PCT
`PCT
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 97/29131
`WO 97/29131
`
`(11) International Publication Number: (cid:9)
`(11) International Publication Number: (cid:9)
`
`(51) International Patent Classification 6 :
`(51) International Patent Classification 6 :
`CO7K 16/24, C12N 15/13, 15/64, 5/10,
`CO7K 16/24, C12N 15/13, 15/64, 5/10,
`1/21, A61K 39/395, GO1N 33/68
`1/21, A61K 39/395, GO1N 33/68
`
`Al
`Al
`
`(43) International Publication Date: (cid:9)
`(43) International Publication Date: (cid:9)
`
`14 August 1997 (14.08.97)
`14 August 1997 (14.08.97)
`
`(21) International Application Number: (cid:9)
`(21) International Application Number: (cid:9)
`
`PCT/US 97/02219
`PCT/US 97/02219
`
`(22) International Filing Date: (cid:9)
`(22) International Filing Date: (cid:9)
`
`10 February 1997 (10.02.97)
`10 February 1997 (10.02.97)
`
`(30) Priority Data:
`(30) Priority Data:
`08/599,226 (cid:9)
`08/599,226 (cid:9)
`60/031,476 (cid:9)
`60/031,476 (cid:9)
`
`9 February 1996 (09.02.96) (cid:9)
`9 February 1996 (09.02.96) (cid:9)
`25 November 1996 (25.11.96) (cid:9)
`25 November 1996 (25.11.96) (cid:9)
`
`
`US US
`US
`US
`
`(60) Parent Applications or Grants
`(60) Parent Applications or Grants
`(63) Related by Continuation
`(63) Related by Continuation
`
`US (cid:9)US (cid:9)
`Filed on (cid:9)
`Filed on (cid:9)
`US (cid:9)
`US (cid:9)
`Filed on (cid:9)
`Filed on (cid:9)
`
`08/599,226 (CIP)
`08/599,226 (CIP)
`9 February 1996 (09.02.96)
`9 February 1996 (09.02.96)
`
`60/031,476 (CIP) 60/031,476 (CIP)
`25 November 1996 (25.11.96)
`25 November 1996 (25.11.96)
`
`(71) Applicant (for all designated States except US): BASF AK-
`(71) Applicant (for all designated States except US): BASF AK-
`TIENGESELLSCHAFT [DE/DE]; Carl-Bosch-Strasse 38,
`TIENGESELLSCHAFT [DE/DE]; Carl-Bosch-Strasse 38,
`
`D-67056 Ludwigshafen (DE). D-67056 Ludwigshafen (DE).
`
`(72) Inventors; and
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): (cid:9) SALFELD, Jochen, G.
`(75) Inventors/Applicants (for US only): (cid:9) SALFELD, Jochen, G.
`
`[DE/US]; 177 Old Westboro Road, North Grafton, MA [DE/US]; 177 Old Westboro Road, North Grafton, MA
`
`01536 (US). ALLEN, Deborah, J. [GB/GB]; 143a Shel- 01536 (US). ALLEN, Deborah, J. [GB/GB]; 143a Shel-
`bourne Road, London N17 9YD (GB). KAYMAKCALAN,
`bourne Road, London N17 9YD (GB). KAYMAKCALAN,
`
`Zehra [TR/US]; 4 Piccadilly Way, Westboro, MA 01581 Zehra [TR/US]; 4 Piccadilly Way, Westboro, MA 01581
`
`(US). LABKOVSKY, Boris [US/US]; (cid:9) Apartment 532,
`(US). LABKOVSKY, Boris [US/US]; (cid:9) Apartment 532,
`1630 Worcester Road, Framingham, MA 01701 (US).
`1630 Worcester Road, Framingham, MA 01701 (US).
`MANKOVICH, John, A. [US/US]; (cid:9) 416 Lowell Street,
`MANKOVICH, John, A. [US/US]; (cid:9) 416 Lowell Street,
`Andover, (cid:9) MA 01810 (US). (cid:9) MCGUINESS, (cid:9) Brian, (cid:9) T.
`Andover, (cid:9) MA 01810 (US). (cid:9) MCGUINESS, (cid:9) Brian, (cid:9) T.
