes used to prepare the phage library), it may be desireable to "backmutate" thealtered framework residues of the selected antibodies to the germline configuration (i.e.,change the framework amino acid sequences of the selected antibodies so that they arethe same as the germline framework amino acid sequences). Such "backmutation" (or"germlining") of framework residues can be accomplished by standard molecularbiology methods for introducing specific mutations (e.g., site-directed mutagenesis;PCR-mediated mutagenesis, and the like).

Following screening and isolation of an anti-hTNFa antibody of the inventionfrom a recombinant immunoglobulin display library, nucleic acid encoding the selectedantibody can be recovered from the display package (e.g., from the phage genome) andsubcloned into other expression vectors by standard recombinant DNA techniques. Ifdesired, the nucleic acid can be further manipulated to create other antibody forms of theinvention (e.g., linked to nucleic acid encoding additional immunoglobulin domains,such as additional constant regions). To express a recombinant human antibody isolatedby screening of a combinatorial library, the DNA encoding the antibody is cloned into arecombinant expression vector and introduced into a mammalian host cells, as describedin further detail in Section II above. IV. Pharmaceutical Compositions and Pharmaceutical Administration 20 The antibodies and antibody-portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, thepharmaceutical composition comprises an antibody or antibody portion of the inventionand a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptablecarrier" includes any and all solvents, dispersion media, coatings, antibacterial and 25 antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers includeone or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol andthe like, as well as combinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium 30 chloride in the composition. Pharmaceutically acceptable carriers may further compriseminor amounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectiveness of the antibody orantibody portion.

The compositions of this invention may be in a variety of forms. These include, 35 for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders,

ATTORNEYD' TNO: BBI-043CPPC f -28- liposomes and suppositories. The preferred form depends on the iritended mode ofadministration and therapeutic application. Typical preferred compositions are in theform of injectable or infusible solutions, such as compositions similar to those used forpassive immunization of humans with other antibodies. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In a preferred embodiment, the antibody is administered by intravenousinfusion or injection. In another preferred embodiment, the antibody is administered byintramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditionsof manufacture and storage. The composition can be formulated as a solution,microemulsion, dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared by incorporating the activecompound into a sterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the use of a coating suchas lecithin, by the maintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectable compositions can be broughtabout 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 administeredby a variety of methods known in the art, although for many therapeutic applications, thepreferred route/mode of administration is intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapid release, such as a ) controlled release formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatible polymers can beused, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparation of suchformulations are patented or generally known to those skilled in the art. See, e.g.,

ATTORNEYDOl TNO: BBI-043CPPC -29-

Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the invention may beorally administered, for example, with an inert diluent or an assimilable edible carrier.The compound (and other ingredients, if desired) may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, wafers, and the like. To administer a compound of theinvention by other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion of the inventionis coformulated with and/or coadministered with one or more additional therapeuticagents that are useful for treating disorders in which TNFa activity is detrimental. Forexample, an anti-hTNFa antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additional antibodies that bindother targets (e.g., antibodies that bind other cytokines or that bind cell surfacemolecules), one or more cytokines, soluble TNFa receptor (see e.g., PCT PublicationNo. WO 94/06476) and/or one or more chemical agents that inhibit hTNFa productionor activity (such as cyclohexane-ylidene derivatives as described in PCT Publication No.WO 93/19751). Furthermore, one or more antibodies of the invention may be used incombination with two or more of the foregoing therapeutic agents. Such combinationtherapies may advantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associated with the variousmonotherapies.

Nonlimiting examples of therapeutic agents for rheumatoid arthritis with whichan antibody, or antibody portion, of the invention can be combined include thefollowing: non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFaantibody; Celltech/Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g, Arthritis &amp;amp;Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see e g.,

ΓΝΟ: BBI-043CPPC -30- 4rthritis &amp;amp; Rheumatism (1995) Vol. 38. S185); DAB 486-IL-2 and/or DAB 389-IL-24L-2 fusion proteins; Seragen; see e.g., Arthritis &amp;amp; Rheumatism (1993) Vol. 36, 1223);Anti-Tac (humanized anti-IL-2Ra; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g., agonistantibodies); IL-IRA (IL-1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNFR(soluble TNF binding protein; see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39. No. 9(supplement), S284; Amer. J. Physiol. - Heart and Circulatory Physiology (1995) Vol.268. pp. 37-42); R973401 (phosphodiesterase Type IV inhibitor; see e.g., Arthritis &amp;amp;Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; seee.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39, -No. 9 (supplement), S81); Iloprost (seee.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39. No. 9 (supplement), S82); methotrexate;thalidomide (see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S282) and thalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatory andcytokine inhibitor; see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39, No. 9 (supplement),S131; Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid (inhibitorof plasminogen activation; see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39. No. 9(supplement), S284); T-614 (cytokine inhibitor; see e.g., Arthritis &amp;amp; Rheumatism (1996)Vol. 39. No. 9 (supplement), S282); prostaglandin El (see e.g., Arthritis &amp;amp; Rheumatism(1996) Vol. 39, No. 9 (supplement), S282); Tenidap (non-steroidal anti-inflammatorydrug; see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39. No. 9 (supplement), S280);Naproxen (non-steroidal anti-inflammatory drug; see e.g., Neuro Report (1996) Vol. 7,pp. 1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug);Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39, No.9 (supplement), S281); Azathioprine (see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39,No. 9 (supplement), S281); ICE inhibitor (inhibitor of the enzyme interleukin-1 βconverting enzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitos of vascular endothelial cellgrowth factor or vascular endothelial cell growth factor receptor; inhibitors ofangiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies; interleukin-11 (see e.g., Arthritis &amp;amp;Rheumatism (1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see e.g.,Arthritis &amp;amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis &amp;amp; Rheumatism (1996) Vol. 39, No. 9 (supplement), SI20);

ATTORNEYDCX TJO: BBI-043CPPC -31- gold; penicillamine; chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation; anti-thymocyte globulin;anti-CD4 antibodies; CD5-toxins; orally-administered peptides and collagen; lobenzaritdisodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (HoughtenPharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides(ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (ΤΡΙΟ; T CellSciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline;anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; seee.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777): auranofin;phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin);amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.

Nonlimiting examples of therapeutic agents for inflammatory bowel disease withwhich an antibody, or antibody portion, of the invention can be combined include thefollowing: budenoside; epidermal growth factor; corticosteroids; cyclosporin,sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-Ιβ monoclonal antibodies; anti-IL-6monoclonal antibodies; growth factors; eiastase inhibitors; pyridinyl-imidazolecompounds; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody;

Celltech/Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75kD TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis &amp;amp; Rheumatism (1994)Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein; Hoffmann-LaRoche); interleukin-10 (SCH 52000; ScheringPlough); IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); interleukin-11;glucuronide- or dextran-conjugated prodrugs of prednisolone, dexamethasone orbudesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302;

Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (ΤΡΙΟ; T Cell Sciences, Inc.);slow-release mesalazine; methotrexate; antagonists of Platelet Activating Factor (PAF);ciprofloxacin; and lignocaine.

Nonlimiting examples of therapeutic agents for multiple sclerosis with which anantibody, or antibody portion, of the invention can be combined include the following:corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide;cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β la (Avonex™;Biogen); interferon-β lb (Betaseron™; Chiron/Berlex); Copolymer 1 (Cop-1;Copaxone™; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous

TNO: BBI-043CPPC 72

R Q 7/2 -32- immunoglobulin; clabribine; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody;Celltech/Bayer); cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75kD TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis &amp;amp; Rheumatism (1994)Vol. 37, S295; J. Invest. Med. (1996) Vol. 44,235A); 55 kdTNFR-lgG (55 kD TNFreceptor-IgG fusion protein; Hofftnann-LaRoche); IL-10; IL-4; and IL-10 and/or IL-4agonists (e.g., agonist antibodies).

Nonlimiting examples of therapeutic agents for sepsis with which an antibody, orantibody portion, of the invention can be combined include the following: hypertonicsaline solutions; antibiotics; intravenous gamma globulin; continuous hemofiltration;carbapenems (e.g., meropenem); antagonists of cytokines such as TNFa, IL-Ιβ, IL-6and/or IL-8; CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer);cA2 (chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis &amp;amp; Rheumatism (1994) Vol. 37. S295; J.Invest. Med. (1996) Vol. 44. 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusionprotein; Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466(Houghten Pharmaceuticals, Inc.); SK&amp;amp;F 107647 (low molecular peptide; SmithKlineBeecham); tetravalent guanylhydrazone CNI-1493 (Picower Institute); Tissue FactorPathway Inhibitor (TFPI; Chiron); PHP (chemically modified hemoglobin; APEXBioscience); iron chelators and chelates, including diethylenetriamine pentaacetic acid -iron (III) complex (DTPA iron (III); Molichem Medicines); lisofylline (synthetic smallmolecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan (aqeuous solubleβΙ^Κιοηη; Alpha-Beta Technology); apolipoprotein A-l reconstituted with lipids;chiral hydroxamic acids (synthetic antibacterials that inhibit lipid A biosynthesis); anti-endotoxin antibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.); rBPfyi(recombinant N-terminal fragment of human Bactericidal/Pernieability-IncreasingProtein); and Synthetic Anti-Endotoxin Peptides (SAEP; BiosYnth ResearchLaboratories);

Nonlimiting examples of therapeutic agents for adult respiratory distresssyndrome (ARDS) with which an antibody, or antibody portion, of the invention can becombined include the following: anti-IL-8 antibodies; surfactant replacement therapy;CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer); cA2(chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgGfusion protein; Immunex; see e.g., Arthritis &amp;amp; Rheumatism (1994) Vol. 37, S295; J.Invest. Med. (1996) Vol. 44, 235A); and 55 kdTNFR-IgG (55 kD TNF receptor-IgGfusion protein; Hoffmann-LaRoche).

ATTORNEYDOt ΓΝΟ: BBI-043CPPC -33-

The use of the antibodies, or antibody portions, of the invention in combinationwith other therapeutic agents is discussed further in subsection IV.

