`Heavner et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,250,165 B2
`Jul. 31, 2007
`
`US007250165B2
`
`(54)
`
`(75)
`
`ANTI-TNF ANTIBODIES, COMPOSITIONS,
`METHODS AND USES
`
`Inventors: George Heavner, Malvem, PA (US);
`David M. Knight, BerWyn, PA (US);
`Jill Giles-Komar, DoWningtoWn, PA
`(US); Bernard Scallon, Collegeville,
`PA (US); David Shealy, DoWningtoWn,
`PA (US)
`
`(73)
`
`Assignee: Centocor, Inc., Malvern, PA (US)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`USC 154(b) by 916 days.
`
`(21)
`
`(22)
`
`(65)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Appl. No.: 09/920,137
`
`Filed:
`
`Aug. 1, 2001
`
`Prior Publication Data
`
`US 2003/0049725 A1
`
`Mar. 13, 2003
`
`Related U.S. Application Data
`
`Provisional application No. 60/236,826, ?led on Sep.
`29, 2000, provisional application No. 60/223,360,
`?led on Aug. 7, 2000.
`
`Int. Cl.
`(2006.01)
`A61K 39/395
`(2006.01)
`C07K 16/28
`U.S. Cl. ............................. .. 424/145.1; 424/130.1;
`424/133.1; 530/388.23; 530/3873
`Field of Classi?cation Search ........... .. 530/387.3;
`424/133.1, 145.1
`See application ?le for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`7/1986 Cerami et al. ............... .. 435/7
`4,603,106 A
`4/1989 Cerami et al. .............. .. 514/21
`4,822,776 A
`7/1993 Moeller et a1. .
`. 435/24027
`5,231,024 A
`8/1994 Creaven et al. .......... .. 424/85.1
`5,342,613 A
`8/1997 Boyle et a1.
`5,654,407 A
`8/1997 Le et al. ................ .. 424/133.1
`5,656,272 A *
`8/1997 Newman et al.
`424/1841
`5,658,570 A
`5,698,195 A 12/1997 Le et al. ................ .. 424/133.1
`5,698,419 A 12/1997 Wolpe et al.
`5,750,105 A
`5/1998 Newman et al. ....... .. 424/133.1
`5,888,511 A
`3/1999 Skurkovich et a1.
`424/1451
`5,919,452 A
`7/1999 Le et al. ................ .. 424/133.1
`5,958,413 A
`9/1999 Anagnostopulos
`et al. ..................... .. 424/1781
`5,993,833 A 11/1999 DeLacharriere et al.
`424/401
`6,190,691 B1
`2/2001 Mak ......................... .. 424/449
`6,277,969 B1
`8/2001 Le et al.
`.. 536/231
`6,284,471 B1
`9/2001 Le et al. .................... .. 435/7.1
`
`FOREIGN PATENT DOCUMENTS
`
`AU
`EP
`EP
`EP
`EP
`
`B-17649/92
`0212489 B1
`0218868
`0260610 B1
`0288088 B1
`
`5/1996
`3/1987
`4/1987
`3/1988
`10/1988
`
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`WO
`WO
`WO
`W0
`WO
`WO
`WO
`WO
`WO
`W0
`W0
`
`3/1989
`0308378 B1
`1/1990
`0350690 A2
`1/1990
`0351789 B1
`8/1990
`0380068 A1
`0393438 A2 10/1990
`0398327 B1
`11/1990
`0412486 B1
`2/1991
`0486526 B2
`3/1991
`0433900 A1
`6/1991
`0 526 905 A3
`8/1992
`0525570 A2
`2/1993
`0526905 A3
`2/1993
`0 610 210 B1
`5/2001
`WO90/ 00902
`2/1990
`WO91/02078
`2/1991
`WO91/09967
`7/1991
`WO 91/09967 A1
`7/1991
`WO92/16553
`3/1992
`WO92/ 07076
`4/1992
`WO92/11383
`7/1992
`WO92/13095
`8/1992
`WO93/02108
`2/1993
`WO 96/33735 A1 10/1996
`WO 97/29131 A1
`8/1997
`
`OTHER PUBLICATIONS
`
`Beutler, B. et al., “Identity of tumour necrosis factor and the
`macrophage-secreted factor cachectin,” Nature, 316:552-554
`(1985).
`Beutler, B. et al., “Passive Immunization Against Cachectin/Tumor
`Necrosis Factor Protects Mice from Lethal Effect of Endotoxin,”
`Science, 229:869-871 (1985).
`Morrison, Sherie L., “Transfectomas Provide Novel Chimeric Anti
`bodies,” Science, 229:1202-1207 (1985).
`Aggarwal, Bharat B. et al., “Human Tumor Necrosis Factor Pro
`duction, Puri?cation and Characterization,” J. of Biol. Chem.,
`260(4):2345-2354 (1985).
`Beutler, B. et al., “Puri?cation of Cachectin, A Lipoprotein Lipase
`Suppressing Hormone Secreted by EndotoXin-induced RAW 264.7
`Cells,” J. Exp. Med., 161:984-995 (1985).
`Paulus, H., APreparation and Biomedical Applications of Bispeci?c
`Antibodies@, Behring Inst. Mitt, No. 78:118-132 (1985).
`Hayashi, H. et al., “An Enzyme-linked Immunosorbent Assay for
`Recombinant Human Tumor Necrosis Factor Using Monoclonal
`Antibody,” Recent Adv. Chemother, 820-821 (1985).
`Liang, Chi-Ming et al., “Production and Characterization of
`Monoclonal Antibodies Against Recombinant Human Tumor
`Necrosis Factor/Cachectin,” Biochem. & Biophy. Res. C0mm.,
`137(2):847-854 (1986).
`Hirai, Makoto et al., “Production and characterization of
`monoclonal antibodies to human tumor necrosis factor,” J. of
`Immun. Methods, 96:57-62 (1987).