`[GB/GB]; 22 The Lane, Hauxton, Cambridge CB2 5HP
`[GB/GB]; 22 The Lane, Hauxton, Cambridge CB2 5HP
`(GB). ROBERTS, Andrew, J. [GB/GB]; (cid:9) 15 Cavendish
`(GB). ROBERTS, Andrew, J. [GB/GB]; (cid:9) 15 Cavendish
`Road, Cambridge CB1 3AE (GB). SAKORAFAS, Paul
`Road, Cambridge CB1 3AE (GB). SAKORAFAS, Paul
`[US/US]; 6114 Arbor Drive, Shrewsbury, MA 01545 (US).
`[US/US]; 6114 Arbor Drive, Shrewsbury, MA 01545 (US).
`HOOGENBOOM, Hendricus, R., J., M. [NL/BE]; Muggen-
`HOOGENBOOM, Hendricus, R., J., M. [NL/BE]; Muggen-
`straat 45, Bus 12, B-3500 Hasselt (BE). SCHOENHAUT,
`straat 45, Bus 12, B-3500 Hasselt (BE). SCHOENHAUT,
`
`David [US/US]; 55 East Ninth Street, Clifton, NJ 07011 David [US/US]; 55 East Ninth Street, Clifton, NJ 07011
`(US). VAUGHAN, Tristan, J. [GB/GB]; 9 Villa Road,
`(US). VAUGHAN, Tristan, J. [GB/GB]; 9 Villa Road,
`Impington, Cambridge CB4 4NZ (GB). WHITE, Michael
`Impington, Cambridge CB4 4NZ (GB). WHITE, Michael
`[US/US]; (cid:9) 30 Angelica Drive, Framingham, MA 01701
`[US/US]; (cid:9) 30 Angelica Drive, Framingham, MA 01701
`(US). WILTON, Alison, J. [GB/GB]; 46 Huntingdon Road,
`(US). WILTON, Alison, J. [GB/GB]; 46 Huntingdon Road,
`Cambridge CB3 OHH (GB).
`Cambridge CB3 OHH (GB).
`
`(74) Agents: DECONTI, Giulio, A., Jr. et al.; Lahive & Cockfield
`(74) Agents: DECONTI, Giulio, A., Jr. et al.; Lahive & Cockfield
`
`L.L.P., 60 State Street, Boston, MA 02109 (US). L.L.P., 60 State Street, Boston, MA 02109 (US).
`
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, (81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU,
`BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU,
`IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT,
`IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT,
`
`LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT,
`
`RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, Tr, UA, UG, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, Tr, UA, UG,
`US, UZ, VN, ARIPO patent (KE, LS, MW, SD, SZ, UG),
`US, UZ, VN, ARIPO patent (KE, LS, MW, SD, SZ, UG),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, DE, DK, ES, FI, FR, GB,
`European patent (AT, BE, CH, DE, DK, ES, FI, FR, GB,
`
`GR, 1E, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, GR, 1E, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ,
`CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG).
`CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG).
`
`Published
`Published
`With international search report.
`With international search report.
`Before the expiration of the time limit for amending the
`Before the expiration of the time limit for amending the
`
`claims and to be republished in the event of the receipt of claims and to be republished in the event of the receipt of
`amendments. (cid:9)
`amendments. (cid:9)
`.
`.
`
`
`
`(54) Title: HUMAN ANTIBODIES THAT BIND HUMAN TNFa (54) Title: HUMAN ANTIBODIES THAT BIND HUMAN TNFa
`
`(57) Abstract
`(57) Abstract
`
`Human antibodies, preferably recombinant human antibodies, that specifically bind to human tumor necrosis factor a (hTNFa) are
`Human antibodies, preferably recombinant human antibodies, that specifically bind to human tumor necrosis factor a (hTNFa) are
`
`disclosed. These antibodies have high affinity for hTNFa(e.g., Kd - 10.8 M or less), a slow off rate for hTNFa dissociation (e.g., Koff disclosed. These antibodies have high affinity for hTNFa(e.g., Kd - 10.8 M or less), a slow off rate for hTNFa dissociation (e.g., Koff
`
`= 10-3sec-I or less) and neutralize hTNFa activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an = 10-3sec-I 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 hTNFa and for inhibiting
`antigen-binding portion thereof. The antibodies, or antibody portions, of the invention are useful for detecting hTNFa and for inhibiting
`
`hTNFa activity, e.g., in a human subject suffering from a disorder in which hTNFa activity is detrimental. Nucleic acids, vectors and host hTNFa activity, e.g., in a human subject suffering from a disorder in which hTNFa activity is detrimental. Nucleic acids, vectors and host
`
`cells for expressing the recombinant human antibodies of the invention, and methods of synthesizing the recombinant human antibodies, cells for expressing the recombinant human antibodies of the invention, and methods of synthesizing the recombinant human antibodies,
`are also encompassed by the invention.
