(12) United States Patent
`Daugherty et al.
`
`(54) METHOD l•OR REDUCING THE
`IMMUNOGENICITY OF ANTIBODY
`VARIABLE DOMAINS
`
`(75)
`
`Inventors: Bruce L. Daugherty, South Orange, NJ
`(US); ~orge E. Mark, IIl, Princeton
`Junction, NJ (US); Eduardo A. Padlan,
`Kensington, MD (US)
`
`(73) Assignees: Merck & Co., Inc., Rahway, NJ (US);
`United States of America, Washington,
`DC (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21)
`
`Appl. No.: 09/810,502
`
`(22)
`
`Filed:
`
`Mar. 16, 2001
`
`(65)
`
`Prior PubUcatlon Data
`
`US 2002/0034765 Al Mar. 21, 2002
`
`Related U.S. Application Data
`
`(63) Co ntinuation of application No. 08/905,280, filed on Aug. I,
`l997, now abandoned, which is a continuation of application
`No. 08/609,218, fi led on Mar. 1, 1996, oow abandoned,
`which is a continual ion of application No. 08/ 109, 187, 6lcd
`on Aug. 19, 1993, now abandoned, which is a conlinuation(cid:173)
`io-part of application No. 07/702,217, filed on May 17,
`1991, now abandoned.
`Int. C l.7
`................................................ C12N 15/00
`(51)
`(52) U.S. C l .
`................. 435/ 69.6; 530/387.1; 530/387.3
`(58) F ield of Search ........................... 530/387.1, 387.3;
`435/69.6, 70.21; 424/130.1, 133.1
`
`(56)
`
`References C ited
`
`U.S. PJX:rENT DOCUMENTS
`
`4,816,567 A
`
`3/ 1989 Cabilly et al.
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`GB
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`239400
`255694
`274394
`328404
`323806
`327000
`332424
`434257
`438312
`2 188 638
`WO 87/02671
`WO 89/00999
`WO 91/08298
`WO 91/09967
`WO 9t/ !0742
`WO 91/11198
`WO 91/11534
`
`9/1987
`2/1988
`7/ 1988
`6/1989
`7/1989
`8/ t989
`9/1989
`6/1991
`7/ t991
`10/1987
`5/1987
`2/ 1989
`6/1991
`7/1991
`7/ t99!
`8/1991
`8/1991
`
`011-IER PUBLICATIONS
`
`Novotny et al., PNAS 83:226-30, 1986.*
`Hunter, W.M. ct al., Preparation of Iodine-131 Labelled
`Human Growth Hormone of High Specific Activity, 1962,
`Nature, 194, pp. 495-496 (c11mu1ative).
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`
`US006797492B2
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,797,492 B2
`Sep.28,2004
`
`English, D. et al., Single Separation of Red Blood Cells:
`Granulocytes and Mononuclear Leukocytes on Discontinu(cid:173)
`ous Density Gradcnts of Ficoll-Hypaque, 1974, J. Immu(cid:173)
`nological Methods, 5, pp. 249-252 (cumulative).
`Bernstein, F.C. el al., The Protein Data Bank: A Computer(cid:173)
`based Archival File for Macromolecular Structures, 1977, J.
`Mo!. Biol., 112, pp. 535- 542 (cumu lative).
`Rodbard, D. et al., Improved Curve-Filling, Parallelism
`Testing, Characterization Of Sensitivity And Specificity,
`Validation, And Optimization For Radioligand Assays,
`1978, lo: Radioimmuooassay & Related Procedures In
`Medicine, International Atomic Agency, Vienna, vol. 1, pp.
`469-504 (cumulative).
`Chirgwin, J. et al., Isolation of Biologically Active Ribo(cid:173)
`nucleic Acid from Sources Enriched in Ribonuclease, 1979,
`Biochemistry, 18, pp. 5294-5299 (cumulative).
`Flanagan, J.G. cl al., Arrangement of human immnoglobulin
`heavy chain constant region genes implies evolutionary
`duplication of a segment containing y, €and a. genes, 1982,
`Nature, 300, pp. 709-813 (cumulative).