`(Continued)
`Primary Examiner4Christine J. Saoud
`Assistant Examinerilegatheesan Seharaseyon
`
`(57)
`
`ABSTRACT
`
`The present invention relates to at least one novel anti-TNF
`antibodies, including isolated nucleic acids that encode at
`least one anti-TNF antibody, TNF, vectors, host cells, trans
`genic animals or plants, and methods of making and using
`thereof, including therapeutic compositions, methods and
`devices.
`
`5 Claims, 26 Drawing Sheets
`
`Ex. 1006 - Page 1 of 98
`
`
`
`US 7,250,165 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Piguet, Pierre-Francois et al., “Tumor Necrosis Factor/Cachectin is
`an Effector of Skin and Gut Lesions of the Acute Phase of
`Graft-vs.-Host Disease,” J Exp. Med., 166:1280-1289 (1987).
`Meager, Anthony et al., “Preparation and Characterization of
`Monoclonal Antibodies Directed Against Antigenic Determinants of
`Recombinant Human Tumour Necrosis Factor (rTNF),”
`Hybridoma, 6(3):305-311 (1987).
`Fendly, Brian M. et al., “Murine Monoclonal Antibodies De?ning
`Neutralizing Epitopes on Tumor Necrosis Factor,” Hybridoma,
`6(4):359-370 (1987).
`Bringman, Timothy S. and Aggarwal, Bharat B., “Monoclonal
`Antibodies to Human Tumor Necrosis Factors Alpha and Beta:
`Applications for Al?nity Puri?cation, Immunoassays, and as Struc
`tural Probes,” Hybridoma, 6(5):489-507 (1987).
`Tracey, Kevin J. et al., “Anti-cachectin/TNF monoclonal antibodies
`prevent septic shock during lethal bacteraemia,” Nature, 330:662
`664 (1987).
`Nagai, M. et al., “Antibody to tumor necrosis factor (TNF) reduces
`endotoxin fever,” Experientia, 44:606-607 (1988).
`Shimamoto, Yoshinori et al., “Monoclonal antibodies against
`human recombinant tumor necrosis factor: prevention of endotoxic
`shock,” Immunology Letters, 17:311-318 (1988).
`Di Giovine, Francesco, S. et al., “Tumour necrosis factor in synovial
`exudates,” Annals ofthe Rheumatic Diseases, 47:768-772 (1988).
`Sunahara, N. et al., “Simple enzyme immunoassay methods for
`recombinant human tumor necrosis factor . and its antibodies using
`a bacterial cell wall carrier,” J Immunol Methods, 109:203-214
`(1988).
`Exley, A.R. et al., “Monoclonal Antibody (Mab) to Recombinant
`Human Tumour Necrosis Factor (rhTNF) in the Prophylaxis and
`Treatment of Endotoxic Shock in Cynomolgus Monkeys,” Medical
`Research Society, Abstract 184, p. 50 (1989).
`Cross, A.S. et al., “Pretreatment with Recombinant Murine Tumor
`Necrosis Factor 01 Cachectin and Murine Interleukin 1 0t Protects
`Mice from Lethal Bacterial Infection,” J of Exp Med., 169:2021
`2027 (1989).
`Whittle, Nigel, et al., “Construction and Expression of a CDR
`Grafted Anti-TNF Antibody,” J Cell Biochem, Supl. 13A:96
`(1989).
`Duncombe, Andrew S. et al., “Tumor Necrosis Factor Mediates
`Autocrine Growth Inhibition in a Chronic Leukemia,” J Immunol,
`143:3828-3834 (1989).
`Aderka, Dan et al., “IL-6 Inhibits Lipopolysaccharide-Induced
`tumor Necrosis Factor Production in Cultured Human Monocytes,
`U937 Cells, and in Mice,” JImmunol, 143:3517-3523 (1989).
`Eck, Michael J. and Sprang, Stephen R., “The Structure of Tumor
`Necrosis Factor-. at 2.6 El Resolution,” JBiol Chem, 264:17595
`17605 (1989).
`Gillies, Stephen D. et al., “High-level expression of chimeric
`antibodies using adapted cDNA variable region cassettes,” J
`Immunol Methods, 125:191-202 (1989).
`Engelmann, Hartmut et al., “A Tumor Necrosis Factor-binding
`Protein Puri?ed to Homogeneity from Human Urine Protects Cells
`from Tumor Necrosis Factor Toxicity,” J of Bio. Chem.,
`264(20):11974-11980 (1989).
`Kawasaki, Hajime et al., “Analysis of Endotoxin Fever in Rabbits
`by Using a Monoclonal Antibody to Tumor Necrosis Factor
`(Cachectin),” Infection and Immunity, 57(10):3131-3135 (1989).
`Fong, Yuman et al., “Antibodies to Cachectin/Tumor Necrosis
`Factor Reduce Interleukin 15 and Interleukin 6 Appearance During
`Lethal Bacteremia,” J Exp. Med., 170:1627-1633 (1989).
`Collins, M.S. et al., “Immunoprophylaxis of Polymicrobic Cellulitis
`with a Human Monoclonal Antibody Against Lipopolysaccharide
`Antigen of Pseudomonas aeruginosa,” Abstract E-63, Abstracts of
`Annual Meeting 1989.
`Kameyama, Koh-zoh, et al., “Convenient plasmid vectors for con
`struction of chimeric mouse/human antibodies,” FEBS Lett,
`244:301-306 (1989).
`Genebank Accession, No. N90300 (Nov. 1, 1989).
`
`Engelmann, Hartmut et al., “Two Tumor Necrosis Factor-binding
`Proteins Puri?ed from Human Urine,” J of Bio. Chem.,
`265(3):1531-1536 (1990).
`Tavernier, Jan et al., “Analysis of the Structure-Function Relation
`ship of Tumour Necrosis Factor. Human/Mouse Chimeric TNF
`Proteins: General Properties and Epitope Analysis,” J Mol. Biol.,
`211:493-501 (1990).
`Lucas, R. et al., “Generation and characterization of a neutralizing
`rat anti-rm TNF-0t monoclonal antibody,” Immunology, 71 :218-223
`(1990).