`are also encompassed by the invention.
`
`Ex. 1006 - Page 1
`
`

`

`
`
`FOR THE PURPOSES OF INFORMATION ONLY FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`applications under the PCT.
`
`AM
`AM
`
`AT AT
`
`AU AU
`
`BB BB
`
`BE BE
`BF
`BF
`BC
`BC
`BJ
`BJ
`
`BR BR
`BY
`BY
`
`CA CA
`CF
`CF
`
`CG CG
`CH
`CH
`
`CI CI
`CM
`CM
`
`CN CN
`CS
`CS
`
`CZ CZ
`
`DE DE
`
`DK DK
`
`EE EE
`
`ES ES
`
`Fl Fl
`FR
`FR
`
`GA GA
`
`Armenia
`Armenia
`
`Austria Austria
`
`Australia Australia
`
`Barbados Barbados
`
`Belgium Belgium
`Burkina Faso
`Burkina Faso
`
`Bulgaria Bulgaria
`Benin
`Benin
`
`Brazil Brazil
`Belarus
`Belarus
`
`Canada Canada
`Central African Republic
`Central African Republic
`
`Congo Congo
`Switzerland
`Switzerland
`
`Certe d'Ivoire Certe d'Ivoire
`Cameroon
`Cameroon
`
`China China
`Czechoslovakia
`Czechoslovakia
`
`Czech Republic Czech Republic
`
`Germany Germany
`
`Denmark Denmark
`
`Estonia Estonia
`
`Spain Spain
`
`Finland Finland
`France
`France
`
`Gabon Gabon
`
`
`GB GB
`
`GE GE
`
`GN GN
`GR
`GR
`
`HU HU
`IE
`IE
`
`IT IT
`JP
`JP
`
`KE KE
`KG
`KG
`
`KP KP
`
`
`KR KR
`KZ
`KZ
`
`LI LI
`
`LK LK
`
`LR LR
`
`LT LT
`
`LU LU
`
`LV LV
`
`MC MC
`
`MD MD
`MG
`MG
`
`ML ML
`MN
`MN
`
`MR MR
`
`
`United Kingdom United Kingdom
`
`Georgia Georgia
`
`Guinea Guinea
`Greece
`Greece
`
`Hungary Hungary
`Ireland
`Ireland
`
`Italy Italy
`Japan
`Japan
`
`Kenya Kenya
`Kyrgystan
`Kyrgystan
`
`Democratic People's Republic Democratic People's Republic
`of Korea
`of Korea
`
`Republic of Korea Republic of Korea
`Kazakhstan
`Kazakhstan
`
`Liechtenstein Liechtenstein
`
`Sri Lanka Sri Lanka
`
`Liberia Liberia
`
`Lithuania Lithuania
`
`Luxembourg Luxembourg
`
`Latvia Latvia
`Monaco
`Monaco
`
`Republic of Moldova Republic of Moldova
`Madagascar
`Madagascar
`
`Mali Mali
`Mongolia
`Mongolia
`
`Mauritania Mauritania
`
`
`MW MW
`MX
`MX
`
`NE NE
`NL
`NL
`
`NO NO
`NZ
`NZ
`
`PL PL
`PT
`PT
`
`RO RO
`RU
`RU
`
`SD SD
`SE
`SE
`
`SG SG
`SI
`SI
`
`SK SK
`
`SN SN
`
`SZ SZ
`
`TD TD
`TG
`TG
`
`TJ TJ
`Tr
`Tr
`
`UA UA
`UG
`UG
`
`US US
`
`UZ UZ
`
`VN VN
`
`
`Malawi Malawi
`Mexico
`Mexico
`
`Niger Niger
`Netherlands
`Netherlands
`
`Norway Norway
`New Zealand
`New Zealand
`
`Poland Poland
`Portugal
`Portugal
`
`Romania Romania
`Russian Federation
`Russian Federation
`
`Sudan Sudan
`Sweden
`Sweden
`
`Singapore Singapore
`Slovenia
`Slovenia
`
`Slovakia Slovakia
`
`Senegal Senegal
`
`Swaziland Swaziland
`
`Chad Chad
`Togo
`Togo
`
`Tajikistan Tajikistan
`Trinidad and Tobago
`Trinidad and Tobago
`
`Ukraine Ukraine
`Uganda
`Uganda
`
`United States of America United States of America
`Uzbekistan
`Uzbekistan
`
`Viet Nam Viet Nam
`
`Ex. 1006 - Page 2
`
`

`

`WO (cid:9) 97/29131
`WO (cid:9) 97/29131
`
`PCT/US97/02219
`PCT/US97/02219
`
`
`
`- 1 - - 1 -
`
`HUMAN ANTIBODIES THAT BIND HUMAN TNFa
`HUMAN ANTIBODIES THAT BIND HUMAN TNFa
`
`5 (cid:9)
`5 (cid:9)
`
`
`
`20 (cid:9)20 (cid:9)
`
`Background of the Invention
`Background of the Invention
`Tumor necrosis factor a (TNFa) is a cytokine produced by numerous cell types,
`Tumor necrosis factor a (TNFa) is a cytokine produced by numerous cell types,
`including monocytes and macrophages, that was originally identified based on its
`including monocytes and macrophages, that was originally identified based on its
`capacity to induce the necrosis of certain mouse tumors (see e.g., Old. L. (1985) Science
`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 230:630-632). Subsequently, a factor termed cachectin, associated with cachexia, was
`