The pharmaceutical compositions of the invention may include a "therapeuticallyeffective amount" or a "prophylactically effective amount" of an antibody or antibodyportion of the invention. A "therapeutically effective amount" refers to an amounteffective, at dosages and for periods of time necessary, to achieve the desired therapeuticresult. A therapeutically effective amount of the antibody or antibody portion may varyaccording to factors such as the disease state, age, sex, and weight of the individual, andthe ability of the antibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighed by thetherapeutically beneficial effects. A "prophylactically effective amount" refers to anamount effective, at dosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used in subjects prior to or atan earlier stage of disease, the prophylactically effective amount will be less than thetherapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response (e.g.,a therapeutic or prophylactic response). For example, a single bolus may beadministered, several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity of active'compoundcalculated to produce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms of the invention aredictated by and directly dependent on (a) the unique characteristics of the activecompound 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 thetreatment 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 typeand severity of the condition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted over time according tothe individual need and the professional judgment of the person administering or

X

ATTORNEYDOt TNO: BBI-043CPPC -34- supervising the administration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope or practice of theclaimed composition. IV. Uses of the Antibodies of the Invention

Given their ability to bind to hTNFa, the anti-hTNFa antibodies, or portionsthereof, of the invention can be used to detect hTNFa (e.g., in a biological sample, suchas serum or plasma), using a conventional immunoassay, such as an enzyme linkedimmunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method for detecting hTNFa in a biological sample comprising contacting a biological sample with an antibody, or antibody portion, of the invention and detecting either the antibody (or antibody portion) bound to hTNFa or unbound antibody (or antibody portion), to thereby detect hTNFa in the biological sample. The antibody is directly or indirectly labeled with a detectable 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, β-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 125 131 35 3 luminol; and examples of suitable radioactive material include I, I, S or H.

Alternative to labeling the antibody, hTNFa can be assayed in biological fluidsby a competition immunoassay utilizing rhTNFa standards labeled with a detectablesubstance and an unlabeled anti-hTNFa antibody. In this assay, the biological sample,the labeled rhTNFa standards and the anti-hTNFa antibody are combined and theamount of labeled rhTNFa standard bound to the unlabeled antibody is determined. Theamount of hTNFa in the biological sample is inversely proportional to the amount oflabeled rhTNFa standard bound to the anti-hTNFa antibody. A D2E7 antibody of the invention can also be used to detect TNFas from speciesother than humans, in particular TNFas from primates (e.g., chimpanzee, baboon,marmoset, cynomolgus and rhesus), pig and mouse, since D2E7 can bind to each ofthese TNFas (discussed further in Example 4, subsection E).

The antibodies and antibody portions of the invention are capable of neutralizinghTNFa activity both in vitro and in vivo (see Example 4). Moreover, at least some of -35- 125697/2 the antibodies of the invention, such as D2E7, can neutralize TNFa activity from otherspecies. Accordingly, the antibodies and antibody portions of the invention can be usedto inhibit TNFa activity, e.g.,, in a cell culture containing hTNFa, in human subjects orin other mammalian subjects having TNFas with which an antibody of the inventioncross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse).Preferably, the TNFa is human TNFa. For example, in a cell culture containing, orsuspected of containing hTNFa, an antibody or antibody portion of the invention can beadded to the culture medium to inhibit hTNFa activity in the culture. TNFa has been implicated in the pathophysiology of a wide variety of disorders(see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Patent No. 5,231,024 toMoeller et al.·, European Patent Publication No. 260 610 BI by Moeller, A.). Preferably, the TNFa is human TNFa and the subject is a human subject. Alternatively, the subjectcan be a mammal expressing a TNFa with which an antibody of the invention cross-reacts. Still further the subject can be a mammal into which has been introduced hTNFa(e.g., by administration of hTNFa or by expression of an hTNFa transgene). Anantibody of the invention can be administered to a human subject for therapeuticpurposes (discussed further below). Moreover, an antibody oflhe invention can beadministered to a non-human mammal expressing a TNFa with which the antibodycross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal modelof human disease. Regarding the latter, such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosagesand time courses of administration).

As used herein, the term "a disorder in which TNFa activity is detrimental" isintended to include diseases and other disorders in which the presence of TNFa in asubject suffering from the disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFa activity isdetrimental is a disorder in which inhibition of TNFa activity is expected to alleviate the 01124304X33-01 .

ATTORNEYDOt ΓΝΟ: BBI-043CPPC -36- symptoms and/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNFa in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration of TNFa in serum,plasma, synovial fluid, etc. of the subject), which can be detected, for example, using ananti-TNFa antibody as described above. There are numerous examples of disorders inwhich TNFa activity is detrimental. The use of the antibodies and antibody portions ofthe 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, vascularleakage syndrome, organ necrosis, stimulation of the release of toxic secondarymediators and activation of the clotting cascade (see e.g., Moeller, A., et al. (1990)Cytokine 2:162-169; U.S. Patent No. 5,231,024 to Moeller et al.·, European PatentPublication No. 260 610 BI by Moeller, A.; 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 inventioncan 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 theinvention can be coadministered with one or more additional therapeutic agents that mayfurther alleviate sepsis, such as an interleukin-1 inhibitor (such as those described inPCT Publication Nos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (seee.g., PCT Publication No. WO 93/11793) or an antagonist of platelet activating factor(see e.g., European Patent Application Publication No. EP 374 510). Other combinationtherapies for the treatment of sepsis are discussed further in subsection III.

Additionally, in a preferred embodiment, an anti-TNFa antibody or antibodyportion of the invention is administered to a human subject within a subgroup of sepsispatients having a serum or plasma concentration of IL-6 above 500 pg/ml, and morepreferably 1000 pg/ml, at the time of treatment (see PCT Publication No. WO 95/20978by Daum, L., et al.}. B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in thepathophysiology of a variety of autoimmune diseases. For example, TNFa has beenimplicated in activating tissue inflammation and causing joint destruction in rheumatoid

ATTORNEYDOt ~ΎνΟ: BBI-043CPPC -37- arthritis (see eg., Moeller, A., 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, A.; Tracey and Cerami, supra·, Arend, W.P. and Dayer, J-M. (1995) Arth. Rheum.38:151-160; Fava, R.A., et al. (1993) Clin. Exp. Immunol. 94:261-266). TNFa also hasbeen implicated in promoting the death of islet cells and in mediating insulin resistancein diabetes (see e.g., Tracey and Cerami, supra·, PCT Publication No. WO 94/08609).TNFa also has been implicated in mediating cytotoxicity to oligodendrocytes andinduction of inflammatory plaques in multiple sclerosis (see e.g., Tracey and Cerami,supra). Chimeric and humanized murine anti-hTNFa antibodies have undergoneclinical testing for treatment of rheumatoid arthritis (see e.g., Elliott, M.J., et al. (1994)Lancet 344:1125-1127; Elliot, M.J., et al. (1994) Lancet 344Λ105-1110; Rankin, E.C.,etal. (1995) Br. J. Rheumatol. 34:334-342).

The human antibodies, and antibody portions of the invention can be used totreat autoimmune diseases, in particular those associated with inflammation, includingrheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy,multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome.Typically, the antibody, or antibody portion, is administered systemically, although forcertain disorders, local administration of the antibody or antibody portion at a site ofinflammation may be beneficial (e.g., local administration in the joints in rheumatoidarthritis or topical application to diabetic ulcers, alone or in combination with acyclohexane-ylidene derivative as described in PCT Publication No. WO 93/19751). Anantibody, or antibody portion, of the invention also can be administered with one ormore additional therapeutic agents useful in the treatment of autoimmune diseases, asdiscussed further in subsection III. C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biological effectsobserved in a variety of infectious diseases. For example, TNFa has been implicated inmediating brain inflammation and capillary thrombosis and infarction in malaria. TNFaalso has been implicated in mediating brain inflammation, inducing breakdown of theblood-brain barrier, triggering septic shock syndrome and activating venous infarction in'meningitis. TNFa also has been implicated in inducing cachexia, stimulating viralproliferation and mediating central nervous system injury in acquired immunedeficiency syndrome (AIDS). Accordingly, the antibodies, and antibody portions, of theinvention, can be used in the treatment of infectious diseases, including bacterialmeningitis (see e.g., European Patent Application Publication No. EP 585 705), cerebral

ATTORNEYDOl ~VNO: BBI-043CPPC -38- malaria, AIDS and AIDS-related complex (ARC) (see e.g., European Patent ApplicationPublication No. EP 230 574), as well as cytomegalovirus infection secondary totransplantation (see e.g., Fietze, E., et al. (1994) Transplantation 58:675-680). Theantibodies, and antibody portions, of the invention, also can be used to alleviatesymptoms associated with infectious diseases, including fever and myalgias due toinfection (such as influenza) and cachexia secondary to infection (e.g., secondary toAIDS or ARC). D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograftrejection and graft versus host disease (GVHD) and in mediating an adverse reactionthat has been observed when the rat antibody OKT3, directed against the T cell receptorCD3 complex, is used to inhibit rejection of renal transplants (see e.g., Eason, J.D., et al.(1995) Transplantation 59:300-305; Suthanthiran, M. and Strom, T.B. (1994) New Engl.J. Med. 331:365-3751 Accordingly, the antibodies, and antibody portions, of the.invention, can be used to inhibit transplant rejection, including rejections of allograftsand xenografts and to inhibit GVHD. Although the antibody or antibody portion may beused alone, more preferably it is used in combination with one or more other agents thatinhibit the immune response against the allograft or inhibit GVHD. For example, in oneembodiment, an antibody or antibody portion of the invention is used in combinationwith OKT3 to inhibit OKT3-induced reactions. In another embodiment, an antibody orantibody portion of the invention is used in combination with one or more antibodiesdirected at other targets involved in regulating immune responses, such as the cellsurface molecules CD25 (interleukin-2 receptor-α), CD1 la (LFA-1), CD54 (ICAM-1),CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2J. In yet anotherembodiment, an antibody or antibody portion of the invention is used in combinationwith one or more general immunosuppressive agents, such as cyclosporin A or FK506. E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia, stimulatingtumor growth, enhancing metastatic potential and mediating cytotoxicity in; malignancies. Accordingly, the antibodies, and antibody portions, of the invention, canbe used in the treatment of malignancies, to inhibit tumor growth or metastasis and/or toalleviate cachexia secondary to malignancy. The antibody, or antibody portion, may beadministered systemically or locally to the tumor site.

