United States Patent [19]
`Salfeld et al.
`
`US006090382A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,090,382
`*Jul. 18,2000
`
`[54] HUMAN ANTIBODIES THAT BIND HUMAN
`TNFO.
`
`B Lymphocytes”, Abstract 2904, Proc. Am. Assoc. Cancer
`Res., vol. 34, p. 487, Mar. 1993.
`
`[75] Inventors: Jochen G. Salfeld, North Grafton,
`Mass.; Deborah J. Allen, London,
`United Kingdom; Hendricus R. J. M.
`Hoogenboom, Hasselt, Belgium; Zehra
`Kaymakcalan, Westboro, Mass.; Boris
`Labkovsky, Framingham, Mass.; John
`A. Mankovich, Andover, Mass.; Brian
`T. McGuinness, Hauxton; Andrew J.
`Roberts, Cambridge, both of United
`Kingdom; Paul Sakorafas, ShreWsbury,
`Mass.; David Schoenhaut, Clifton,
`N.J.; Tristan J. Vaughan, Impington,
`United Kingdom; Michael White,
`Framingham, Mass.; Alison J. Wilton,
`Cambridge, United Kingdom
`
`[73] Assignee: BASF Aktiengesellschaft, Germany
`
`1*] Notice?
`
`This patent issued on a continued pros
`ecution application ?led under 37 CFR
`1.53(d), and is subject to the tWenty year
`patent term provisions of 35 U.S.C.
`154(a)(2).
`
`[21] Appl. No.1 08/599,226
`[22] Filed:
`Feb. 9, 1996
`
`[51] Int. Cl.7 ....................... .. A61K 39/395; C07K 16/24
`[52] U.S. Cl. ................................... .. 424/1331; 424/1351;
`424/142.1; 424/145.1; 530/387.3; 530/388.15;
`530/388.23
`[58] Field of Search ............................ .. 424/130.1, 133.1,
`424/1351, 142.1, 145.1; 350/387.3, 388.23,
`388.24, 388.15
`
`[56]
`
`References Cited
`
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`
`5,231,024
`5,654,407
`5,795,967
`
`7/1993 Moeller et al. .................. .. 435/24027
`8/1997 Boyle et al.
`.. 530/38815
`8/1998 Aggarwal et al. ............... .. 530/38823
`
`FOREIGN PATENT DOCUMENTS
`
`351 789 A2 1/1990 European Pat. Off. .
`366 043 A1 5/1990 European Pat. Off. .
`492 448 A1 7/1992 European Pat. Off. .
`186 833 B1 8/1992 European Pat. Off. .
`614 984 A2 9/1994 European Pat. Off. .
`212 489 B1 11/1994
`European Pat. Off. .
`101 681 B1 12/1994
`European Pat. Off. .
`659 766 A1 6/1995
`European Pat. Off. .
`2 279 077 12/1994
`United Kingdom .
`WO 91/01078 2/1991 WIPO .
`WO 92/11383
`7/1992 WIPO .
`WO 92/16553 10/1992 WIPO .
`WO 93/06213
`4/1993 WIPO .
`WO 94/29347 12/1994 WIPO .
`95/23813
`9/1995 WIPO .
`
`OTHER PUBLICATIONS
`
`Bendtzen, K. et al. “Auto—Antibodies to IL—1(X and TNFO. in
`Nornal Individuals and in Infectious and Immunoin?amma
`tory Disorders”, in The Physiological and Pathological
`Effects of Cytokines, pp. 447—452, Wiley—Liss, Inc., 1990.
`
`Boyle, P. et al. “A Novel Monoclonal Human IgM Autoan
`tibody Which Binds Recombinant Human and Mouse Tumor
`Necrosis Factor—ot”, Cell. Immunol., vol. 152, pp. 556—568,
`(1993).
`Boyle, P. et al. “The B5 Monoclonal Human Autoanitibody
`Binds to Cell Surface TNFO. on Human Lymphoid Cells and
`Cell Lines and Aappears to Recognize a Novel Epitope”,
`Cell. Immunol., vol. 152, pp. 569—581, (1993).