`shown to be the same molecule as TNFa. TNFa has been implicated in mediating shown to be the same molecule as TNFa. TNFa has been implicated in mediating
`
`shock (see e.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518; shock (see e.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518;
`
`10 (cid:9) Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655). Furthermore, 10 (cid:9) Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655). Furthermore,
`
`TNFa has been implicated in the pathophysiology of a variety of other human diseases TNFa has been implicated in the pathophysiology of a variety of other human diseases
`
`and disorders, including sepsis, infections. autoimmune diseases. transplant rejection and disorders, including sepsis, infections. autoimmune diseases. transplant rejection
`
`and graft-versus-host disease (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169: and 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. 260 610
`U.S. Patent No. 5,231,024 to Moeller et al.; European Patent Publication No. 260 610
`15 (cid:9) B1 by Moeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey,
`15 (cid:9) B1 by Moeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey,
`K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).
`K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).
`Because of the harmful role of human TNFa (hTNFa) in a variety of human
`Because of the harmful role of human TNFa (hTNFa) in a variety of human
`disorders, therapeutic strategies have been designed to inhibit or counteract hTNFa
`disorders, therapeutic strategies have been designed to inhibit or counteract hTNFa
`activity. In particular, antibodies that bind to, and neutralize. hTNFa have been sought
`activity. In particular, antibodies that bind to, and neutralize. hTNFa have been sought
`
`as a means to inhibit hTNFa activity. Some of the earliest of such antibodies were as a means to inhibit hTNFa activity. Some of the earliest of such antibodies were
`
`mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from
`
`lymphocytes of mice immunized with hTNFa (see e.g., Hahn T; et al., (1985) Proc Natl lymphocytes of mice immunized with hTNFa (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. 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, Commun. 137:847-854; Hirai, M., et al. (1987) J. Immunol. Methods 96:57-62; Fendly,
`25 (cid:9) B.M., et al. (1987) Hybridoma 6:359-370; Moeller, A.. et al. (1990) Cytokine 2:162-
`25 (cid:9) 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. 186
`169; U.S. Patent 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 Al by 833 B1 by Wallach, D.; European Patent Application Publication No. 218 868 Al by
`
`Old et al.; European Patent Publication No. 260 610 B1 by Moeller, A., et al.). While Old et al.; European Patent Publication No. 260 610 B1 by Moeller, A., et al.). While
`
`these mouse anti-hTNFa antibodies often displayed high affinity for hTNFa (e.g., Kd these mouse anti-hTNFa antibodies often displayed high affinity for hTNFa (e.g., Kd
`
`10-9M) and were able to neutralize hTNFa activity, their use in vivo may be limited by 10-9M) and were able to neutralize hTNFa activity, their use in vivo may be limited by
`
`problems associated with administration of mouse antibodies to humans, such as short 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 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-
`an unwanted immune response against the mouse antibody in a human (the "human anti-
`mouse antibody" (HAMA) reaction).