ATTORNEYDOk ΓΝΟ: BBI-043CPPC η -39- F. Pulmonary Disorders

Tumor necrosis factor has been implicated in the pathophysiology of adultrespiratory distress syndrome (ARDS), including stimulating leukocyte-endothelialactivation, directing cytotoxicity to pneumocytes and inducing vascular leakagesyndrome. Accordingly, the antibodies, and antibody portions, of the invention, can beused to treat various pulmonary disorders, including adult respiratory distress syndrome(see e.g., PCT Publication No. WO 91/04054), shock lung, chronic pulmonaryinflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. Theantibody, or antibody portion, may be administered systemically or locally to the lungsurface, for example as an aerosol. An antibody, or antibody portion, of the inventionalso can be administered with one or more additional therapeutic agents useful in thetreatment of pulmonary disorders, as discussed further in subsection III. G. Intestinal Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofinflammatory bowel disorders (see e.g., Tracy, K.J., et al. (1986) Science 234:470-474:Sun, X-M., et al. (1988) J. Clin. Invest. 81.: 1328-1331; MacDonald, T.T., et al. (1990)Clin. Exp. Immunol. 81.:301-305). Chimeric murine anti-hTNFa antibodies haveundergone clinical testing for treatment of Crohn’s disease (van Dullemen, H.M., 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 idiopathicinflammatory bowel disease, which includes two syndromes, Crohn's disease andulcerative colitis. An antibody, or antibody portion, of the invention also can beadministered with one or more additional therapeutic agents useful in the treatment ofintestinal disorders, as discussed further in subsection III. H. Cardiac Disorders

The antibodies, and antibody portions, of the invention, also can be used to treatvarious cardiac disorders, including ischemia of the heart (see e.g., European PatentApplication Publication No. EP 453 898) and heart insufficiency (weakness of the heartmuscle)(see e.g., PCT Publication No. WO 94/20139). I. Others

The antibodies, and antibody portions, of the invention, also can be used to treatvarious other disorders in which TNFa activity is detrimental. Examples of otherdiseases and disorders in which TNFa activity has been implicated in the

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10.02.1997 ' ATTORNEYDoi NO: BBI-043CPPC . ‘ ~ - i 2 569 7/ 2 -40- pathophysiology, and thus which can be treated using an antibody, or antibody portion,of the invention, include inflammatory bone disorders and bone resorption disease (seee.g., Bertolini, D.R., et al. (1986) Nature 319:516-518: Konig, A., et al. (1988)7. BoneMiner. Res. 3:621-627; Lemer, U.H. and Ohlin, A. (1993) J. Bone Miner. Res. 8:147- 5 155; and Shankar, G. and Stem, P.H. (1993) Bone 14:871 -876), hepatitis, including alcoholic hepatitis (see e.g., McClain, C.J. and Cohen, D.A. (1989) Hepatology 9-.349-351; Felver, M.E., et al. (1990) Alcohol. Clin. Exp. Res. 14:255-259; and Hansen, J., etal. (1994) Hepatology 20:461 -474), viral hepatitis (Sheron, N., et al. (1991) J. Hepatol. 12:241 -245; and Hussain, M. J., et al. (1994) J. Clin. Pathol. 47:1112-1115), and 10 fulminant hepatitis; coagulation disturbances (see e.g., van der Poll, T., etal. (1990) N.

Engl. J. Med. 322:1622-1627; and van der Poll, T., et al. (1991) Prog Clin. Biol. Res.367:55-60), bums (see e.g., Giroir, B.P., et al. (1994) Am. J. Physiol. 267:H118-124:and Liu, X.S., et al. (1994) Burns 20:40-44), reperfusion injury (see e.g, Scales, W.E.,et al. (1994) Am. J. Physiol. 267:G1122-1127; Serrick, C., et al. (1994) Transplantation 15 58:1158-1162; and Yao, Y.M., et al. (1995) Resuscitation 29:157-168), keloid formation (see e.g., McCauley, R.L., et al. (1992) J. Clin. Immunol. 12:300-308), scar tissueformation; pyrexia; periodontal disease; obesity and radiation toxicity.

This invention is further illustrated by the following examples which should not 20 be construed as limiting. The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporated by reference. EXAMPLE 1: Kinetic Analysis of Binding of Human Antibodies to hTNFa 25 Real-time binding interactions between ligand (biotinylated recombinant human TNFa (rhTNFa) immobilized on a biosensor matrix) and analyte (antibodies insolution) were measured by surface plasmon resonance (SPR) using the BIAcore system(Pharmacia Biosensor, Piscataway, NJ). The system utilizes the optical properties ofSPR to detect alterations in protein concentrations within a dextran biosensor matrix. 30 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 35 sensorgram. ATTORNEYDOt TNO: BBI-043CPPC ( -41 -

To facilitate immobilization of biotinylated rhTNFa on the biosensor matrix,streptavidin is covalently linked via free amine groups to the dextran matrix by firstactivating carboxyl groups on the matrix with 100 mM N-hydroxysuccinimide (NHS)and 400 mM N-ethyl-N'-(3-diethylaminopropyl) carbodiimide hydrochloride (EDC).Next, streptavidin is injected across the activated matrix. Thirty-five microliters ofstreptavidin (25 pg/ml), diluted in sodium acetate, pH 4.5, is injected across theactivated biosensor and free amines on the protein are bound directly to the activatedcarboxyl groups. Unreacted matrix EDC-esters are deactivated by an injection of 1 Methanolamine. 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 of biotin (D-biotinyl-s-aminocaproic acid N-hydroxysuccinimide ester; Boehringer Mannheim Cat.No. 1008 960) in 500 μΐ dimethylsulfoxide to make a 10 mg/ml solution. Tenmicroliters of biotin was added per ml of rhTNFa (at 2.65 mg/ml) for a 2:1 molar ratioof biotin to rhTNFa. The reaction was mixed gently and incubated for two hours atroom temperature in the dark. A PD-10 column, Sephadex G-25M (Pharmacia CatalogNo. 17-0851-01) was equilibrated with 25 ml of cold PBS and loaded with 2 ml ofrhTNFa-biotin per column. The column was eluted with 10 χ 1 ml cold PBS. Fractionswere collected and read at OD280 (1.0 OD = 1.25 mg/ml). The appropriate fractionswere pooled and stored at -80° C until use. Biotinylated rhTNFa also is commerciallyavailable (R &amp;amp; D Systems Catalog No. FTA00, Minneapolis, MN).

Biotinylated rhTNFa to be immobilized on the matrix via streptavidin wasdiluted in PBS running buffer (Gibco Cat. No. 14190-144, Gibco BRL, Grand Island,NY) supplemented with 0.05% (BIAcorej-surfactant P20 (Pharmacia BR-1000-54,Pharmacia Biosensor, Piscataway, NJ). To determine the capacity of rhTNFa-specificantibodies to bind immobilized rhTNFa, a binding assay was conducted as follows.Aliquots of biotinylated rhTNFa (25 nM; 10 μΐ aliquots) were injected through thestreptavidin-coupled dextran matrix at a flow rate of 5 μΐ/min. Before injection of theprotein and immediately afterward, PBS buffer alone flowed through each flow cell.

The net difference in signal between baseline and approximately 30 sec. aftercompletion of biotinylated rhTNFa injection was taken to represent the binding value-(approximately 500 RU). Direct rhTNFa-specific antibody binding to immobilizedbiotinylated rhTNFa was measured. Antibodies (20 pg/ml) were diluted in PBSrunning buffer and 25 μΐ aliquots were injected through the immobilized proteinmatrices at a flow rate of 5 μΐ/min. Prior to injection of antibody, and immediatelyafterwards, PBS buffer alone flowed through each flow cell. The net difference in 42-

ATTORNEYDOt ’’NO: BBI-043CPPC 7 2 5 6 9 7/2 baseline signal and signal after completion of antibody injection was taken to representthe binding value of the particular sample. Biosensor matrices were regenerated using100 mM HCI before injection of the next sample. To determine the off rate (Κθ^·), onrate (K^), association rate (K.J and dissociation rate (Kd) constants, BIAcore kinetic 5 evaluation software (version 2.1) was used.