`CoX, J.P.L. et al., “A directory of human germ—line Vx
`segments reveals a strong bias in their usage”, Eur. J.
`Immunol., vol. 24, pp. 827—836 (1994).
`
`Elliot, M.J. et al., “Treatment of rheumatoid arthritis With
`chimeric monoclonal antibodies to tumor necrosis factor 00”,
`Arthritis & Rheumatism, vol. 36, No. 12, pp. 1681—1690
`(1993).
`Fomsgaard, A. et al. “Auto—antibodies to Tumor Necrosis
`Factor (X in Healthy Humans and Patients With In?ammatory
`Diseases and Gram—Negative Bacterial Infections”, Scand.
`J. Immunol., vol. 30, pp. 219—223, (1989).
`Huse, W.D. et al., “Generation of a large combinatorial
`library of the immunoglobulin repertoire in phage lambda”,
`Science, vol. 246, pp. 1275—1281 (1989).
`
`Lerner, RA. et al., “Antibodies Without immunization”,
`Science, vol. 258, pp. 1313—1314 (1992).
`
`Leusch, H—G. et al. “Failure to demonstrate TNFot—speci?c
`autoantibodies in human sera by ELISA and Western blot”,
`J. Immunol. Methods, vol. 139, pp. 145—147, (1991).
`
`(List continued on neXt page.)
`
`Primary Examiner—David Saunders
`Attorney, Agent, or Firm—Lahive & Cock?eld, LLP;
`Catherine J. Kara; Elizabeth A. Hanley, Esq.
`
`[57]
`
`ABSTRACT
`
`Human antibodies, preferably recombinant human
`antibodies, that speci?cally bind to human tumor necrosis
`factor (X (hTNFot) are disclosed. These antibodies have high
`affinity for hTNFa (e.g., Kd=10_8 M or less), a sloW off rate
`for hTNFO. dissociation (e.g., K0,]=10_3 sec“1 or less) and
`neutralize hTNFa activity in vitro and in vivo. An antibody
`of the invention can be a full-length antibody or an antigen
`binding portion thereof. The antibodies, or antibody
`portions, of the invention are useful for detecting hTNFO.
`and for inhibiting hTNFO. activity, e.g., in a human subject
`suffering from a disorder in Which hTNFa activity is detri
`mental. Nucleic acids, vectors and host cells for expressing
`the recombinant human antibodies of the invention, and
`methods of synthesizing the recombinant human antibodies,
`are also encompassed by the invention.
`
`Barbuto, J .A.M., et al. “Production of Neutralizing Antibod
`ies to Tumor Necrosis Factor by Human Tumor—In?ltrating
`
`30 Claims, 10 Drawing Sheets
`
`Ex. 1005 - Page 1 of 41
`
`

`

`6,090,382
`Page 2
`
`OTHER PUBLICATIONS
`
`Marks, J .D. et al., “By—passing immunization: Human anti
`bodies from V—gene libraries displayed on phage”, J. M01.
`Biol., vol. 222, pp. 581—597 (1991).
`Moller, et al., “Monoclonal antibodies to human tumor
`necrosis factor 0t: in vitro and in vivo application”, Cytok
`ine, vol. 2, No. 3, pp. 162—169 (1990).
`Tomlinson, I.M. et al., “The structural repertoire of the
`human Vk domain”, EMBO, vol. 14, No. 18, pp. 4628—4638
`(1995)
`
`Tomlinson, I.M. et al., “The repertoire of human germline
`VH sequences reveals about ?fty groups of VH segments
`With different hypervariable loops”, J. Mol. Biol., vol. 227,
`pp. 776—698 (1992).
`Tracey, K.J. et al., “Tumor necrosis factor: A pleiotropic
`cytokine and therapuetic target”, Annu. Rev. Med., vol. 45,
`pp. 491—503 (1994).
`Winter, G. et al., “HumaniZed antibodies”, Immunology
`Today, vol. 14, No. 6, pp. 243—246 (1993).