`mouse antibody" (HAMA) reaction).
`In an attempt to overcome the problems associated with use of fully-murine
`In an attempt to overcome the problems associated with use of fully-murine
`
`antibodies in humans, murine anti-hTNFa antibodies have been genetically engineered antibodies in humans, murine anti-hTNFa antibodies have been genetically engineered
`
`30 (cid:9)
`30 (cid:9)
`
`35 (cid:9)
`35 (cid:9)
`
`
`
`SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)
`
`Ex. 1006 - Page 3
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`WO (cid:9) 97/29131
`WO (cid:9) 97/29131
`
`PCT/US97/02219
`PCT/US97/02219
`
`
`
`10 (cid:9)10 (cid:9)
`
`15 (cid:9)
`15 (cid:9)
`
`
`
`20 (cid:9)20 (cid:9)
`
`to be more "human-like." For example, chimeric antibodies, in which the variable
`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
`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 antibody chains are human-derived, have been prepared (Knight, D.M, et al. (1993) MoL
`
`Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 by Daddona, P.E., et al.). Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 by Daddona, P.E., et al.).
`
`5 (cid:9) Additionally, humanized antibodies, in which the hypervariable domains of the antibody 5 (cid:9) Additionally, humanized antibodies, in which the hypervariable domains of the antibody
`
`variable regions are murine-derived but the remainder of the variable regions and the 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 antibody constant regions are human-derived, have also been prepared (PCT Publication
`
`No. WO 92/11383 by Adair, J.R., et al.). However, because these chimeric and No. WO 92/11383 by Adair, J.R., et al.). However, because these chimeric and
`
`humanized antibodies still retain some murine sequences, they still may elicit an humanized antibodies still retain some murine sequences, they still may elicit an
`unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction,
`unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction,
`especially when administered for prolonged periods, e.g., for chronic indications, such
`especially when administered for prolonged periods, e.g., for chronic indications, such
`as rheumatoid arthritis (see e.g., Elliott, M.J., et al. (1994) Lancet 344:1125-1127; Elliot.
`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).
`M.J., et al. (1994) Lancet 344:1105-1110).
`A preferred hTNFa inhibitory agent to murine mAbs or derivatives thereof (e.g.,
`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,
`chimeric or humanized antibodies) would be an entirely human anti-hTNFa antibody,
`since such an agent should not elicit the HAMA reaction, even if used for prolonged
`since such an agent should not elicit the HAMA reaction, even if used for prolonged
`
`periods. Human monoclonal autoantibodies against hTNFa have been prepared using periods. Human monoclonal autoantibodies against hTNFa have been prepared using
`
`human hybridoma techniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568; human hybridoma techniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568;
`
`Boyle, P., et al. (1993) Cell. Immunol. 152:569-581; European Patent Application 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
`Publication No. 614 984 A2 by Boyle, et al.). However, these hybridoma-derived
`monoclonal autoantibodies were reported to have an affinity for hTNFa that was too
`monoclonal autoantibodies were reported to have an affinity for hTNFa that was too
`low to calculate by conventional methods, were unable to bind soluble hTNFa and were
`low to calculate by conventional methods, were unable to bind soluble hTNFa and were
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`unable to neutralize hTNFa-induced cytotoxicity (see Boyle, et al.; supra). Moreover, unable to neutralize hTNFa-induced cytotoxicity (see Boyle, et al.; supra). Moreover,
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`the success of the human hybridoma technique depends upon the natural presence in the success of the human hybridoma technique depends upon the natural presence in
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`human peripheral blood of lymphocytes producing autoantibodies specific for hTNFa. human peripheral blood of lymphocytes producing autoantibodies specific for hTNFa.
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`Certain studies have detected serum autoantibodies against hTNFa in human subjects Certain studies have detected serum autoantibodies against hTNFa in human subjects
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`(Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al.