Representative results of D2E7 (IgG4 full-length antibody) binding to biotinylated rhINFa, as Compared to the mouse mAb MAK 195 (F(ab')2 fragment), areshown below in Table 1. 10

Table 1: Binding of D2E7 IgG4 or MAK 195 to Biotinylated rhTNFa

Antibody [Ab], nM rhTNFa,bound, RUs Ab, bound,RUs rhTNFa/Ab Koff, sec*1,(Avg) D2E7 267 373 1215 1.14 8.45 x 10-5 133 420 1569 1.30 5.42 x 10-5 67 434 1633 1.31 4.75 x IO-5 33 450 1532 1.19 4.46 x IO-5 17 460 1296 0.98 3.47 x IO’5 8 486 936 0.67 2.63 x IO"5 4 489 536 0.38 2.17 x IO'5 2 470 244 0.18 3.68 x IO'5 (4.38 x IO'5) MAK 195 400 375 881 1.20 5.38 x IO’5 200 400 1080 1.38 4.54 x 10-* 100 419 1141 1.39 3.54 x 10-5 50 427 1106 1.32 3.67 x IO"5 25 446 957 1.09 4.41 x IO'5 13 464 708 0.78 3.66 x 10-5 6 474 433 0.47 7.37 x 10-5 3 451 231 0.26 6.95 x IO-5 (4.94 x 10-5)“ 10.02.1997

In a second series of experiments, the molecular kinetic interactions between anIgGl full-length form of D2E7 and biotinylated rhTNF was quantitatively analyzedusing BIAcore technology, as described above, and kinetic rate constants were derived,summarized below in Tables 2, 3 and 4. 15

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ATTORNEYDCX *ΊΓΝΟ: BBI-043CPPC 725 ./ -43 -

Table 2: Apparent dissociation rate constants of the interaction between D2E7 and

biotinylated rhTNF

Experiment Kd(s-1) 1 9.58 x 10-5 2 9.26xl0-5 3 7.60x10-5 Average 8.81 ± 1.06 x 10-5

Table 3: Apparent association rate constants of the interaction between D2E7 and

biotinylated rhTNF

Experiment Ka(M-l,s-l) 1 1.33x105 2 1.05 x IO5 3 3.36x105 Average 1.91 ± 1.26 χ 105 10.02.1997

Table 4: Apparent kinetic reate and affinity constants of D2E7 and biotinylated rhTNF

Experiment Ka(M-l,s-i) Kd (s·1) Kd(M) 1 1.33x105 9.58 x IO’5 7.20 x 10-10 2 1.05 χ 105 9.26 x 10-5 8.82 x 10-10 3 3.36 x IO5 7.60 x IO-5 2.26 x 10-1° Average 1.91 ±1.26x105 8.81 ± 1.06 x IO’5 6.09 ± 3.42 x 10"l° 10 15 20

Dissociation and association rate constants were calculated by analyzing the dissociationand association regions of the sensorgrams by BIA analysis software. Conventionalchemical reaction kinetics were assumed for the interaction between D2E7 andbiotinylated rhTNF molecule: a zero order dissociation and first order associationkinetics. For the sake of analysis, interaction only between one arm of the bivalentD2E7 antibody and one unit of the trimeric biotinylated rhTNF was considered inchoosing molecular models for the analysis of the kinetic data. Three independentexperiments were performed and the results were analyzed separately. The averageapparent dissociation rate constant (kj) of the interaction between D2E7 and biotinylatedrhTNF was 8.81 ± 1.06 χ 10'5 s’1, and the average apparent association rate constant, kawas 1.91 ± 1.26 χ 105 M’1 s’1. The apparent intrinsic dissociation constant (BQ) was thencalculated by the formula: BQ= ky ka. Thus, the mean BQ of D2E7 antibody for rhTNF

ATTORNEYDOt TNO: BBI-043CPPC 2 S S3?/p

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-44- 10.02.1997 derived from kinetic parameters was 6.09 ± 3.42 χ IO'10 M. Minor differences in thekinetic values for the IgGl form of D2E7 (presented in Tables 2, 3 and 4) and the IgG4form of D2E7 (presented in Table 1 and in Examples 2 and 3) are not thought to be truedifferences resulting from the presence of either an IgGl or an IgG4 constant regions butrather are thought to be attributable to more accurate antibody concentrationmeasurements used for the IgGl kinetic analysis. Accoringly, the kinetic values for theIgGl form of D2E7 presented herein are thought to be the most accurate kineticparameters for the D2E7 antibody. 10 EXAMPLE 2: Alanine Scanning Mutagenesis of D2E7 CDR3 Domains A series of single alanine mutations were introduced by standard methods alongthe CDR3 domain of the D2E7 VL and the D2E7 VH regions. The light chainmutations are illustrated in Figure IB (LD2E7*.A1, ED2E7*.A3, LD2E7*.A4, 15 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 mutationsare illustrated in Figure 2B (HD2E7*.A1, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4,HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8 and HD2E7*.A9, having analanine mutation at position 2, 3, 4, 5, 6, 8,9, 10 or 11, respectively, of the D2E7 VH 20 CDR3 domain). The kinetics of rhTNFa interaction with an antibody composed ofwild-type D2E7 VL and VH was compared to that of antibodies composed of 1) a wild-type D2E7 VL paired with an alanine-substituted D2E7 VH; 2) a wild-type D2E7 VHpaired with an alanine-substituted D2E7 VL; or 3) an alanine-substituted D2E7 VLpaired with an alanine-substituted D2E7 VH. All antibodies were tested as full-length, 25 IgG4 molecules. ‘

Kinetics of interaction of antibodies with rhTNFa was determined by surface plasmon resonance as described in Example 1. The Koff rates for the different VH/VLpairs are summarized below in Table 5: 30

Table 5: Binding of D2E7 Alanine-Scan Mutants to Biotinylated rhTNFa VH VL Kofffsec-1) D2E7 VH D2E7 VL 9.65 x IO'5 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 IO'4 HD2E7*.A5 D2E7 VL 2.35 x 10-4

ATTORNEYDOi TNO: BBI-043CPPC -45- HD2E7*.A6 D2E7 VL 2.9 x 10-4 HD2E7*.A7 D2E7 VL 1.0 x 10-4 HD2E7*.A8 D2E7 VL 3.1 x 10-4 HD2E7*.A9 D2E7 VL 8.1 x 10-4 D2E7 VH LD2E7*.A1 6.6 χ IO'5 D2E7 VH LD2E7*.A3 NOT DETECTABLE D2E7 VH LD2E7*.A4 1.75 x 10-4 D2E7 VH LD2E7*.A5 1.8 x IO*4 D2E7 VH LD2E7*.A7 1.4 x IO-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 ofthe D2E7 VL region and VH region are amenable to substitution with a single alanineresidue. Substitution of a single alanine at position 1,4, 5, or 7 of the D2E7 VL CDR3 5 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 parentalD2E7 antibody. Substitution of alanine at position 8 of the D2E7 VL CDR3 or atposition 3 of the D2E7 VH CDR3 gives a 4-fold faster K^ff and an alanine substitutionat position 4 or 11 of D2E7 VH CDR3 gives an 8-fold faster K^, indicating that these 10 positions are more critical for binding to hTNFa. However, a single alanine substitutionat position 1,4,5,7 or 8 of the D2E7 VL CDR3 domain or at position 2,3,4, 5, 6, 8,9,10 or 11 of the D2E7 VH CDR3 domain still results in an anti-hTNFa antibody having aKofj· of 1 x IO-3 sec*1 or less. 15 EXAMPLE 3: Binding Analysis of D2E7-Related Antibodies

A series of antibodies related in sequence to D2E7 were analyzed for theirbinding to rhTNFa, as compared to D2E7, by surface plasmon resonance as described inExample 1. The amino acid sequences of the VL regions tested are shown in Figures 1A 20 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 full-length IgGl or IgG4 antibody or as a scFv) are summarized below in Table 6:

Table 6: Binding of D2E7-Related Antibodies to Biotinylated rhTNFa VH VL Format KoffCsec-1) D2E7 VH D2E7 VL IgGl/IgG4 1 9.65 x 10-5

ATTORNEYDCh ''’'NO: BBI-043CPPC -46- VH1-D2 LOE7 IgGl/IgG4 7.7 x 10-5 VH1-D2 LOE7 scFv 4.6 x 10-4 VH1-D2.N LOE7.T IgG4 2.1 x 10-5 VH1-D2.Y LOE7.A IgG4 2.7x10-5 VH1-D2.N LOE7.A IgG4 3.2x10-5 VH1-D2 EPB12 scFv 8.0 x 10-4 VH1-D2 2SD4 VL scFv 1.94 x IO-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 VH1A11 2SD4 VL scFv 1.34 x 10-2 VH1B12 2SD4 VL scFv 1.01 x io-2 VH1B11 2SD4 VL scFv 9.8 x 10-3 VH1E4 2SD4 VL scFv 1.59x10-2 VH1F6 2SD4 VL scFv 2.29 x IO*2 VH1D8 2SD4 VL scFv 9.5 x 10-3 VH1G1 2SD4 VL scFv 2.14 x IO*2 2SD4 VH EPB12 scFv 6.7 x 10-3 2SD4 VH VL10E4 scFv 9.6 x IO’3 2SD4 VH VL100A9 scFv 1.33 x 10-2 2SD4 VH VL100D2 scFv 1.41 x IO-2 2SD4 VH VL10F4 scFv 1.11 x io-2 2SD4 VH VLLOE5 scFv 1.16 x IO-2 2SD4 VH VLL0F9 scFv 6.09 x 10-3 2SD4 VH VLL0F10 scFv 1.34x10-2 2SD4 VH VLLOG7 scFv 1.56 x IO-2 2SD4 VH VLLOG9 scFv 1.46 x IO*2 2SD4 VH VLLOH1 scFv 1.17 x IO-2 2SD4 VH VLLOHIO scFv 1.12 x IO*2 2SD4 VH VL1B7 scFv 1.3 x 10-2 2SD4 VH VL1C1 scFv 1.36x10-2 2SD4 VH VL1C7 scFv 2.0 x IO*2 2SD4 VH VL0.1F4 scFv 1.76x10-2 2SD4 VH VL0.1H8 scFv 1.14 x 10-2

The slow off rates (i.e., Koff &lt; 1 x IO"4 sec*1) for full-length antibodies (i.e., IgGί 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 5 CDR3 can be occupied by either of these two residues without substantially affecting the IQff. Accordingly, a consensus motif for the D2E7 VL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3). Furthermore, the slow off rates

(i.e., K-off- 1 x Ιθ"4 sec*1) for antibodies having a VH selected from D2E7, VH1-D2.N

ATTORNEYDOi "NO: BBI-043CPPC -47- and VH1-D2.Y, which have either a tyrosine or an asparagine at position 12, indicatethat position 12 of the D2E7 VH CDR3 can be occupied by either of these two residueswithout substantially affecting the Koff. Accordingly, a consensus motif for the D2E7VH CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQID NO: 4).