`Hinshaw, L.B. et al., “Survival of Primates in LD100 Septic Shock
`Following Therapy with Antibody to Tumor Necrosis Factor
`(TNFOt),” Circulatory Shock, 30:279-292 (1990).
`Nophar, Yaron et al., “Soluble forms of tumor necrosis factor
`receptors (TNF-Rs). The cDNA for the type 1 TNF-R, cloned using
`amino acid sequence data of its soluble form, encodes both the cell
`surface and a soluble form of the receptor,” The EMBO Journal,
`9(10):3269-3278 (1990).
`Verhoef, J. and Torensma, R., “Prospects for Monoclonal Antibod
`ies in the Diagnosis and Treatment of Bacterial Infections,” Eur J
`Clin. Microbiol. Dis., 9(4):247-250 (1990).
`Loetscher, Hansruedi et al., “Molecular Cloning and Expression of
`the Human 55 kd Tumor Necrosis Factor Receptor,” Cell, 61:351
`359 (1990).
`Schall, Thomas J. et al., “Molecular Cloning and Expression of a
`Receptor for Human Tumor Necrosis Factor,” Cell, 61:361-370
`(1990).
`Akama, Hideto et al., “Mononuclear Cells Enhance Prostaglandin
`E2 Production of Polymorphonuclear Leukocytes via Tumor Necro
`sis Factor ot,” Biochemical and Biophysical Research Comm.,
`168(2):857-862 (1990).
`Exley, A.R. et al., “Monoclonal antibody to TNF in severe septic
`shock,” The Lancet, 335:1275-1277 (1990).
`Moller, Achim et al., “Monoclonal Antibodies to Human Tumor
`Necrosis Factor 012 In Vitro and In Vivo Application,” Cytokine,
`2(3):162-169 (1990).
`Gorman, SD. and Clark, M.R., “Humanisation of monoclonal
`antibodies for therapy,” Sem Immunol, 2:457-466 (1990).
`Echtenacher, Bernd et al., “Requirement of Endogenous Tumor
`Necrosis Factor/Cachectin for Recovery from Experimental Peri
`tonitis,” J ofImmunology, 145(11):3762-3766 (1990).
`Ruddle, Nancy H. et al., “An Antibody to Lymphotoxin and Tumor
`Necrosis Factor Prevents Transfer of Experimental Allergic
`Encephalomyelitis,” J Exp. Med., 172:1193-1200 (1990).
`Von Asmuth, E.J.U. et al., “Tumour Necrosis Factor Alpha (TNF-0t)
`and Interleukin 6 in a Zymosan-Induced Shock Model,” Scand. J
`Immunol., 32:313-319 (1990).
`Herve, P et al., “Monoclonal Anti TNF 0t Antibody for the Treat
`ment of Severe Acute GvHD in Humans,” Abstract 3.25, Lymphoma
`Res. 9:591 (1990).
`Silva, Ayona T. et al., “Prophylactic and Therapeutic Effects of a
`Monoclonal Antibody to Tumor Necrosis Factor-0t in Experimental
`Gram-Negative Shock,” J of Infectious Diseases, 162:421-427
`(1990).
`Opal, Steven M. et al., “Efficacy of a Monoclonal Antibody Directed
`Against Tumor Necrosis Factor in Protecting Neutropenic Rats from
`Lethal Infection with Pseudomonas aeruginosa, ” J of Infectious
`Diseases, 161:1148-1152 (1990).
`Fong, Yuman and Lowry, Stephen F., “Tumor Necrosis Factor in the
`Pathophysiology of Infection and Sepsis,” Clin Immunol
`Immunopathol, 55:157-170 (1990).
`Starnes, H. Fletcher, Jr., et al., “ANTI-IL-6 Monoclonal Antibodies
`Protect Against Lethal Escherichia coli Infection and Lethal Tumor
`Necrosis Factor. Challenge in Mice,” JImmunol, 145:4185-4191
`(1990).
`Genebank Accession, No. M32046 (Jun. 15, 1990).
`Smith, Craig R., “Human and Chimeric Antibodies to LPS and
`TNF,” 4Abstract, Endotoxemia & Sepsis Conference (1991).
`Bodmer, Mark, “Humanized Antibodies for Anti-TNF Therapy,”
`Abstract, Endotoxemia & Sepsis Conference (1991).
`Aderka, Dan, “Role of Tumor Necrosis Factor in the Pathogenesis
`of Intravascular Coagulopathy of Sepsis: Potential New Therapeutic
`Implications,” Isr JMed Sci, 27:52-60 (1991).
`
`Ex. 1006 - Page 2 of 98
`
`
`
`US 7,250,165 B2
`Page 3
`
`Galloway, Cynthia J. et al., “Monoclonal anti-tumor necrosis factor
`(TNF) antibodies protect mouse and human cells from TNF
`cytotoXicity,” J. oflmmunological Methods, 140:37-43 (1991).
`Waldmann, Thomas A., “Monoclonal Antibodies in Diagnosis and
`Therapy,” Science, 252:1657-1662 (1991).
`Lassalle, Ph., et al., “Potential Implication of Endothelial Cells in
`Bronchial Asthma,” Int Arch Allergy Appl Immunol, 94:233-238
`(1991).
`Aderka, Dan et al., “The Possible Role of Tumor Necrosis Factor
`(TNF) and Its Natural Inhibitors, The Soluble-TNF Receptors, In
`Autoimmune Diseases,” Israel]. Med. Sci., 28(2):126-130 (1992).
`Pennington, James, “TNF: Therapeutic Target in Patients With
`Sepsis,” ASM News, 58(9):479-482 (1992).
`Harris, William J. and Emery, Steven, “Therapeutic antibodiesithe
`coming of age,” TBTECH, 11:42-44 (1993).
`Parrillo, Joseph E., “Pathogenetic Mechanisms of Septic Shock,”
`NE. Journal ofMedicine, 328(20):1471-1477 (1993).