`Grif?ths et al., The EMBO J. 12(2):725, 1993.
`LeWis et al., J. Cell. Biochem., 18Dz215, 1994.
`
`Ex. 1005 - Page 2 of 41
`
`

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`US. Patent
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`Jul. 18, 2000
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`US. Patent
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`Jul. 18, 2000
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`U.S. Patent
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`Jul. 18,2000
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`U.S. Patent
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`Jul. 18, 2000
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`Ex. 1005 - Page 11 of 41
`
`

`

`US. Patent
`
`Jul. 18, 2000
`
`Sheet 10 0f 10
`
`6,090,382
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`
`Ex. 1005 - Page 12 of41
`
`Ex. 1005 - Page 12 of 41
`
`

`

`1
`HUMAN ANTIBODIES THAT BIND HUMAN
`TNFO.
`
`BACKGROUND OF THE INVENTION
`
`Tumor necrosis factor 0t (TNFot) is a cytokine produced
`by numerous cell types, including monocytes and
`macrophages, that Was originally identi?ed based on its
`capacity to induce the necrosis of certain mouse tumors (see
`e.g., Old, L. (1985) Science 230:630—632). Subsequently, a
`factor termed cachectin, associated With cacheXia, Was
`shoWn to be the same molecule as TNFot. TNFO. has been
`implicated in mediating shock (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, TNFO. has been implicated in the
`pathophysiology of a variety of other human diseases and
`disorders, including sepsis, infections, autoimmune
`diseases, transplant rejection and graft-versus-host disease
`(see e.g., Vasilli, P. (1992)Annu. Rev Immunol. 10:411—452;
`Tracey, K. J. and Cerami, A. (1994) Annu. Rev Med.
`45:491—503).
`Because of the harmful role of human TNFO. (hTNFot) in
`a variety of human disorders, therapeutic strategies have
`been designed to inhibit or counteract hTNFO. activity. In
`particular, antibodies that bind to, and neutraliZe, hTNFO.
`have been sought as a means to inhibit hTNFO. activity.
`Some of the earliest of such antibodies Were mouse mono
`clonal antibodies (mAbs), secreted by hybridomas prepared
`from lymphocytes of mice immuniZed With hTNFO. (see
`e.g., Hahn T; et al., (1985) Proc Natl Acad 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)
`Hybria'oma 6:359—370; Moller, A., et al. (1990) Cytokine
`2:162—169; US. Pat. No. 5,231,024 to Moeller et al.;
`European Patent Publication No. 186 833 B1 by Wallach,
`D.; European Patent Application Publication No. 218 868
`A1 by Old et al.; European Patent Publication No. 260 610
`B1 by Moeller, A., et al.). While these mouse anti-hTNFot
`antibodies often displayed high af?nity for hTNFO. (e.g.,
`Kd§10_9M) and Were able to neutraliZe hTNFO. activity,
`their use in vivo may be limited by problems associated With
`administration of mouse antibodies to humans, such as short
`serum half life, an inability to trigger certain human effector
`functions and elicitation of an unWanted immune response
`against the mouse antibody in a human (the “human anti
`mouse antibody” (HAMA) reaction).
`In an attempt to overcome the problems associated With
`use of fully-murine antibodies in humans, murine anti
`hTNFO. antibodies have been genetically engineered to be
`more “human-like.” For eXample, chimeric antibodies, in
`Which the variable regions of the antibody chains are
`murine-derived and the constant regions of the antibody
`chains are human-derived, have been prepared (Knight, D.
`M, et al. (1993) Mol. Immunol. 30:1443—1453; PCT Publi
`cation No. WO 92/16553 by Daddona, P. E., et al.).