The results shown in Table 6 demonstrate that, in scFv format, antibodiescontaining the 2SD4 VL or VH CDR3 region exhibit a faster Κθβ· (i.e., K^ff &gt; 1 χ 10*3sec'1) as compared to antibodies containing the D2E7 VL or VH CDR3 region. Withinthe VL CDR3,2SD4 differs from D2E7 at positions 2,5 and 9. As discussed above,however, position 9 may be occupied by Ala (as in 2SD4) or Thr (as in D2E7) withoutsubstantially affecting the K^. Thus, by comparison of 2SD4 and D2E7, positions 2and 5 of the D2E7 VL CDR3, both arginines, can be identified as being critical for theassociation of the antibody with hTNFa. These residues could be directly involved ascontact residues in the antibody binding site or could contribute critically to maintainingthe scaffolding architecture of the antibody molecule in this region. Regarding theimportance of position 2, replacement of Arg (in LOE7, which has the same VL CDR3as D2E7) with Lys (in EP B12) accelerates the off rate by a factor of two. Regarding theimportance of position 5, replacement of Arg (in D2E7) with Ala (in LD2E7*.A5), asdescribed in Example 2, also accelerates the off rate two-fold. Furthermore, withouteither Arg at positions 2 and 5 (in 2SD4), the off rate is five-fold faster. However, itshould be noted that although position 5 is important for improved binding to hTNFa, achange at this position can be negated by changes at other positions, as seen inVLLOE4, VLLOH1 orVL0.1H8.

Within the VH CDR3,2SD4 differs from D2E7 at positions 1, 7 and 12. Asdiscussed above, however, position 12 may be occupied by Ash (as in 2SD4) or Tyr (asin D2E7) without substantially affecting the Koff. Thus, by comparison of 2SD4 andD2E7, positions 1 and 7 of the D2E7 VH CDR3 can be identified as being critical forbinding to hTNFa. As discussed above, these residues could be directly involved ascontact residues in the antibody binding site or could contribute critically to maintainingthe scaffolding architecture of the antibody molecule in this region. Both positions areimportant 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 a3-fold faster off rate than when the D2E7 VH CDR3 is used but this off rate is still fourtimes slower than when the 2SD4 VH CDR3 is used (which has changes at bothpositions 1 and 7 with respect to the D2E7 VH CDR3).

ATTORNEYDOi ΓΝΟ: BBI-043CPPC -48- EXAMPLE 4: Functional Activity of D2E7

To examine the functional activity of D2E7, the antibody was used in severalassays that measure the ability of the antibody to inhibit hTNFa activity, either in vitroor in vivo. A. Neutralization of TNFa-Induced Cytotoxicity in L929 Cells

Human recombinant TNFa (rhTNFa) causes cell cytotoxicity to murine L929cells after an incubation period of 18-24 hours. Human anti-hTNFa antibodies wereevaluated in L929 assays by coincubation of antibodies with rhTNFa and the cells asfollows. A 96-well microtiter plate containing 100 μΐ of anti-hTNFa Abs was seriallydiluted 1/3 down the plate in duplicates using RPMI medium containing 10% fetalbovine serum (FBS). Fifty microliters of rhTNFa was added for a final concentration of500 pg/ml in each sample well. The plates were then incubated for 30 minutes at roomtemperature. Next, 50 μΐ of TNFa-sensitive L929 mouse fibroblasts cells were addedfor 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 aTNFa standard curve, ranging from 2 ng/ml to 8.2 pg/ml, were used to determine thequality of the assay and provide a window of neutralization. The plates were thenincubated overnight (18-24 hours) at 37° C in 5% CO2.

One hundred microliters of medium was removed from each well and 50 μΐ of5 mg/ml 3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT;commercially available from Sigma Chemical Co., St. Louis, MO) in PBS was added.The plates were then incubated for 4 hours at 37° C. Fifty microliters of 20% sodiumdodecyl sulfate (SDS) was then added to each well and the plates were incubatedovernight at 37° C. The optical density at 570/630 nm was measured, curves wereplotted for each sample and IC5QS were determined by standard methods.

Representative results for human antibodies having various VL and VH pairs, ascompared to the murine MAK 195 mAb, are shown in Figure 3 and in Table 7 below. 1 Table 7: Neutralization of TNFa-Induced L929 Cytotoxicity VH VL Structure IC50xM D2E7 D2E7 scFv 1.1 x IO'10 D2E7 D2E7 IgG4 4.7 χ 10*11 2SD4 2SD4 scFv/IgGl/IgG4 3.0 x IO’7

ATTORNEYDOt "NO: BBI-043CPPC

<img img-format="tif" img-content="drawing" file="IL125697AD00027.tif" id="idf0007" />

7 / z -49- 2SD4 LOE7 scFv 4.3 x 10-8 VH1-D2 2SD4 scFv 1.0 x 10-8 VH1-D2 LOE7 scFv/IgGl/IgG4 3.4 x 10-1° VH1.D2.Y LOE7.T IgG4 8.1 x IO’11 VH1-D2.N LOE7.T IgG4 1.3 x 10-10 VH1-D2.Y LOE7.A IgG4 2.8 x 10-11 VH1-D2.N LOE7.A IgG4 6.2 x IO*11 MAK 195 MAK 195 scFv 1.9 x 10-8 MAK 195 MAKI 95 F(ab')2 6.2X10-11

The results in Figure 3 and Table 7 demonstrate that the D2E7 human anti-hTNFaantibody, and various D2E7-related antibodies, neutralize TNFa-induced L929cytotoxicity with a capacity approximately equivalent to that of the murine anti-hTNFa 5 mAb MAK 195. 10

In another series of experiments, the ability of the IgGl form of D2E7 toneutralize TNFa-induced L929 cytotoxicity was examined as described above. Theresults from three independent experiments, and the average thereof, are summarizedbelow in Table 8: 10.02.1997

Table 8: Neutralization of TNFa-induced L929 Cytotoxicity by D2E7 IgGl

Experiment IC50 [M] 1 1.26 x IO'10 2 1.33 x 10-10 3 1.15 xlO-10 Average 1.25 ± 0.01 x 10-10 15

This series of experiments confirmed that D2E7, in the full-length IgGl form,neutralizes TNFa-induced L929 cytotoxicity with an average IC50 [M] of 1.25 ± 0.01 x 10-&gt;0. B. Inhibition of TNFa Binding to TNFa Receptors on U-937 Cells 20 The ability of human anti-hTNFa antibodies to inhibit the binding of hTNFa to-

hTNFa receptors on the surface of cells was examined using the U-937 cell line (ATCC

No. CRL 1593), a human histiocytic cell line that expresses hTNFa receptors. U-937-

<img img-format="tif" img-content="drawing" file="IL125697AD00028.tif" id="idf0008" />

10.02.1997

ATTORNEYDCK TNO: BBI-043CPPC ί 1 2 5 β Q J -50- cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum(Hyclone A-l 111, Hyclone Laboratories, Logan, UT), L-glutamine (4 nM), HEPESbuffer solution (10 mM), penicillin (100 μg/ml) and streptomycin (100 pg/ml). Toexamine the activity of full-length IgG antibodies, U-937 cells were preincubated with 5 PBS supplemented with 1 mg/ml of human IgG (Sigma 1-4506, Sigma Chemical Co., St.Louis, MO) for 45 minutes on ice and then cells were washed three times with bindingbuffer. For the receptor binding assay, U-937 cells (5 χ 106 cells/well) were incubatedin a binding buffer (PBS supplemented with 0.2% bovine serum albumin) in 96-wellmicrotiter plates (Costar 3799, Costar Corp., Cambridge, MA) together with 125I-labeled

10 rhTNFa (3 x 10-10 M; 25 pCi/ml; obtained from NEN Research Products, Wilmington,DE), with or without anti-hTNFa antibodies, in a total volume of 0.2 ml. The plateswere incubated on ice for 1.5 hours. Then, 75 μΐ of each sample was transferred to 1.0ml test tubes (Sarstedt 72.700, Sarstedt Corp., Princeton, NJ) containing dibutylphthalate(Sigma D-2270, Sigma Chemical Co., St. Louis, MO) and dinonylphthalate (ICN 15 210733, ICN, Irvine, CA). The test tubes contained a 300 μΐ mixture of dibutylphthalate and dinonylphthalate, 2:1 volume ratio, respectively. Free (/. e., unbound) 125I-labeledrhTNFa was removed by microcentrifugation for five minutes. Then, each test tube endcontaining a cell pellet was cut with the aid of a microtube scissor (Bel-Art 210180001,Bel-Art Products, Pequannock, NJ). The cell pellet contains 125I-labeled rhTNFa bound 20 to the p60 or p80 TNFa receptor, whereas the aqueous phase above the oil mixturecontains excess free l^I-labeled rhTNFa. All cell pellets were collected in a countingtube (Falcon 2052, Becton Dickinson Labware, Lincoln Park, NJ) and counted in ascintillation counter.

Representative results are shown in Figure 4. The 1C5Q value for D2E7 25 inhibition of hTNFa binding to hTNFa receptors on U-937 cells is approximately 3 x10-10 m in these experiments. These results demonstrate that the D2E7 human anti-hTNFa antibody inhibits rhTNFa binding to hTNFa receptors on U-937 cells atconcentrations approximately equivalent to that of the murine anti-hTNFa mAb MAK195. 30

In another series of experiments, the ability of the IgGl form of D2E7 to inhibitrhTNFa binding to hTNFa receptors on U-937 cells was examined as described above.The results from three independent experiments, and the average thereof, aresummarized below in Table 9: 35

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ATTORNEYDOC 'TNO: BBI-043CPPC 7 2 5 6 s 7 / -51-

Table 9: Inhibition of TNF Receptor Binding on U-937 Cells by D2E7 IgGl

Exneriment IC50 [M] 1 1.70 x 10'10 2 1.49 x IO'10 3 1.50 x 10'1θ Average 1.56± 0.12 x 10-10 10.02.1997

This series of experiments confirmed that D2E7, in the full-length IgGl form,inhibits TNF receptor binding on U-937 cells with an average IC50 [M] of 1.56 ±0.12 5 x IO'10.