`Daugherty, B.L et al.,Polymerase chain reaction facilitates
`the cloning, CDR-grafting and rapid expression of a murine
`monoclonal antibody directed against the CD18 component
`of leukocyte iotegrins, 1991, Nucleic Acids Research, 19,
`pp. 2471-2476 (cumulative).
`Beatty, P.O. ct al., Definition of a Common Leukocyte
`Cell-Surface Antigen )Lo95-150) Associated with Diverse
`Cell-Mediated Immune Functions, 1983, J. Immunol., 131,
`pp. 2913-1918 (cumulative).
`Sanchez-Madrid, r. et al., A Human Leukocyte Differen(cid:173)
`tiation Antigen Family With Distinct a-Subunits and a
`Common B-Subunit, 1983, J. Exp. Med. 158, pp.
`1785- 1803 (cumulative).
`Van Voorhis, W.C. et al., Specific Aotimononuclear Phago(cid:173)
`cyte Monoclonal Antibodies, 1983, J. Exp. Med., 158, pp.
`126-145 (cumulative).
`Wright, S.D. ct al., Identification of the C3bi receptor of
`human monocytes and macrophages by using monoclonal
`antibodies (cumulative).
`Gritz, Let al., Plasmid-encoded bygromycin B resistance:
`the sequence of hygromycin B phosphotransferase gene and
`its expression in Escherichia coli and Saccharomyces cer(cid:173)
`evisiae, 1983, Gene, 25, pp. 179-188 (cumulative).
`
`(List continued on next page.)
`
`Primary Examiner-Larry R. Helms
`(74) Allorney, Agent, or Firm-Yang Xu; Jack L. Tribble
`
`(57)
`
`ABSTRACT
`
`A unique method is disclosed for identifying and replacing
`immunoglobulin surface amino acid residues which con(cid:173)
`verts the antigenicity of a fi rst mammalian species to that of
`a second mammalian species. The method will simulta(cid:173)
`neously change immuoogenicity and strict ly preserve ligind
`binding properties. The judicious replacement o( exterior
`amino acid residues bas no effect on the ligind binding
`properties but greatly alters immunogenicity.
`
`3 C laims, 24 Drawing Sheets
`
`1 of 45
`
`BI Exhibit 1119
`
`

`

`US 6, 797 ,492 B2
`Page 2
`
`ffnIER PUBLICATIONS
`
`Bcrkner, K.L. et al., Effect of the tripartite leader on syn(cid:173)
`thesis of a non-viral protein in an adenovirus 5 recombinant,
`J 985 Nucleic Acids, Re.s., 13, pp. 841-857 (cumulative).
`Riechmann, L. et al., Reshaping human antibodies for
`therapy, 1988, Nature, 332, pp. 323-327 (cumulative).
`LoBuglio, A.F. ct al., Mouse/human chimeric monoclonal
`antibody in man: Kinetics and immune response, 1989,
`Proc. Nat' I Acad. Sci. USA, 86, pp. 422~224 (cumula(cid:173)
`tive).
`Sheriff, S., An1ibody-An1igen Complexesl, 1990, Annu.
`Rev. Biochcm., 59, pp. 439-473 (cumulative).
`Padan, E., On the Nature of Antibody Combining Sites:
`Unusual Structural Features T hat May Confer on These Sites
`an Enhanced Capacity for Bind ing Ligands, 1990, Proteins:
`Struc., Funct., Genet., 7, pp. 1L2-124 (cumulative).
`llakimi, J. ct al., Reduce lmmunogenicity And Improved
`Pharmacokinclics Of I lumani1,ed Anti-Tac In Cynomolgus
`Monkeys, 1991, J. Immunol., 147, pp. 1352-1359 (cumu(cid:173)
`lative).
`Lo, S.K. et al., Transient Adhesion of Neu1rophils To Endot(cid:173)
`helium, J. l:.xp. Med., 169, pp. 1779-1793 (cumulative).
`Sequences of Proteins of lmmunol. lat., 4th Edition, U.S.
`Dept. of Ileahh and Iluman Services (cumulative).
`Gorman, S.D. et al., Reshaping a therapeutic C04 antibody,
`1991, Proc. Nat'I. /\cad. Sci., 88, pp. 4181-4185 (cumula(cid:173)
`tive).