`Additionally, humaniZed antibodies, in Which the hypervari
`able domains of the antibody variable regions are murine
`derived but the remainder of the variable regions and the
`antibody constant regions are human-derived, have also
`been prepared (PCT Publication No. W0 92/ 11383 by Adair,
`J. R., et al.). HoWever, because these chimeric and human
`iZed antibodies still retain some murine sequences, they still
`may elicit an unWanted immune reaction, the human anti
`chimeric antibody (HACA) reaction, especially When
`administered for prolonged periods, e.g., for chronic
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`indications, such as 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 hTNFO. inhibitory agent to murine mAbs or
`derivatives thereof (e.g., chimeric or humaniZed antibodies)
`Would be an entirely human anti-hTNFot antibody, since
`such an agent should not elicit the HAMA reaction, even if
`used for prolonged periods. Human monoclonal autoanti
`bodies against hTNFO. have been prepared using human
`hybridoma techniques (Boyle, P., et al. (1993) Cell. Immu
`nol. 152:556—568; Boyle, P., et al. (1993) Cell. Immunol.
`152:569—581; European Patent Application Publication No.
`614 984 A2 by Boyle, et al.). HoWever, these hybridoma
`derived monoclonal autoantibodies Were reported to have an
`af?nity for hTNFO. that Was too loW to calculate by conven
`tional methods, Were unable to bind soluble hTNFO. and
`Were unable to neutraliZe hTNFot-induced cytotoXicity (see
`Boyle, et al.; supra). Moreover, the success of the human
`hybridoma technique depends upon the natural presence in
`human peripheral blood of lymphocytes producing autoan
`tibodies speci?c for hTNFot. Certain studies have detected
`serum autoantibodies against hTNFO. 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.,
`et al. (1991) J. Immunol. Methods 139:145—147).,
`Alternative to naturally-occurring human anti-hTNFot
`antibodies Would be a recombinant hTNFO. antibody.
`Recombinant human antibodies that bind hTNFO. With rela
`tively loW af?nity (i.e., KJ~10_7M) and a fast off rate (i.e.,
`K0?~10_2 sec_1) have been described (Grif?ths, A. D., et al.
`(1993) EMBO J. 12:725—734). HoWever, because of their
`relatively fast dissociation kinetics, these antibodies may not
`be suitable for therapeutic use. Additionally, a recombinant
`human anti-hTNFot has been described that does not neu
`traliZe hTNFO. activity, but rather enhances binding of
`hTNFO. to the surface of cells and enhances internaliZation
`of hTNFO. (Lidbury, A., et al. (1994) Biotechnol. T her.
`5:27—45; PCT Publication No. WO 92/03145 by Aston, R. et
`al.)
`Accordingly, human antibodies, such as recombinant
`human antibodies, that bind soluble hTNFO. With high
`af?nity and sloW dissociation kinetics and that have the
`capacity to neutraliZe hTNFO. activity, including hTNFot
`induced cytotoXicity (in vitro and in vivo) and hTNFot
`induced cell activation, are still needed.
`
`SUMMARY OF THE INVENTION
`
`This invention provides human antibodies, preferably
`recombinant human antibodies, that speci?cally bind to
`human TNFot. The antibodies of the invention are charac
`teriZed by binding to hTNFO. With high af?nity and sloW
`dissociation kinetics and by neutraliZing hTNFO. activity,
`including hTNFot-induced cytotoXicity (in vitro and in vivo)
`and hTNFot-induced cellular activation. The antibodies can
`be full-length (e.g., an IgG1 or IgG4 antibody) or can
`comprise only an antigen-binding portion (e.g., a Fab,
`F(ab‘)2 or scFv fragment). The most preferred recombinant
`antibody of the invention, termed D2E7, has a light chain
`CDR3 domain comprising the amino acid sequence of SEQ
`ID NO: 3 and a heavy chain CDR3 domain comprising the
`amino acid sequence of SEQ ID NO: 4. Preferably, the D2E7
`antibody has a light chain variable region (LCVR) compris
`ing the amino acid sequence of SEQ ID NO: 1 and a heavy
`chain variable region (HCVR) comprising the amino acid
`sequence of SEQ ID NO: 2.
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`In one embodiment, the invention provides an isolated
`human antibody, or an antigen-binding portion thereof, that
`dissociates from human TNFO. With a Kd of 1x10“8 M or less
`and a Ko?rate constant of 1x10‘3 s-1 or less, both determined
`by surface plasmon resonance, and neutraliZes human TNFO.