To investigate the inhibitory potency of D2E7 in the binding of 125I-rhTNFbinding to individual p55 and p75 receptors, a solid phase radioimmunoassay wasperformed. To measure the IC5q values of D2E7 for separate TNF receptors, varying 10 concentrations of the antibody were incubated with 3 x 10'10 concentration of 125I-rhTNF. The mixture was then tested on separate plates containing either the p55 or thep75 TNF receptors in a dose dependent manner. The results are summarized below inTable 10: 15

Table 10: Inhibition of TNF Receptor Binding to p55 and p75 TNFR by D2E7 IgGl IC50 [M] Reagent p55 TNFR p 75TNFR D2E7 1.47 x IO'9 1.26 x IO'9 rhTNF 2.31 x IO'9 2.70 x IO'9

Inhibition of 125I-rhTNF binding to the p55 and p75 TNF receptors on U937 cells byD2E7 followed a simple sigmoidal curve, indicating similar IC50 values for eachreceptor. In the solid phase radioimmunoassay (RIA) experiments with recombinant 20 TNF receptors, IC50 values for inhibition of l25I-rhTNF binding to the p55 and the p75receptors by D2E7 were calculated as 1.47 x 10*9 and 1.26 x 10'9 M, respectively. Thedecrease in IC50 values in the solid phase was probably due to higher density of receptorsin the RIA format, as unlabeled rhTNF also inhibited with similar IC5o values. The IC50values for inhibition of l25I-rhTNF binding to the p55 and the p75 receptors by 25 unlabeled rhTNF were 2.31 x 10'9 and 2.70 x IO'9 M, respectively

ATTORNEYDCX TNO: BBI-043CPPC - 52 -

C. Inhibition of ELAM-1 Expression on HUVEC

Human umbilical vein endothelial cells (HUVEC) can be induced to expressendothelial cell leukocyte adhesion molecule 1 (ELAM-1) on their cell-surface by 5 treatment with rhTNFa, which can be detected by reacting rhTNFa-treated HUVECwith an mouse anti-human ELAM-1 antibody. The ability of human anti-hTNFaantibodies to inhibit this TNFa-induced expression of ELAM-1 on HUVEC wasexamined as follows: HUVEC (ATCC No. CRL 1730) were plated in 96-well plates (5χ 104 cells/well) and incubated overnight at 37 °C. The following day, serial dilutions 10 of human anti-hTNFa antibody (1:10) were prepared in a microtiter plate, starting with20-100 μg/ml of antibody. A stock solution of rhTNFa was prepared at 4.5 ng/ml,aliquots of rhTNFa were added to each antibody-containing well and the contents weremixed well. Controls included medium alone, medium plus anti-hTNFa antibody andmedium plus rhTNFa. The HUVEC plates were removed from their overnight 15 incubation at 37° C and the medium gently aspirated from each well. Two hundredmicroliters of the antibody-rhTNFa mixture were transferred to each well of theHUVEC plates. The HUVEC plates were then further incubated at 37° C for 4 hours.Next, a murine anti-ELAM-1 antibody stock was diluted 1:1000 in RPMI. The mediumin each well of the HUVEC plate was gently aspirated, 50 μΐ/well of the anti-ELAM-1 20 antibody solution was added and the HUVEC plates were incubated 60 minutes at roomtemperature. An 125I-labeled anti-mouse Ig antibody solution was prepared in RPMI(approximately 50,000 cpm in 50 μΐ). The medium in each well of the HUVEC plateswas gently aspirated, the wells were washed twice with RPMI and 50 μΐ of the 125I-labeled anti-mouse Ig solution was added to each well. The plates were incubated for 25 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. Thecell lysate from each well was then transferred to a tube and counted in a scintillationcounter.

Representative results are shown in Figure 5. The IC50 value for D2E7 30 inhibition of hTNFa-induced expression of ELAM-1 on HUVEC is approximately 6 x10*11 M in these experiments. These results demonstrate that the D2E7 human anti-i hTNFa antibody inhibits the hTNFa-induced expression of ELAM-1 on HUVEC atconcentrations approximately equivalent to that of the murine anti-hTNFa mAb MAK195. 35

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ATTORNEYDCM &gt;' ^TNO: BBI-043CPPC -53 - 10.02.1997 r·* 0 2/2

In another series of experiments, the ability of the IgGl form of D2E7 to inhibithTNFa-induced expression of ELAM-1 on HUVEC was examined as described above.The results from three independent experiments, and the average thereof, aresummarized below in Table 11:

Table 11: Inhibition of TNFa-Induced ELAM-1 Expression by D2E7 IgGl Receptor

Experiment IC50 [M] 1 1.95 x 10-10 2 1.69χ10-1θ 3 1.90 x IO'10 Average 1.85 ± 0.14 x 10-10 10

This series of experiments confirmed that D2E7, in the full-length IgGl form,inhibits TNFa-induced ELAM-1 expression on HUVEC with an average IC50 [M] of1.85 ± 0.14 x IO-10. 15 20 25

The neutralization potency of D2E7 IgGl was also examined for the rhTNFinduced expression of two other adhesion molecules, ICAM-1 and VCAM-1. Since therhTNF titration curve for ICAM-1 expression at 16 hours was very similar to the curveof ELAM-1 expression, the same concentration of rhTNF was used in the antibodyneutralization experiments. The HUVEC were incubated with rhTNF in the presence ofvarying concentrations of D2E7 in a 37°C CO2 incubator for 16 hours, and the ICAM-1expression was measured by mouse anti-ICAM-1 antibody followed by I-labeledsheep anti-mouse antibody. Two independent experiments were performed and the IC50values were calculated. An unrelated human IgGl antibody did not inhibit the ICAM-1expression.

The experimental procedure to test inhibition of VCAM-1 expression was thesame as the procedure for ELAM-1 expression, except anti-VCAM-1 MAb was usedinstead of anti-ELAM-1 MAb. Three independent experiments were performed and theIC50 values were calculated. An unrelated human IgGl antibody did not inhibit VCAM-1 expression.

The results are summarized below in Table 12:

ATTORNEYDOl ΓΝΟ: BBI-043CPPC 7 2 5 6 9 7 / 2 -54-

Table 12: Inhibition of ICAM-1 and VCAM-I Expression bvD2E7 IgGl ICAM-1 Inhibition IC50 [M] Experiment IC50 [M] Experiment IC50 [M] 1 1.84 x 10-10 1 1.03x10-1° 2 2.49x10-1° 2 9.26 x ΊΟ-H 3 1.06 x 10-10 Average 2.17 ± 0.46 x 10-1° Average 1.01 ±0.01 x io-’0 10.02.1997 10 15

These experiments demonstrate that treatment of primary human umbilical veinendothelial cells with rhTNF led to optimum expression of adhesion molecules: ELAM-1 and VCAM-1 at four hours, and the maximum up-regulated expression ofICAM-1 at 16 hours. D2E7 was able to inhibit the expression of the three adhesionmolecules in a dose dependent manner. The IC50 values for the inhibition of ELAM-1,ICAM-1 and VCAM-1 were 1.85 x IO'10, 2.17 x 10‘10 and 1.01 x IO’10 M, respectively.These values are very similar, indicating similar requirements for the dose of rhTNFactivation 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-1expression. The ICAM-1 inhibition assay required 16 hours of co-incubation of rhTNFand D2E7 with HUVEC as opposed to 4 hours required for the ELAM-1 and theVCAM-1 inhibition assays. Since D2E7 has a slow off-rate for rhTNF, it is conceivablethat during the 16 hour co-incubation period there was no significant competition by theTNF receptors on the HUVEC. D. In Vivo Neutralization of hTNFa 20 Three different in vivo systems were used to demonstrate that D2E7 is effective at inhibiting hTNFa activity in vivo. I. Inhibition of TNF-Induced Lethality in D-Galactosamine-Sensitized Mice 25 Injection of recombinant human TNFa (rhTNFa) to D-galactosamine sensitized mice causes lethality within a 24 hour time period. TNFa neutralizing agents have been shown to prevent lethality in this model. To examine the ability of human anti-hTNFa antibodies to neutralize hTNFa in vivo in this model, C57B1/6 mice were injected with varying concentrations of D2E7-IgGl, or a control protein, in PBS intraperitoneally 30 (i.p.). Mice were challenged 30 minutes later with 1 pg of rhTNFa and 20 mg of D-

ATTORNEYDOt TNO: BBI-043CPPC 125697/2 -55 - galactosamine. in PBS i.p., and observed 24 hours later. These amount of rhTNFa andD-galactosamine were previously determined to achieve 80-90% lethality in these mice.