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`(1990) Prog. Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H-G., et (1990) Prog. Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H-G., et
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`(1991),I. Immunol. Methods 139:145-147). (1991),I. Immunol. Methods 139:145-147).
`Alternative to naturally-occurring human anti-hTNFa antibodies would be a
`Alternative to naturally-occurring human anti-hTNFa antibodies would be a
`recombinant hTNFa antibody. Recombinant human antibodies that bind hTNFa with
`recombinant hTNFa antibody. Recombinant human antibodies that bind hTNFa with
`relatively low affinity (i.e., Kd --10-7M) and a fast off rate (i.e., Koff — 10-2 sec-1) have
`relatively low affinity (i.e., Kd --10-7M) and a fast off rate (i.e., Koff — 10-2 sec-1) have
`been described (Griffiths, A.D., et al. (1993) EMBO I 12:725-734). However, because
`been described (Griffiths, A.D., et al. (1993) EMBO I 12:725-734). However, because
`of their relatively fast dissociation kinetics, these antibodies may not be suitable for
`of their relatively fast dissociation kinetics, these antibodies may not be suitable for
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`therapeutic use. Additionally, a recombinant human anti-hTNFa has been described therapeutic use. Additionally, a recombinant human anti-hTNFa has been described
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`that does not neutralize hTNFa activity, but rather enhances binding of hTNFa to the that does not neutralize hTNFa activity, but rather enhances binding of hTNFa to the
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`25 (cid:9)
`25 (cid:9)
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`30 (cid:9)
`30 (cid:9)
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`35 (cid:9)
`35 (cid:9)
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`SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)
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`Ex. 1006 - Page 4
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`WO (cid:9) 97/29131 WO (cid:9) 97/29131
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`PCT/US97/02219
`PCT/US97/02219
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`- 3 -
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`surface of cells and enhances internalization of hTNFa (Lidbury. A.. et al. (1994) surface of cells and enhances internalization of hTNFa (Lidbury. A.. et al. (1994)
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`Biotechnol. Ther. 5:27-45; PCT Publication No. WO 92/03145 by Aston, R. et al.) Biotechnol. Ther. 5:27-45; PCT Publication No. WO 92/03145 by Aston, R. et al.)
`Accordingly, human antibodies, such as recombinant human antibodies, that bind
`Accordingly, human antibodies, such as recombinant human antibodies, that bind
`soluble hTNFcc with high affinity and slow dissociation kinetics and that have the
`soluble hTNFcc with high affinity and slow dissociation kinetics and that have the
`capacity to neutralize hTNFa activity, including hTNFa-induced cytotoxicity (in vitro
`capacity to neutralize hTNFa activity, including hTNFa-induced cytotoxicity (in vitro
`and in vivo) and hTNFa-induced cell activation, are still needed.
`and in vivo) and hTNFa-induced cell activation, are still needed.
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`5 (cid:9)
`5 (cid:9)
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`10 (cid:9)
`10 (cid:9)
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`15 (cid:9)15 (cid:9)
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`Summary of the Invention
`Summary of the Invention
`This invention provides human antibodies, preferably recombinant human
`This invention provides human antibodies, preferably recombinant human
`antibodies, that specifically bind to human TNFa. The antibodies of the invention are
`antibodies, that specifically bind to human TNFa. The antibodies of the invention are
`characterized by binding to hTNFa with high affinity and slow dissociation kinetics and
`characterized by binding to hTNFa with high affinity and slow dissociation kinetics and
`by neutralizing hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in
`by neutralizing hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in
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`vivo) and hTNFa-induced cellular activation. Antibodies of the invention are further vivo) and hTNFa-induced cellular activation. Antibodies of the invention are further
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`characterized by binding to hTNFa but not hTNFI3 (lymphotoxin) and by having the characterized by binding to hTNFa but not hTNFI3 (lymphotoxin) and by having the
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`ability to bind to other primate TNFas and non-primate TNFas in addition to human ability to bind to other primate TNFas and non-primate TNFas in addition to human
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`TNFa. TNFa.