`M. Mendez et al., “Functional transplant of megabase human
`immunoglobulin loci recapitulates human antibody response in
`
`mice”, Nature Genetics, vol. 15, No. 2, Feb. 1997, pp. 146-156
`XP002067603.
`S. Stephens et al., “Comprehensive pharmacokinetics of a human
`iZed antibody and analysis of residual anti-idiotypic responses”,
`Immunology, vol. 85, No. 4, Aug. 1995, pp. 668-674,
`XP000881488.
`S. Siegel et al., “The mouse/human chimeric monoclonal antibody
`cA2 neutralizes TNF in vitro and protects transgenic mice from
`cacheXia and TNF lethality in vivo”, Cytokine, vol. 7, No. 1, Jan.
`1995, pp. 15-25, XP000990566.
`E. Rankin et al., “The therapeutic effects of an engineered human
`anti-tumour necrosis factor alpha antibody (CDP571) in rheumatoid
`arthritis”, British Journal of Rheumatology, vol. 34, No. 4, Apr.
`1995, pp. 334-342, XP000674590.
`PCT Search Report PCT/US 01/24785 dated Jun. 28, 2002.
`
`* cited by examiner
`
`Ex. 1006 - Page 3 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 1 of 26
`
`US 7,250,165 B2
`
`NNEEIDI
`
`w¢F>ZF.InYI
`
`omF>ZF.IATI
`
`€9.55m<oIll
`
`asw_o::oo0T
`
`
`
`€505cozgcoocoon<E
`
`OS72We
`
`mb_,
`
`o
`
`Ex. 1006 - Page 4 of 98
`
`0000
`
`ooom
`
`ooov
`
`Doom
`
`Doom
`
`ooor
`
`Ego
`
`6:302
`
`Ex. 1006 - Page 4 of 98
`
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 2 0f 26
`
`US 7,250,165 B2
`
`FIG. 2A
`
`TNVS
`
`ATGGGGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGA
`
`germline
`
`Q V Q L V E S G G G V
`CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG
`
`TNVS
`
`GGTGTCCAGTGT . . . . . . . . . . . .
`
`. . . . . . . . . . . . . . . . . . . . .
`
`TNV148 (B) GGTGTCCAGTGT. . . . .A . . .
`
`. . .
`
`. . . . . . . . . . . . . . . . . . . . .
`
`V Q P G R S L R L S C A A S G
`I germline GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA
`
`TNVS
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`F S S Y A M H W V R Q A P
`F 'I‘
`germline TTCACCTTCAGTAGC'I‘A'I‘GCTA'I‘GCACTGGG'I‘CCGCCAGGCTCCA
`
`TNVléL, 15 . . . . . . . . . . . . . . . . .
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`TNV148 (B) . . . .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`'I‘NV196
`
`. . . . . . . . . . . . . . . . . . . .C . . . . . . . . . . . . . . . . . . . . . . . .
`
`G K G L E W V A V I S Y D G S
`germline GGCAAGGGGCTGGAGTGGG‘I‘GGCAGTTATATCA'I‘ATGATGGAAGC
`
`TNV14
`
`. . . . . . . . . . . . . . . . . . . . . . . .A. . . .C.T . . . . . . . . . . . .T
`
`TNVl5
`
`. . . . . . . . . . . . . . . . . . . . . . . .T . . . . . .T . . . . . . . . . . . .‘I‘
`
`TNV148 (B) . . . . .C . . . . . . . . . . . . . . . . . .T. . . .G . . . . . . . . . . . . . . .
`
`TNV196
`
`. . . . . . . . . . . . . . . . . . . . . . . .‘I‘ . . . . . . . . . . . . .
`
`. . . . . . .
`
`N K Y Y A D S V K G R F T I S
`germline AATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCC
`
`TNV14
`
`.GC. . .A.G. . . . .G . . . . . . . . . . . . .A . . . . . . . . . . . . . . . .
`
`TNVl5
`
`. .C. . .A.G . . . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . . . . .
`
`TNV148 (B) . . .
`
`. . .A.G . . . . . . . . . . . . . . . . . . . . . .
`
`. . .
`
`. . . . . . . . . . .
`
`TNV196
`
`. . .
`
`. . .A.G.C . . . . . . . . . . . . . . . . . . . . . . . . . . . .G. . . . .
`
`Ex. 1006 - Page 5 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 3 0f 26
`
`US 7,250,165 B2
`
`FIG. 2B
`
`R D N S K N T L Y L Q M N S L
`germline AGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
`
`TNV14
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`TNV15
`
`.
`
`. . . . . . . . . . . . . . . . .G . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`TNT/148
`
`.
`
`.
`
`. . . . . . .C . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`. . . . . . . . .
`
`TNVl48B
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`TNV196
`
`. . . . . . . . . . . . . . . . . .
`
`. . . . . . .T . . . . . . . . . . . . . . . . . . .
`
`R A E D T A V Y Y C A R
`germline AGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGA
`
`TNVl4, l5
`
`. . . . . . . . . . . . . . . . . . . . . . .
`
`. . . . . . . . . . . . .GATCGAGGT
`
`TNV148 (B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A
`
`TNVl96
`
`. . . . . . . . . . . . . . . . . . . . . .T . . . . . . . . . . . . . . . . . . . . . .
`
`germline
`TNV14
`
`Y Y Y Y Y G M D V W
`TACTACTACTACTACGGTATGGACGTCTGG
`
`ATATCAGCAGGTGGAA . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`.
`
`TNV15
`
`G.C .
`
`. . . . . . . . . . .A.T. .T . . . . . . . . . . . . . . . . . . . . . . . .
`
`TNV148 (B) . . .G . . . . . . . . . . .A . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`TNV196
`
`. .TGG . . . . . . . . . .A . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`G Q G T T V T V S S
`germline GGGCAAGGGACCACGGTCACCGTCTCCTCAG
`
`TNV14
`
`. .C . . . . . .
`
`.
`
`. . . . . . . . . . . . . . . . . . . . .