`cytotoXicity in a standard in vitro L929 assay With an IC5O
`of 1><10_M or less. More preferably, the isolated human
`antibody, or antigen-binding portion thereof, dissociates
`from human TNFO. With a K017 of 5x10“4 s'1 or less, or even
`more preferably, With a K017 of 1x10‘4 s-1 or less. More
`preferably, the isolated human antibody, or antigen-binding
`portion thereof, neutraliZes human TNFO. cytotoXicity in a
`standard in vitro L929 assay With an IC5O of 1x10“8 M or
`less, even more preferably With an IC5O of 1x10“9 M or less
`and still more preferably With an IC5O of 1x10‘10 M or less.
`In another embodiment, the invention provides a human
`antibody, or antigen-binding portion thereof, With the fol
`loWing characteristics:
`a) dissociates from human TNFO. With a Ko?of 1><10_3 s'1
`or less, as determined by surface plasmon resonance;
`b) has a light chain CDR3 domain comprising the amino
`acid sequence of SEQ ID NO: 3, or modi?ed from SEQ
`ID NO: 3 by a single alanine substitution at position 1,
`4, 5, 7 or 8 or by one to ?ve conservative amino acid
`substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
`c) has a heavy chain CDR3 domain comprising the amino
`acid sequence of SEQ ID NO: 4, or modi?ed 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 ?ve conservative
`amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,
`10, 11 and/or 12.
`More preferably, the antibody, or antigen-binding portion
`thereof, dissociates from human TNFU. With a K017 of 5 x10-4
`s-_1 or less. Still more preferably, the antibody, or antigen
`binding portion thereof, dissociates from human TNFO. With
`a K077 of 1x10“4 s'1 or less.
`In yet another embodiment, the invention provides a
`human antibody, or an antigen-binding portion thereof, With
`an LCVR having CDR3 domain comprising the amino acid
`sequence of SEQ ID NO: 3, or modi?ed from SEQ ID NO:
`3 by a single alanine substitution at position 1, 4, 5, 7 or 8,
`and With an HCVR having a CDR3 domain comprising the
`amino acid sequence of SEQ ID NO: 4, or modi?ed from
`SEQ ID NO: 4 by a single alanine substitution at position 2,
`3, 4, 5, 6, 8, 9, 10 or 11. More preferably, the LCVR further
`has a CDR2 domain comprising the amino acid sequence of
`SEQ ID NO: 5 and the HCVR further has a CDR2 domain
`comprising the amino acid sequence of SEQ ID NO: 6. Still
`more preferably, the LCVR further has CDR1 domain
`comprising the amino acid sequence of SEQ ID NO: 7 and
`the HCVR has a CDR1 domain comprising the amino acid
`sequence of SEQ ID NO: 8.
`In still another embodiment, the invention provides an
`isolated human antibody, or an antigen binding portion
`thereof, With an LCVR comprising the amino acid sequence
`of SEQ ID NO: 1 and an HCVR comprising the amino acid
`sequence of SEQ ID NO: 2. In certain embodiments, the
`antibody has an IgG1 heavy chain constant region or an
`IgG4 heavy chain constant region. In yet other
`embodiments, the antibody is a Fab fragment, an F(ab‘)2
`fragment or a single chain Fv fragment.
`In still other embodiments, the invention provides
`antibodies, or antigen-binding portions thereof, With an
`LCVR having CDR3 domain comprising an amino acid
`sequence selected from the group consisting of SEQ ID NO:
`3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
`ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
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`17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ
`ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
`24, SEQ ID NO: 25, SEQ ID NO: 26 or With an HCVR
`having a CDR3 domain comprising an amino acid sequence
`selected from the group consisting of SEQ ID NO: 4, SEQ
`ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:
`30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ
`ID NO: 34 and SEQ ID NO: 35.