`The antibodies of the invention can be full-length (e.g., an IgG1 or IgG4
`The antibodies of the invention can be full-length (e.g., an IgG1 or IgG4
`antibody) or can comprise only an antigen-binding portion (e.g., a Fab, F(ab1)2 or scFv
`antibody) or can comprise only an antigen-binding portion (e.g., a Fab, F(ab1)2 or scFv
`fragment). The most preferred recombinant antibody of the invention, termed D2E7, has
`fragment). The most preferred recombinant antibody of the invention, termed D2E7, has
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`a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and a a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and a
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`heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4. heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4.
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`Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the
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`amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)
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`comprising the amino acid sequence of SEQ ID NO: 2. comprising the amino acid sequence of SEQ ID NO: 2.
`In one embodiment, the invention provides an isolated human antibody, or an
`In one embodiment, the invention provides an isolated human antibody, or an
`antigen-binding portion thereof, that dissociates from human TNFot with a Kd of 1 x
`antigen-binding portion thereof, that dissociates from human TNFot with a Kd of 1 x
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`10-8 M or less and a Koff rate constant of 1 x 10-3 s'i or less, both determined by surface 10-8 M or less and a Koff rate constant of 1 x 10-3 s'i or less, both determined by surface
`plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitro
`plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitro
`L929 assay with an IC50 of 1 x 10-7 M or less. More preferably, the isolated human
`L929 assay with an IC50 of 1 x 10-7 M or less. More preferably, the isolated human
`antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koff
`antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koff
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`of 5 x 10-4 s-1 or less, or even more preferably, with a Koff of 1 x 10-4 s-1 or less. More of 5 x 10-4 s-1 or less, or even more preferably, with a Koff of 1 x 10-4 s-1 or less. More
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`preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes
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`human TNFa cytotoxicity in a standard in vitro L929 assay with an IC50 of 1 x 10-8 M human TNFa cytotoxicity in a standard in vitro L929 assay with an IC50 of 1 x 10-8 M
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`or less, even more preferably with an IC50 of 1 x 10-9 M or less and still more preferably or less, even more preferably with an IC50 of 1 x 10-9 M or less and still more preferably
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`35 (cid:9) with an IC50 of 5 x 10-10 M or less. 35 (cid:9) with an IC50 of 5 x 10-10 M or less.
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`20 (cid:9)
`20 (cid:9)
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`25 (cid:9)
`25 (cid:9)
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`30 (cid:9)
`30 (cid:9)
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`SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)
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`Ex. 1006 - Page 5
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`WO 97/29131 (cid:9)
`WO 97/29131 (cid:9)
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`PCT/US97/02219
`PCT/US97/02219
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`4 4
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`In another embodiment, the invention provides a human antibody, or antigen-
`In another embodiment, the invention provides a human antibody, or antigen-
`binding portion thereof, with the following characteristics:
`binding portion thereof, with the following characteristics:
`a) dissociates from human TNFa with a Koff of 1 x 10-3 s-1 or less, as
`a) dissociates from human TNFa with a Koff of 1 x 10-3 s-1 or less, as
`determined by surface plasmon resonance;
`determined by surface plasmon resonance;
`b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ
`b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ
`ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1,
`ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1,
`4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6,
`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;
`7, 8 and/or 9;
`c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ
`c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ
`ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2,
`ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2,
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`3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at
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`positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12. positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
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`More preferably, the antibody, or antigen-binding portion thereof, dissociates More preferably, the antibody, or antigen-binding portion thereof, dissociates
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`from human TNFa with a Koff of 5 x 10-4 s-1 or less. Still more preferably, the from human TNFa with a Koff of 5 x 10-4 s-1 or less. Still more preferably, the
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`antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koff antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Koff
`of 1 x 10-4 s-1 or less.
`of 1 x 10-4 s-1 or less.
`In yet another embodiment, the invention provides a human antibody, or an
`In yet another embodiment, the invention provides a human antibody, or an
`antigen-binding portion thereof, with an LCVR having CDR3 domain comprising the
`antigen-binding portion thereof, with an LCVR having CDR3 domain comprising the
`amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
`amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
`alanine substitution at position 1, 4, 5, 7 or 8, and with an HCVR having a CDR3
`alanine substitution at position 1, 4, 5, 7 or 8, and with an HCVR having a CDR3
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`domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ
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`ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. More 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
`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
`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
`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
`comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDR1
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`domain comprising the amino acid sequence of SEQ ID NO: 8. domain co