`
`TNVl5
`
`. .C . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`TNV148 (B) . .C . . . . . . . . . . . . . . . . . . . . . . .
`
`. . . .
`
`.
`
`TNV196
`
`. .C. .G . . . .
`
`. . . . . . . . . . . . . . . .
`
`.
`
`. . . .
`
`Ex. 1006 - Page 6 of 98
`
`
`
`U.S. Patent
`
`Jul. 31,2007
`
`Sheet 4 0f 26
`
`US 7,250,165 B2
`
`FIG. 3
`
`TNVs
`
`ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTC
`
`germline
`TNVS
`
`E I V L T Q S P A T
`GAAATTGTGTTGACACAGTCTCCAGCCACC
`
`CCAGATACCACCGGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`germline
`TNVs
`
`L S L S P G E R A T L S C R A
`CTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC
`
`u u n Q u q ~ u a ~ u o s u u ~ a - Q o ¢ o ¢ v u u u - 1 I c o n u n u u a I n o . n 0 a
`
`germline
`TNV14, l5
`TNV148,196
`
`S Q S V S S Y L A W Y Q Q K P
`AGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCT
`
`u u o a u o a n a n a a n c u a a a o a u . u - o a n o n u u Q u Q p o u a n a o a o a o
`
`.ov
`
`. . . . . .
`
`germline
`TNVs
`
`G Q A P R L L I Y D A S N R A
`GGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCC
`
`~ v ¢ ~ u v a . u . o v o » a u v Q o a o 0 o u u - . . u Q ~ c - Q a I o u . - o a - ¢ 0
`
`germline
`TNVs
`
`'1'_ G I P A R F s G s G s G T D
`ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAC
`
`y n n o n n a n o a o o - a - Q . u u ¢ . . . a v v o u u o e n u u o a q n a u ‘ a ' a C
`
`germline
`TNVS
`
`F T L T I S S L E P E D F A V
`TTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTT
`
`v - ¢ a I u u u - - a n o - a - u . n
`
`n n . - u . u u o u
`
`v Q ‘ ~ u ¢ - 1 c c v u v a o n
`
`Germline
`TNVS
`
`Y Y c Q Q R g N W P P F '1' F G
`TATTACTGTCAGCAGCGTAGCAACTGGCCTCCATTCACTTTCGGC
`
`. - a u - - Q c o - 0 - . o u - . v ‘ ~ u - - I u - c o u Q u o p - n a . a a o u Q u - -
`
`gerline
`TNVs
`
`P G T K V D I K R
`CCTGGGACCAAAGTGGATATCAAACGT
`
`a o u . u a u Q - Q n a - - - . u u n a v ~ . o u 1
`
`Ex. 1006 - Page 7 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 5 0f 26
`
`US 7,250,165 B2
`
`FIG. 4
`
`MGFGLSWVFLVALLRGVQC
`
`signal
`
`FRl
`
`CDRl
`
`PR2
`
`CDRZ
`
`u o a a o a a a o u . n a o v u a a o o a
`
`n o a n o u o 0 0
`
`germline
`TNVS
`
`germline
`TNVS
`TNV148 (B)
`
`germline
`TNVS
`
`SYAMH
`
`germline
`TNVs
`TNV148 (B)
`
`WVRQAPGKGLEWVA
`
`. . . . . . .N. . . . . .
`
`germline
`TNVl4
`TNV15
`TNV148 (B)
`TNV196
`
`VISYDGSNKYYADSVKG
`
`I .L. . . .S.K . . . . . .D
`
`F.L . . . . . .K . . . . . . .
`
`FM . . . . . . .K . . . .
`
`. . .
`
`F . . . . . . . .KS . . .
`
`.
`
`. .
`
`germline
`TNV14
`TNV15
`TNV148
`TNVl48B
`TNV196
`
`germline
`TNV14
`TNV15
`TNVl48 (B)
`TNV196
`
`RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
`
`FR3
`
`. . . . . . . . . . .A. .
`
`. . . . . . . . . . . . . .
`
`. . . .
`
`. . . . . . . . P. . . . .
`
`. . . . . . .
`
`. . . . . . . . . . .
`
`V . . . . . . . . .F . . . . . . . . . . . . .F
`
`— — — — — - ——YYYYYGMDV
`
`DRGISAGGN . . . . . .
`
`. .
`
`.V.
`
`N . . . . .
`
`. . .
`
`.A.
`
`N . . . . . . . .
`
`.G.
`
`N . . . . . . . .
`
`CDR3
`
`germline
`TNVS
`
`WGQGTTVTVSS
`
`¢ u u u a u ¢ a a 0 a
`
`Ex. 1006 - Page 8 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 6 0f26
`
`US 7,250,165 B2
`
`FIG. 5
`
`TNVS
`
`MEAPAQLLFLLLLWLPDTTG
`
`signal
`
`EIVLTQSPATLSLSPGERATLSC
`
`FRl
`
`CDRl
`
`FR2
`
`CDRZ
`
`PR3
`
`CDR3
`
`germline
`TNVS
`
`germline
`TNV14
`TNV15
`TNV148 (B)
`TNV196
`
`germline
`TNVS
`
`germline
`TNVS
`
`germline
`TNVS
`
`germline
`TNVS
`
`germline
`TNVS
`
`Ex. 1006 - Page 9 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 7 0f 26
`
`US 7,250,165 B2
`
`0 Enhancer
`
`Ex. 1006 - Page 10 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 8 0f 26
`
`US 7,250,165 B2
`
`mm
`
`_ _ _
`
`om w? or 3 NF o__ w w v N
`
`$0
`
`@553
`cozmbcmocoo Q<E -
`
`ow I
`
`25205.5
`
`E25 :8
`
`
`
`awe <83 H 8.?83
`
`QR 6E
`
`llllullllllllllll-ll'll-ll'llllll
`
`lmé
`
`2:8 =00 lnT
`
`.ocoo Q<E IOI
`
`WON
`
`Ex. 1006 - Page 11 of 98
`
`
`
`U.S. Patent
`
`3
`
`m
`
`S
`
`M
`
`U
`
`w
`
`2B5
`
`m,23
`
`am2553omwcozSEmocoon<Emow
`
`03
`
`Mk.GE
`
`:59380u5-852.“
`
`S0
`
`ow
`
`om
`
`n,mmvmwmomwwmrvwmrowmméu
`m,om?.ocooQ<E|OI
`
`
`moi:58:8ID!