Representative results, depicted as a bar graph of % survival versus antibodyconcentration, are shown in Figure 6. The black bars represent D2E7, whereas the 5 hatched bars represent MAK 195. Injection of 2.5-25 gg of D2E7 antibody per mouseprotected the animals from TNFa-induced lethality. The ED50 value is approximately1-2.5 gg/mouse. The positive control antibody, MAK 195, was similar in its protectiveability. Injection of D2E7 in the absence of rhTNFa did not have any detrimental effecton the mice. Injection of a non-specific human IgGl antibody did not offer any 10 protection from TNFa-induced lethality. 15

In a second experiment, forty-nine mice were divided into 7 equal groups. Eachgroup received varying doses of D2E7 thirty minutes prior to receiving an LDgo dose ofrhTNF/D-galactosamine mixture (1.0 gg rhTNF and 20 mg D-galactosamine permouse). Control group 7 received normal human IgGl kappa antibody at 25 gg/mousedose. The mice were examined 24 hours later. Survival for each group is summarizedbelow in Table 13. 10.02.1997

Table 13: 24 Hour Survival After Treatment with D2E7

Group Survival (alive/total) Survival (%) 1 (no antibody) 0/7 0 2 (1 gg) 1/7 14 3 (2.6 gg) 5/7 71 4 (5.2 gg) 6/7 86 5 (26 gg) 6/7 86 6 (26 gg; no rhTNF) 7/7 100 7 (25 gg Hu IgGl) 1/7 14 II. Inhibition of TNF-Induced Rabbit Pyrexia 25

The efficacy of D2E7 in inhibiting rhTNF-induced pyrexia response in rabbitswas examined. Groups of three NZW female rabbits weighing approximately 2.5 kgeach were injected intravenously with D2E7, rhTNF, and immune complexes of D2E7and rhTNF. Rectal temperatures were measured by thermistor probes on a Kaye thermal.recorder every minute for approximately 4 hours. Recombinant human INF in saline,injected at 5 gg/kg, elicted a rise in temperature greater than 0.4°C at approximately 45

ATTORNEYDOt TNO: BBI-043CPPC -56- 25597/2 4». minutes after injection. The antibody preparation by itself, in saline at a dose of 138pg/kg, did not elicit a rise in temperature in the rabbits up to 140 minutes afteradministration. In all further experiments, D2E7 or control reagents (human IgGl or asaline vehicle) were injected i.v. into rabbits followed 15 minutes later by an injection ofrhTNF in saline at 5 pg/kg i.v. Representative results of several experiments aresummarized below in Table 14:

Table 14: Inhibition of rhTNF-induced Pyrexia with D2E7 in Rabbits

Temp, rise*, °C Molar Ratio Peak Temp. D2E7 dose(pg/kg) rhTNF rhTNF+D2E7 % Inhib.** D2E7: rhTNF minutespost rhTNF 14 0.53 0.25 53 1 60 24 0.43 0.13 70 1.6 40 48 0.53 0.03 94 3.3 50 137 0.53 0.00 100 9.5 60 792 0.80 0.00 100 55 60 *=Peak temperature 10 * *=% inhibition=( 1 - {temperature rise with rhTNF &amp;amp; D2E7/temperature rise with rhTNF10.02.1997 alone}) x 100. 15 20

Intravenous pretreatment with D2E7 at a dose of 14 pg/kg partially inhibited thepyrogenic response, compared to rabbits pre-treated with saline alone. D2E7administered at 137 pg/kg totally suppressed the pyrogenic response of rhTNF in thesame experiment. In a second experiment, D2E7 administered at 24 pg/kg also partiallysuppressed the pyrogenic response, compared to rabbits pretreated with saline alone.

The molar ratio of D2E7 to rhTNF was 1/6:1 in this experiment. In a third experiment,D2E7 injected i.v. at 48 pg/kg (molar ratio D2E7:rhTNF = 3.3:1) totally suppressed thepyrogenic response, compared to rabbits pretreated with the control human IgGl insaline at 30 pg/kg. In the final experiment, rabbits pretreated with D2E7 (792 pg/kg) ata very high molar ratio to rhTNF (55:1) did not develop any rise in temperature at anytime up to 4 hours of observation. Treatment of rabbits with immune complexesgenerated from a mixture of D2E7 and rhTNF incubated at 37°C for 1 hour at a molarratio of 55:1, without subsequent rhTNF administration, also did not elicit any rise intemperature in the same experiment. 25 ATTORNEYDCK ΓΝΟ: BBI-043CPPC ' ί 72 5 6 3 7/2 -57- III. Prevention of Polyarthritis in Tgl97 Transgenic Mice

The effect of D2E7 on disease development was investigated in a transgenicmurine model of arthritis. Transgenic mice (Tgl97) have been generated that expresshuman wild type TNF (modified in the 3' region beyond the coding sequences) andthese mice develop chronic polyarthritis with 100% incidence at 4-7 weeks of age (seeEMBO J (1991) Γ0:4025-4031 for further description of the Tgl97 model ofpolyarthritis).

Transgenic animals were identified by PCR at 3 days of age Litters of transgenicmice were divided into six groups. Transgenic mice were verified by slot-blothybridization analysis at 15 days of age. The treatment protocols for the six groups Wereas follows: Group l=no treatment; Group 2=saline (vehicle); Group 3=D2E7 at 1.5pg/g; Group 4=D2E7 at 15 pg/g; Group 5=D2E7 at 30 pg/g; and Group 6=IgGl isotypecontrol at 30 pg/g. A litter with non transgenic mice was also included in the study toserve as a control (Group 7 - nontransgenic; no treatment). Each group received threei.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. Atthe same time measurements of joint size (in mm) were also taken, as a measurement ofdisease severity. Joint size was established as an average of three measurements on thehind right ankle using a micrometer device. Arthritic scores were recorded weekly asfollows: 0 = No arthritis, (normal appearence and flexion); + = mild arthritis (jointdistortion); ++ = moderate arthritis (swelling, joint deformation) and +++ = heavyarthritis (ankylosis detected on flexion and severely impaired movement).

Histopathological scoring based on haematoxylin/eosin staining of joint sections wasbased 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 j oint size of the Tg 197 transgenicarthritic mice is shown in the graph of Figure 9. The histopathological and arthriticscores of the Tgl97 transgenic mice, at 11 weeks of age, are summarized below in Table15:

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ATTORNEYDO' ΓΝΟ: BBI-043CPPC -58- 10.02.1997

Table 15: Effect of D2E7 on Histopathology and Arthritic Score in Tgl97 Mice

Group Treatment Histopathological Score Arthritic Score 1 none 3 (7/70 +++ (7/7) 2 saline 3 (8/8) +++(8/8) 6 IgGl control 3 (9/9) +++ (7/9) 3 D2E7 at 1.5 pg/g 0 (6/8) 0 (8/8) 4 D2E7 at 15 pg/g 0 (7/8) 0 (8/8) 5 D2E7 at 30 pg/g 0(8/8) 0(8/8)

This experiment demonstrated that the D2E7 antibody has a definite beneficialeffect on transgenic mice expressing the wild-type human TNF (Tgl97) with no arthritisevident after the study period. E. D2E7 Neutralization of TNFas from Other Species

The binding specificity of D2E7 was examined by measuring its ability to10 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 resultsare summarized in Table 16 below: 10.02.1997 10.02.1997

Table 16: Ability of D2E7 to Neutralize TNF from Different Species in the L929 Assay TNFa* Source IC™ for D2E7 Neutralization (M)** Human Recombinant . 7.8 x 10-11 Chimpanzee LPS-stimulated PBMC 5.5 x ΊΟ-U baboon Recombinant 6.0x10-11 marmoset LPS-stimulated PBMC 4.0 x 10-10 cynomolgus LPS-stimulated PBMC 8.0 x 10-n rhesus LPS-stimulated PBMC 3.0 x ΊΟ-U canine LPS-stimulated WBC 2.2x10-1° porcine Recombinant 1.0 x IO*7 murine Recombinant &gt;1.0 x IO’7 10.02.1997 15 — The results in Table 16 demonstrate that D2E7 can neutralize the activity of fiveprimate TNFas approximately equivalently to human TNFa and, moreover, can

ATTORNEYDCX TNO: BBI-043CPPC f neutralize the activity of canine TNFa (about ten-fold less well than human TNFa) and porcine and mouse TNFa (about -1000-fold less well than human TNFa). Moreover, 10.02.1997 the binding of D2E7 to solution phase rhTNFa was not inhibited by other cytokines, such as lymphotoxin (TNFP), IL-la, IL-1 β, IL-2, IL-4, IL-6, IL-8, IFNy and TGFp, 5 indicating that D2E7 is very specific for its ligand TNFa. 10 10.02.1997 15 20 -59- F. Lack of Cytokine Release by Human Whole Blood Incubated with D2E7 /2

In this example, the ability of D2E7 to induce, by itself, normal human bloodcells to secrete cytokines or shed cell surface molecules was examined. D2E7 wasincubated with diluted whole blood from three different normal donors at varyingconcentrations for 24 hours. An LPS positive control was run at the same time, at aconcentration previously determined to stimulate immunocompetent blood cells tosecrete cytokines. The supernatants were harvested and tested in a panel of ten solublecytokine, receptor and adhesion molecule ELISA kits: IL-Ια, IL-1 β, IL-1 receptorantagonist, IL-6, IL-8, TNFa, soluble TNF receptor I, soluble TNF receptor II, solubleICAM-1 and soluble E-selectin. No significant amounts of cytokines or shed cellsurface molecules were measured as a result of D2E7 antibody co-incubation, atconcentrations up to 343 μg/ml. Control cultures without the addition of the antibodyalso did not yield any measurable amounts of cytokines, whereas the LPS co-culturecontrol yielded elevated values in the high picogram to low nanogram range. Theseresults indicate that D2E7 did not induce whole blood cells to secrete cytokines or shedcell surface proteins above normal levels in ex vivo cultures. 25 ATTORNEYDOC · TNO: BBI-043CPPC ) -60-

Forming part of the present disclosure is the appended Sequence Listing, thecontents of which are summarized in the table below: SEQ ID NO: ANTIBODY CHAIN REGION SEQUENCE TYPE 1 D2E7 VL amino acid 2 D2E7 VH amino acid 3 D2E7 VL CDR3 amino acid 4 D2E7 VH CDR3 amino acid 5 D2E7 VL CDR2 amino acid 6 D2E7 VH CDR2 amino acid 7 D2E7 VL CDR1 amino acid 8 D2E7 VH CDR1 amino acid 9 2SD4 VL amino acid 10 2SD4 VH amino acid 11 2SD4 VL CDR3 amino acid 12 EP B12 VL CDR3 amino acid . 13 VL10E4 VL CDR3 amino acid 14 VL100A9 VL CDR3 amino acid 15 VLL100D2 VL CDR3 amino acid 16 VLL0F4 VL CDR3 amino acid 17 LOE5 VL CDR3 amino acid 18 VLLOG7 VL CDR3 amino acid 19 VLLOG9 VL CDR3 amino acid 20 VLLOH1 VL CDR3 amino acid 21 VLLOHIO VL CDR3 amino acid 22 VL1B7 VL CDR3 amino acid 23 VL1C1 VL CDR3 amino acid 24 VLO.1F4 VL CDR3 * amino acid 25 VLO.1H8 VL CDR3 amino acid 26 LOE7.A VL CDR3 amino acid 27 2SD4 VH CDR3 amino acid 28 VH1B11 VH CDR3 amino acid 29 VH1D8 VH CDR3 amino acid 30 VH1A11 VH CDR3 amino acid 31 VH1B12 VH CDR3 amino acid 32 VH1E4 VH CDR3 amino acid 33 VH1F6 VH CDR3 amino acid 34 3C-H2 VH CDR3 amino acid 35 VH1-D2.N VH CDR3 amino acid 36 D2E7 VL nucleic acid 37 D2E7 VH nucleic acid ATTORNEYDCX )NO: BBI-043CPPC ' ) -61 -

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than5 routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the followingclaims.