`In yet another embodiment, the invention provides an
`isolated human antibody, or antigen-binding portion thereof,
`that neutraliZes the activity of human TNFot, chimpanZee
`TNFO. and at least one additional primate TNFO. selected
`from the group consisting of baboon TNFot, marmoset
`TNFot, cynomolgus TNFO. and rhesus TNFot. In one
`subembodiment, the isolated human antibody, or antigen
`binding portion thereof, also neutraliZes the activity of
`mouse TNFot. In another subembodiment, the isolated
`human antibody, or antigen-binding portion thereof, also
`neutraliZes the activity of pig TNFot.
`Another aspect of the invention pertains to nucleic acid
`molecules encoding the antibodies, or antigen-binding
`portions, of the invention. A preferred nucleic acid of the
`invention, encoding a D2E7 LCVR, has the nucleotide
`sequence shoWn in FIG. 7 and SEQ ID NO 36. Another
`preferred nucleic acid of the invention, encoding a D2E7
`HCVR, has the nucleotide sequence shoWn in FIG. 8 and
`SEQ ID NO 37. Recombinant expression vectors carrying
`the antibody-encoding nucleic acids of the invention, and
`host cells into Which such vectors have been introduced, are
`also encompassed by the invention, as are methods of
`making the antibodies of the invention by culturing the host
`cells of the invention.
`Yet another aspect of the invention pertains to methods for
`inhibiting human TNFO. activity using an antibody, or
`antigen-binding portion thereof, of the invention. In one
`embodiment, the method comprises contacting human
`TNFO. With the antibody of the invention, or antigen-binding
`portion thereof, such that human TNFO. activity is inhibited.
`In another embodiment, the method comprises administer
`ing an antibody of the invention, or antigen-binding portion
`thereof, to a human subject suffering from a disorder in
`Which TNFO. activity is detrimental such that human TNFO.
`activity in the human subject is inhibited. The disorder can
`be, for example, sepsis, an autoimmune disease (e.g., rheu
`matoid arthritis, allergy, multiple sclerosis, autoimmune
`diabetes, autoimmune uveitis and nephrotic syndrome), an
`infectious disease, a malignancy, transplant rejection or
`graft-versus-host disease, a pulmonary disorder, a bone
`disorder, an intestinal disorder or a cardiac disorder.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIGS. 1A and 1B shoW the amino acid sequences of the
`light chain variable region of D2E7 (D2E7 VL; also shoWn
`in SEQ ID NO: 1), alanine-scan mutants of D2E7 VL
`(LD2E7*.A1, LD2E7*.A3, LD2E7*.A4, LD2E7*.A5,
`LD2E7*.A7 and LD2E7*.A8), the light chain variable
`region of the D2E7-related antibody 2SD4 (2SD4 VL; also
`shoWn in SEQ ID NO: 9) and other D2E7-related light chain
`variable regions (EP B12, VL10E4, VL100A9, VL100D2,
`VL10F4, LOE5, VLLOF9, VLLOF10, VLLOG7, VLLOG9,
`VLLOH1, VLLOH10, VL1B7, VL1C1, VL1C7, VLO.1F4,
`VLO.1H8, LOE7, LOE7.A and LOE7.T). FIG. 1A shoWs the
`FR1, CDR1, FR2 and CDR2 domains. FIG. 1B shoWs the
`FR3, CDR3 and FR4 domains. The light chain CDR1
`(“CDR L1”), CDR2 (“CDR L2”) and CDR3 (“CDR L3”)
`domains are boXed.
`FIGS. 2A and 2B shoW the amino acid sequences of the
`heavy chain variable region of D2E7 (D2E7 VH; also shoWn
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`in SEQ ID NO: 2), alanine-scan mutants of D2E7 VH
`(HD2E7*.A1, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4,
`HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8 and
`HD2E7*.A9), the heavy chain variable region of the D2E7
`related antibody 2SD4 (2SD4 VH; also shown in SEQ ID
`NO: 10) and other D2E7-related heavy chain variable
`regions (VH1B11, VH1D8, VH1A11, VH1B12, VH1-D2,
`VH1E4, VH1F6, VH1G1, 3C-H2, VH1-D2.N and VH1
`D2.Y). FIG. 2A shoWs the FR1, CDR1, FR2 and CDR2
`domains. FIG. 2B shoWs the FR3, CDR3 and FR4 domains.