`
`
`
`Esoo=8
`
`eéwcgaé
`
`Ex. 1006 - Page 12 of 98
`
`Ex. 1006 - Page 12 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 10 0f 26
`
`US 7,250,165 B2
`
`cothzcwocoo Q<E -
`C553 81
`
`o
`
`mm om MW?
`
`3 3 Q or w w
`
`>8
`
`UN .GE
`
`
`
`8.0.9 895 u 1-26am
`
`2:8 :8 In!
`
`
`
`
`
`.260 94.8 IO!
`
`1-
`
`E80 :8
`
`QERCQEEV
`
`Ex. 1006 - Page 13 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 11 0f 26
`
`US 7,250,165 B2
`
`seas
`couhzcmocoo Q<E -
`
`
`
`0
`
`_
`
`mm ON 2 m: 3 NF 2.
`
`
`
`ocoo Q<E +
`
`$5 $30 0 8.8.33
`
`QM 6E
`
`
`
`
`
`:58 __mo IUI
`
`Ex. 1006 - Page 14 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 12 0f 26
`
`US 7,250,165 B2
`
`cosmbcmocoo Q<E -
`:53 8|
`31
`
`owwl
`
`ONTI
`
`cowl
`
`2:8 =3 lnT
`
`.ocoo Q<E [O]
`
`
`
`same <83 " NNTNTR;
`
`Mk 6E
`
`ION
`
`T
`
`250855
`
`E80 :00
`
`Ex. 1006 - Page 15 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`m .UE
`
`lmm
`
`1cm
`
`
`
`
`
`0 mm woE: @5325
`
`m_.
`
`
`
`1 mm 6505
`
`X6.
`
`
`
`mwownu <ww¢0
`
`
`
`XNd X04. Xwd o "6:00 X15
`
`
`
`
`
`Ex. 1006 - Page 16 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 14 0f 26
`
`US 7,250,165 B2
`
`FIG. 9A
`
`6466C
`
`_
`
`+w|th MHX
`—1:1- without MHX
`
`175w
`150-
`
`125
`
`ug/mlAb 100
`75t
`
`50
`
`25
`
`0
`
`I
`
`I
`
`I
`
`F I
`
`F I
`
`‘i l
`
`l
`
`l
`
`5O
`40
`010 20 30
`Days in culture
`
`60
`
`70 80
`
`175-
`
`C466D
`
`-c|-with MHX
`-0- Without MHX
`
`Days in culture
`
`Ex. 1006 - Page 17 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 15 of 26
`
`US 7,250,165 B2
`
`9.55oEVSEIT
`
`9.3:.72‘SE.In!
`
`355ESE.iT
`
`9:?onE>ZPAT
`
`BEE78SEI»!
`
`
`
`Sag.8-855Iol
`
`35E2&3IT
`
`Bag5%LT
`
`mmméI.T
`
`2..GE
`
`2:92528m
`
`
`
`05.3.3E9:omcmco
`
`
`
`:32965
`
`AmEEQ
`
`Ex. 1006 - Page 18 of 98
`
`Ex. 1006 - Page 18 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 16 6f 26
`
`US 7,250,165 B2
`
`
`
`5E9 $6.69 25E 2&5 9%.: v8 55 m
`
`
`
`
`
`
`
`529 93a 2-655
`
`
`
`66.19 9%.: 2.255 g
`E29 255 2.96526 %
`
`
`
`.52 3:05 wwpocmv M,
`
`cos; @0262 2:62:56
`
`
`
`@326 .wmmé m5 2 @2358
`
`
`
`9m uovcmE 6: 3:96 __<
`
`
`
`S86v9 E8586
`
`
`
`6E9 9&5 72,526 HE
`
`i=9 3&5 Il>zP EH
`
`
`@1129 BEE ¢N<6 I
`$6.19 wmmé U
`
`
`
`a‘: GE
`
`
`
`x22: 0525.14
`
`19.
`
`am
`
`965
`56.22
`
`Ex. 1006 - Page 19 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 17 of 26
`
`US 7,250,165 B2
`
`Eta9:9:78E5m6%..935.5SWEEPE$ng9%,:
`ESEi$5935ETm<oIEggmmEdD
`
`m:GE
`
`
`
`32.025:4.
`
`E
`
`or
`
`965
`
`:82
`
`
`
`
`
`can;AmodvaEmoEcmfi*
`
`
`m1F>z._.2UmfiQEoocmEs
`amm<mmoz_22:85:93
`
`
`
`.95828:888
`
`3wamvM
`
`m
`
`N
`
`.o_
`
`EX. 1006 - Page 20 of 98
`
`Ex. 1006 - Page 20 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 18 of 26
`
`US 7,250,165 B2
`
`8%ngSEEo$2>2Fg$5SEE2-355%35:99.35oti>z+_
`
`.965mmmém52umhmano
`5:3803.09>_Ewo:_cm_m
`
`98noxEEflied8:05=<
`
`.88932855.0,
`
`
`
`._.028:20mmgocov*
`
`
`
`SEE9:9:03%gSEQmmmdflu
`
`\\\\\\\‘\\\\\\\\\\\\\\\\'
`It'll/II:lllllllltllIllllllz
`
`L\\\\\\\\\\\\\\\\\\\\‘
`”
`
`.\\\\\\\\\\\\\\\\\\\~
`
`n\\\\\\\\\\\\\‘
`Illllllll
`
`mm
`
`\x\\\\\\\
`'Illlllll,
`
`0:.GE
`
`
`
`x09:oEEt<
`
`m_.
`
`or
`
`955
`
`:85.