ATTORNEYDCX TNO: BBI-043CPPC •Π -62-

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BASF Aktiengesellschaft (B) STREET: Carl-Bosch Str. 38 (C) CITY: 67056 Ludwigshafen (D) STATE: Rheinland-Pfalz (E) COUNTRY: Federal Republic of Germany (ii) TITLE OF INVENTION: Human Antibodies that Bind Human TNFa (iii) NUMBER OF SEQUENCES: 37 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: LAHIVE &amp;amp; COCKFIELD (B) STREET: 60 State Street, suite 510 (C) CITY: Boston (D) STATE: Massachusetts

(E) COUNTRY: USA (F) ZIP: 02109-1875 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (Vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/599,226 (B) FILING DATE: 09-FEB-1996 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/031,476 (B) FILING DATE: 25-NOV-1996 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: DeConti, Giulio A., Jr. (B) REGISTRATION NUMBER: 31,503

(C) REFERENCE/DOCKET NUMBER: BBI-043CPPC (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (617)227-7400 (B) TELEFAX: (617)227-5941 (2) INFORMATION FOR SEQ ID NO :1:

ATTORNEYDOi TNO: BBI-043CPPC n -63 - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 / -- 40 45 Tyr Ala Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly '50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 /* 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr ’ Phe Gly Gin Gly Thr Lys Val Glu Ile Lys 100 105 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 121 amino acidsIB) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (V) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 1 5 10 15 z . Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Aspl Asp Tyr 20 25 30 *- Ala Met His Trp Val Axg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35' 40 45

ATTORNEYDOC NO: BBI-043CPPC t -64-

Ser Ala Ile'50 Thr Trp Asn Ser 55 Gly His Ile Asp Tyr 60 Ala Asp Ser Val Glu 65 Gly!Arg Phe Thr Ile 70 Ser Arg Asp Asn Ala 75 Lys Asn Ser Leu Tyr 80 Leu Gin Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys Ala Lys Val Ser 100 Tyr Leu Ser Thr Ala 105 Ser Ser Leu Asp Tyr, 110 Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE : (A) NAME/KEY: (B) LOCATION: Modified 9 (D) OTHER INFORMATION: /note= "Xaa is Thr or Ala" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Gin Arg Tyr Asn Arg Ala Pro Tyr Xaa15 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE : (A) NAME/KEY: (B) LOCATION: Modified 12 (D) OTHER INFORMATION: /note= "Xaa is Tyr or Asn"

ATTORNEYDCX "NO: BBI-043CPPC -65- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa1 5 10 (2) INFORMATION FOR SEQ ID NO : 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Ala Ala Ser Thr Leu Gin Ser1 5 (2) INFORMATION FOR SEQ ID NO:6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu1 5 10 15

Gly (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ATTORNEYDOi Γ^ΝΟ: BBI-043CPPC -66-

Arg Ala Ser Gin Gly He Arg Asn Tyr Leu Ala15 10 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Asp Tyr Ala Met His1 5 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 107 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Asp 1 lie Gin Met Thr 5 Gin Ser Pro Ser Ser 10 Leu Ser Ala Ser He 15 Gly Asp Arg Val Thr 20 He Thr Cys Arg Ala 25 Ser Gin Gly He Arg 30 Asn Tyr Leu Ala Trp 35 Tyr Gin Gin Lys Pro 40 Gly Lys Ala Pro Lys 45 Leu Leu He Tyr Ala 50 Ala Ser Thr Leu Gin 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Gly Ser 65 Gly Ser Gly Thr Asp 70 Phe Thr Leu Thr He 75 Ser Ser Leu Gin Pro 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gin Lys Tyr Asn Ser Ala Pro Tyr 85 90 95

Ala Phe Gly Gin Gly Thr Lys Val Glu lie Lys100 105

ATTORNEYDOi ΓΝΟ: BBI-043CPPC

'' J -67- (2) INFORMATION FOR SEQ ID NO :10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 121 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Gin 1 Val Gin Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gin Pro Gly 15 Arg Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Asp 30 Asp Tyr Ala Met His 35 Trp Val Arg Gin Ala 40 Pro Gly Lys Gly Leu 45 Asp Trp Val Ser Ala 50 He Thr Trp Asn Ser 55 Gly His lie Asp Tyr 60 Ala Asp Ser Val Glu 65 Gly Arg Phe Ala Val 70 Ser Arg Asp Asn Ala 75 Lys Asn Ala Leu Tyr 80 Leu Gin Met Asn Ser 85 Leu Arg Pro Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys Thr Lys Ala Ser 100 Tyr Leu Ser Thr Ser 105 Ser Ser Leu Asp Asn 110 Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser - 115 120 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Gin Lys Tyr Asn Ser Ala Pro Tyr Ala1 5 9

ATTORNEYDOC ί ΓΝΟ: BBI-043CPPC η -68- 10 15 (2) INFORMATION FOR SEQ ID NO :12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Gin Lys Tyr Asn Arg Ala Pro Tyr Ala1 5 20 (2) INFORMATION FOR SEQ ID NO:13: 25 30 35 40 45 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Gin Lys Tyr Gin ATg Ala Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Gin Lys Tyr Ser Ser Ala Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:15: 50 55

<img img-format="tif" img-content="drawing" file="IL125697AD000213.tif" id="idf0013" />

ATTORNEYDOC BBI-043CPPC -69- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Gin Lys Tyr Asn Ser Ala Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Gin Lys Tyr Asn Arg Ala Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Gin Lys Tyr Asn Ser Ala Pro Tyr Tyr1 5 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear

ATTORNEYDOG *")ΤΝΟ: BBI-043CPPC -70- (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Gin Lys Tyr Asn Ser Ala Pro Tyr Asn1 5 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Gin Lys Tyr Thr Ser Ala Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Gin Lys Tyr Asn Arg Ala Pro Tyr Asn1 5 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

ATTORNEYDOC ΓΝΟ: BBI-043CPPC

<img img-format="tif" img-content="drawing" file="IL125697AD000214.tif" id="idf0014" />

-71 - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Gin Lys Tyr Asn Ser Ala Ala Tyr Ser1 5 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO-.22:

Gin Gin Tyr Asn Ser Ala Pro Asp Thr1 5 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Gin Lys Tyr Asn Ser Asp Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ATTORNEYDOC ΓΝΟ: BBI-043CPPC

<img img-format="tif" img-content="drawing" file="IL125697AD000215.tif" id="idf0015" />

-72-

Gln Lys Tyr Ile Ser Ala Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO :25:

Gin Lys Tyr Asn Arg Pro Pro Tyr Thr1 5 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Gin Arg Tyr Asn Arg Ala Pro Tyr Ala1 5 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (V) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO :27: Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 1 5 10

ATTORNEYDOC ΓΝΟ: BBI-043CPPC η -73- (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys15 10 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Ala Ser Tyr Leu Ser Thr Ser Ser Ser"Leu Asp Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO :30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp15 10 (2) INFORMATION FOR SEQ ID NO :31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids

ATTORNEYDOi Γ*)ΤΝΟ: BBI-043CPPC

<img img-format="tif" img-content="drawing" file="IL125697AD000216.tif" id="idf0016" />

-74- (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide5 (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 10 Ala Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr 15 10 (2) INFORMATION FOR SEQ ID NO:32: 15 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear 20 (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr15 10 30 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids 35 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 40 (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr45 i 5 io * (2) INFORMATION FOR SEQ ID NO:34: 50 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear 55 (ii) MOLECULE TYPE: peptide

ATTORNEYDOC JTNO: BBI-043CPPC -75- (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr15 10 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Asn15 10 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGGGA CAGAGTCACC 60 ATCACTTGTC GGGCAAGTCA GGGCATCAGA AATTACTTAG CCTGGTATCA GCAAAAACCA 120 GGGAAAGCCC CTAAGCTCCT GATCTATGCT GCATCCACTT TGCAATCAGG GGTCCCATCT 180 CGGTTCAGTG GCAGTGGATC TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTACAGCCT 240 GAAGATGTTG CAACTTATTA CTGTCAAAGG TATAACCGTG CACCGTATAC TTTTGGCCAG 300 GGGACCAAGG TGGAAATCAA A 321 (2) INFORMATION FOR SEQ ID NO 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 363 base pairs

ATTORNEYDOG ~ΎΝΟ: BBI-043CPPC

<img img-format="tif" img-content="drawing" file="IL125697AD000217.tif" id="idf0017" />

-76- (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear

5 (ii) MOLECULE TYPE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GAGGTGCAGC TGGTGGAGTC TGGGGGAGGC TTGGTACAGC CCGGCAGGTC CCTGAGACTC 60 TCCTGTGCGG CCTCTGGATT CACCTTTGAT GATTATGCCA TGCACTGGGT CCGGCAAGCT 120 CCAGGGAAGG GCCTGGAATG GGTCTCAGCT ATCACTTGGA ATAGTGGTCA CATAGACTAT 180 GCGGACTCTG TGGAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCCCTGTAT 240 CTGCAAATGA ACAGTCTGAG AGCTGAGGAT ACGGCCGTAT ATTACTGTGC GAAAGTCTCG 300 TACCTTAGCA CCGCGTCCTC CCTTGACTAT TGGGGCCAAG GTACCCTGGT CACCGTCTCG 360 20 AGT 363