`The heavy chain CDR1 (“CDR H1”), CDR2 (“CDR H2”)
`and CDR3 (“CDR H3”) domains are boxed.
`FIG. 3 is a graph depicting the inhibition of TNFot
`induced L929 cytotoxicity by the human anti-hTNFot anti
`body D2E7, as compared to the murine anti-hTNFot anti
`body MAK 195 (IgG1 or Fab fragment).
`FIG. 4 is a graph depicting the inhibition of rhTNFot
`binding to hTNFO. receptors on U-937 cells by the human
`anti-hTNFot antibody D2E7, as compared to the murine
`anti-hTNFot antibody MAK 195 (IgG1 or Fab fragment).
`FIG. 5 is a graph depicting the inhibition of TNFot
`induced ELAM-1 expression on HUVEC by the human
`anti-hTNFot antibody D2E7, as compared to the murine
`anti-hTNFot antibody MAK 195 (IgG1 or Fab fragment).
`FIG. 6 is a bar graph depicting protection from TNFot
`induced lethality in D-galactosamine-sensitiZed mice by
`administration of the human anti-hTNFot antibody D2E7
`(black bars), as compared to the murine anti-hTNFot anti
`body MAK 195 (hatched bars).
`FIG. 7 shoWs the nucleotide sequence of the light chain
`variable region of D2E7, With the predicted amino acid
`sequence beloW the nucleotide sequence. The CDR L1,
`CDR L2 and CDR L3 regions are underlined.
`FIG. 8 shoWs the nucleotide sequence of the heavy chain
`variable region of D2E7, With the predicted amino acid
`sequence beloW the nucleotide sequence. The CDR H1,
`CDR H2 and CDR H3 regions are underlined.
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`DETAILED DESCRIPTION OF THE
`INVENTION
`
`This invention pertains to isolated human antibodies, or
`antigen-binding portions thereof, that bind to human TNFO.
`With high af?nity, a loW off rate and high neutraliZing
`capacity. Various aspects of the invention relate to antibodies
`and antibody fragments, and pharmaceutical compositions
`thereof, as Well as nucleic acids, recombinant expression
`vectors and host cells for making such antibodies and
`fragments. Methods of using the antibodies of the invention
`to detect human TNFO. or to inhibit human TNFO. 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 ?rst de?ned.
`The term “human TNFot” (abbreviated herein as hTNFot,
`or simply hTNF), as used herein, is intended to refer to a
`human cytokine that exists as a 17 kD secreted form and a
`26 kD membrane associated form, the biologically active
`form of Which is composed of a trimer of noncovalently
`bound 17 kD molecules. The structure of hTNFO. is
`described further in, for example, Pennica, D., et al. (1984)
`Nature 312:724—729; Davis, J. M., et al. (1987) Biochem
`istry 26:1322—1326; and Jones, E. Y., et al. (1989) Nature
`338:225—228. The term human TNFO. is intended to include
`recombinant human TNFO. (rhTNFot), Which can be pre
`pared by standard recombinant expression methods or pur
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`chased commercially (R & D Systems, Catalog No. 210-TA,
`Minneapolis, Minn.).
`The term “antibody”, as used herein, is intended to refer
`to immunoglobulin molecules comprised of four polypep
`tide chains, tWo heavy
`chains and tWo light (L) chains
`inter-connected by disul?de bonds. Each heavy chain is
`comprised of a heavy chain variable region (abbreviated
`herein as HCVR or VH) and a heavy chain constant region.
`The heavy chain constant region is comprised of three
`domains, CH1, CH2 and CH3. Each light chain is comprised
`of a light chain variable region (abbreviated herein as LCVR
`or VL) and a light chain constant region. The light chain
`constant region is comprised of one domain, CL. The VH
`and VL regions can be further subdivided into regions of
`hypervariability, termed complementarity determining
`regions (CDR), interspersed With regions that are more
`conserved, termed frameWork regions
`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