`
`EX. 1006 - Page 21 of 98
`
`Ex. 1006 - Page 21 of 98
`
`
`
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 19 of 26
`
`US 7,250,165 B2
`
`m<o9:05FLT
`
`«<09.3::m+
`
`mmnTDIII
`
`E>2F35:.mIol
`
`m<o35E2lol
`
`2‘EE9:9:mlnT
`
`N“.GE
`
`E90328m5«220
`0.39.9
`
` @535
`
`OODCONCOLOQ'CONV—O
`‘—
`
`292528m
`
`GEEQ
`
`Ex. 1006 - Page 22 of 98
`
`Ex. 1006 - Page 22 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 20 of 26
`
`US 7,250,165 B2
`
`
`
` 965m<o33Eor93935.8555modvq\anewmmmé9umLmQEoocmEsmodva*
`
`*
`
`“v.“
`
`$55BEEmgE>z+95:.mmN<o335EE«<09.35mi«<09.35FImmmdU
`
`<m.F.GE
`
`
`
`3.8.0:252
`
`0.0—.
`
`mN
`
`o.m
`
`md
`
`9.20
`
`:85.
`
`m
`
`v
`
`mN
`
`mxww>>
`
`F.n
`1.00
`O.
`
`Ex. 1006 - Page 23 of 98
`
`Ex. 1006 - Page 23 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 21 0f 26
`
`US 7,250,165 B2
`
`
`
` 985wmmé2uothEoo:mEsmodva*
`
`«<0BEE2g«<0BEEmEN<o9.35FImmméD
`
`*”w*
`
`
`
`mm.“.GE
`
`
`
`ES.0:252
`
`32:58
`
`0dr
`
`mN
`
`Qm
`
`m.N
`
`Q3020
`
`cam:
`
`ow
`
`mxmm>>mw.mNmaJm..
`._.—“.0-0.0
`
`Ex. 1006 - Page 24 of 98
`
`Ex. 1006 - Page 24 of 98
`
`
`
`U.S. Patent
`
`m
`
`2Bm
`
`mN<o33Emimnm,mmnEDm0.0?
`
`
`
`0w”w.mwE>2F93EmEomsE>2F9%.:mI
`mmn_-n_”w*m9nemQEoom985:mEsmodva
`
`
`m,mxmm>>
`
`7,mwmNFos.UIIO0mmWTm_m_mm_m.
`
`m.m
`
`985
`
`58:
`
`
`
`Um.“.GE
`
`x99:—oEEt<
`
`Ex. 1006 - Page 25 of 98
`
`Ex. 1006 - Page 25 of 98
`
`
`
`U.S. Patent
`
`m
`
`2Bm0,n,7m
`
`0305
`
`N<o93EmlnT
`
`mm<oSEEorom,mmad+
`mlo...3352+355ml»!mm<o35Emlar.
`:93,mova*mwi>2k9:9:
`mmEé2UEQQEoo
`
`
`3..GE
`
`592528mE2320
`
`:3game965
`
`Ex. 1006 - Page 26 of 98
`
`Ex. 1006 - Page 26 of 98
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 24 0f 26
`
`US 7,250,165 B2
`
`9.
`'o
`
`9(
`
`6
`
`E
`
`e
`
`e
`
`N>N> 0
`Z
`Z
`0
`
`0,9333 5%
`0053555 39
`D,-EEEE gm
`Dmmmm 0'53
`7'“
`w
`Vn.
`D/
`§
`*0
`
`%
`0..
`
`n\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\‘
`
`- CD
`
`
`|\
`
`
`
`Weeks
`
`FIG.15
`
`NFOCDOONCDLDVC’JN-r—O
`FF?-
`
`GroupMean
`
`Ex. 1006 - Page 27 of 98
`
`Ex. 1006 - Page 27 of 98
`
`
`
`U.S. Patent
`
`m
`
`0
`
`7
`
`05
`
`2B5m,
`
`ch.GE
`
`292528mE0920
`
`
`
`Mnm,moxBEoTN/xololmmmé¢.T*Ma*
`
`
`
`
`mN
`
`
`
`
`
`m9:9:Tm?S2:Iol0.0m9:05vaF>z+JT
`
`93ETm<ol4!
`
`
`
`
`
`mmmd2bmthEoocmEsm.m-%modvBm2w>Qm$5ch*
`
`2,$625
`
`WmNmmwmNFo
`
`Qm-
`
`E82965
`
`3:239
`
`Ex. 1006 - Page 28 of 98
`
`Ex. 1006 - Page 28 of 98
`
`
`
`
`U.S. Patent
`
`Jul. 31, 2007
`
`Sheet 26 of 26
`
`US 7,250,165 B2
`
`8 O
`
`'U)
`onv
`H_n_
`0':
`0'
`%
`mm
`.3307
`BE 22
`EF
`(“'0
`‘73;
`39
`$3 «$2.
`52 {-38
`I—IE
`(1ch
`2:2
`96;
`
`(5
`
`,__
`
`>
`
`U)
`
`ElD-PBS-cA2-10mg/kgmcA2-1mg/kg
`
`I}!
`
`8
`
`[\
`
`mm
`
`9%
`
`— I
`
`IIIIIIIIIIIIIIIIIIIIllllllllllllllllll
`lam
`m (D
`9?
`
`— u
`
`mmmnmu 0‘)m
`
`66 m LO %
`96
`_ (D
`9(-
`mmmnn
`g
`45 m
`as
`“I q-
`
`96
`
`— I
`
`IIIIIIIII
`an
`a- 0')
`
`
`
`FIG.17 ArthriticIndex
`
`N
`
`Ex. 1006 - Page 29 of 98
`
`— I
`
`II!
`
`E l
`
`-
`
`15
`
`0
`,—
`
`LO
`
`0
`
`Q.
`= E
`0 <1)L
`(5 2
`
`Ex. 1006 - Page 29 of 98
`
`
`
`US 7,250,165 B2
`
`1
`ANTI-TNF ANTIBODIES, COMPOSITIONS,
`METHODS AND USES
`
`This application is based in part on, and claims priority to,
`US. Provisional 60/223,360 filed Aug. 7, 2000 and 60/236,
`826 filed Sep. 29, 2000, each of which is entirely incorpo-
`rated herein by reference.
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`
`including
`invention relates to antibodies,
`The present
`specified portions or variants, specific for at least one tumor
`necrosis factor alpha (TNF) protein or fragment thereof, as
`well as nucleic acids encoding such anti-TNF antibodies,
`complementary nucleic acids, vectors, host cells, and meth-
`ods of making and using thereof,
`including therapeutic
`formulations, administration and devices.
`2. Related Art
`
`5
`
`10
`
`15
`
`2
`
`Surg. 762670-671 (1989); Debets et al., Second Vienna
`Shock Forum, p. 463-466 (1989); Simpson et al., Crit. Care
`Clin. 5227-47 (1989)), including fever, malaise, anorexia,
`and cachexia. Endotoxin strongly activates monocyte/mac-
`rophage production and secretion of TNF alpha and other
`cytokines (Kombluth et al., J. Immunol. 13722585-2591
`(1986)). TNF alpha and other monocyte-derived cytokines
`mediate the metabolic and neurohormonal responses to
`endotoxin (Michie et al., New Engl. J. Med. 31821481-1486
`(1988)). Endotoxin administration to human volunteers pro-
`duces acute illness with flu-like symptoms including fever,
`tachycardia,
`increased metabolic rate and stress hormone
`release (Revhaug et al., Arch. Surg. 1232162-170 (1988)).
`Circulating TNF alpha increases in patients suffering from
`Gram-negative sepsis (Waage et al., Lancet 12355-357
`(1987); Hammerle et al., Second Vienna Shock Forum, p.
`715-718 (1989); Debets et al., Crit. Care Med. 172489-497
`(1989); Calandra et al., J. Infect. Dis. 1612982-987 (1990)).
`Thus, TNF alpha has been implicated in inflammatory
`diseases, autoimmune diseases, viral, bacterial and parasitic
`infections, malignancies, and/or neurodegenerative diseases
`and is a useful
`target for specific biological
`therapy in
`diseases, such as rheumatoid arthritis and Crohn’s disease.
`Beneficial effects in open-label trials with a chimeric mono-
`clonal antibody to TNF alpha (cA2) have been reported with
`suppression of inflammation and with successful retreatment
`after relapse in rheumatoid arthritis (Elliott et al., Arthritis
`Rheum. 3621681-1690 (1993); and Elliott et al., Lancet
`34421125-1127 (1994)) and in Crohn’s disease (Van Dulle-
`men et al., Gastroenterology 1092129-135 (1995)). Benefi-
`cial results in a randomized, double-blind, placebo-con-
`trolled trial with cA2 have also been reported in rheumatoid
`arthritis with suppression of inflammation (Elliott et al.,
`Lancet 34421105-1110 (1994)).
`Antibodies to a “modulator” material which was charac-
`
`terized as cachectin (later found to be identical to TNF) were
`disclosed by Cerami et al. (EPO Patent Publication 0212489,
`Mar. 4, 1987). Such antibodies were said to be useful in
`diagnostic immunoassays and in therapy of shock in bacte-
`rial
`infections. Rubin et al.
`(EPO Patent Publication
`0218868, Apr. 22, 1987) disclosed monoclonal antibodies to
`human TNF,
`the hybridomas secreting such antibodies,
`methods of producing such antibodies, and the use of such
`antibodies in immunoassay of TNF. Yone et al. (EPO Patent
`Publication 0288088, Oct. 26, 1988) disclosed anti-TNF
`antibodies, including mAbs, and their utility in immunoas-
`say diagnosis of pathologies,
`in particular Kawasaki’s
`pathology and bacterial infection. The body fluids of patients
`with Kawasaki’s pathology (infantile acute febrile mucocu-
`taneous lymph node syndrome; Kawasaki, T., Allergy
`162178 (1967); Kawasaki, T., Shonica (Pediatrics) 262935
`(1985)) were said to contain elevated TNF levels which were
`related to progress of the pathology (Yone et al., supra).
`Other investigators have described mAbs specific for
`recombinant human TNF which had neutralizing activity in
`vitro (Liang, C-M. et al. (Biochem. Biophys. Res. Comm.
`1372847-854 (1986); Meager, A. et al., Hybridoma 62305-
`311 (1987); Fendly et al., Hybridoma 62359-369 (1987);
`Bringman, T. S. et al., Hybridoma 62489-507 (1987); Hirai,
`M. et al., J. Immunol. Meth. 96:57-62 (1987); Moller, A. et
`al. (Cytokine 22162-169 (1990)). Some of these mAbs were
`used to map epitopes of human TNF and develop enzyme
`immunoassays (Fendly et al., supra; Hirai et al., supra;
`Moller et al., supra) and to assist in the purification of
`recombinant TNF (Bringman et al., supra). However, these
`studies do not provide a basis for producing TNF neutral-
`izing antibodies that can be used for in vivo diagnostic or
`
`Ex. 1006 - Page 30 of 98
`
`20
`
`25
`
`30
`
`35
`
`40
`
`TNF alpha is a soluble homotrimer of 17 kD protein
`subunits
`(Smith et al.,
`J. Biol. Chem. 26226951-6954
`(1987)). A membrane-bound 26 kD precursor form of TNF
`also exists (Kriegler et al., Cell 53:45-53 (1988)). For
`reviews of TNF, see Beutler et al., Nature 3202584 (1986);
`Old, Science 2302630 (1986); and Le et al., Lab. Invest.
`562234 (1987).
`Cells other than monocytes or macrophages also produce
`TNF alpha. For example, human non-monocytic tumor cell
`lines produce TNF alpha (Rubin et al., J. Exp. Med. 164:
`1350 (1986); Spriggs et al., Proc. Natl. Acad. Sci. USA
`8426563 (1987)). CD4+ and CD8+ pe