`Padlan, E. et al., Modeling of Antibody Comining Sites,
`1991, Methods in enzymology, 203, pp. 30-21 (cumula(cid:173)
`tive).
`Padlan, E., A Possible Procedure for Reducing The Immu(cid:173)
`nogenicity of Antibody Variable Domains While Preserving
`Their Ligand- Binding Properties, 1991, Molcc. lmmun.,
`28(4/5), pp. 489-498 (cumulative).
`Vcrhocycn, M. ct al., Reshaping Human Antibodies: Graft(cid:173)
`ing an Antilysozmc Activity, 1988, Science, 239, pp.
`1534-1536 (cumularive).
`Queen, C. c t al., A humanized antibody that binds lo rhe
`interleukin 2 receptor, 1989, PNAS, 86, pp. 10029-10033
`(cumulative).
`Bolger, M.B., ct al., Computer Modeling of C:om~ining Site
`Structure of An1i-hap1cn Monoclonal Anllbod1cs, 1991,
`Methods in Enzy., 203, pp. 21-45 (cumulative).
`
`Lewin, ·When Doe.5 I lomology Mean Something else',
`Science 237: 1570 ( 1987).
`Recek cl al. · Ilomology in Proteins and Nucleic Acids ...
`out of ii", Cell, vol. 50, p 667, 1987.
`Creighton Proteins Structure and Molecular Principals, WI I
`Freeman & Co.NY pp 93-94, 1983.
`Creighton, ·Experimental Studies of Protein Folding and
`Unfolding', Biophys. Mole/Bio, vol. 33, pp 231-233, 19?5.
`Burgess et al. ·Possible Dissociatio~ of th~ Hepar~a­
`bindjog ... by Site directed Mutagenes1s of a Smgle Lys10e
`Residue', J. of Cell Biol., vol. 111, pp 2129-2138, 1990.
`Lazar et al. Transofrming Growht Factor alpha: Mutation ...
`Different ... Biological Activities', Mo! Cell Biol. vol. 8
`8(3), pp 1247-1252, 1988.
`.
`.
`Schwartz e t al. 'A supcractive insulin [Bl0-A5pamc acid]
`insulin(human) ', Proc. Natl. Acacl. Sci. USA, Vo.l, 84, pp.
`6408-6411, 1987.
`Lia ct al. ·s1ruc1ure Function Relatioosbips in Glucagon: ...
`(homoserinc lactonc 27)-glucagon', Biochemistry, vol. 14,
`pp 1559- 1563 1975.
`.
`.
`.
`Rudikoff et al. ·r:unctional antibody lacing a variable region
`disulfide bridge', Proc. Natl. acacl. Sci, USA, vol. 79, p
`1979, 1982.
`Paoka ct al. ·Variable region framework differences . . .
`antibodies', Proc. Natl.. Acad. Sci, USA, vol. 85, pp
`3080-3084, l 988.
`Amit et al. .. lbrcc Dimensional Structure of an Antigen-An(cid:173)
`tibody Complex at 2.8/\ Resolution·, Science, vol. 233, p
`747,753. 1986.
`Ehrlich et al. PCT Technology, Macmillan Publisbers, pp
`80-83, 1989.
`Paul, Fuadamnta l Immunology p 242, Third Edition 1993.
`Ilunter et al., "Preparation of lodine-131 Labeled Human
`Growth llormonc of l!igh Specific Activity" 1962, Nature
`vol. 194, pp. 495-6.
`English ct al., ··Single Step Separation of Red Blood Cells,
`Granulocytcs and Mononuclear Leukocytes on Discontinu(cid:173)
`ous Density Graclicnrs of FicolJ-Hypaque" 1974 J. lmmu(cid:173)
`oological Methods 5, pp. 349-252.
`Bernstein cl al., "The Protein Data Bank: A Computer-based
`Archival File for Macromolecu lar Structures" 1997 J Mol
`Biol, 112, pp. 535- 542.
`* cited by examiner
`
`2 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Scp.28,2004
`
`Sheet 1 of 24
`
`US 6,797,492 B2
`
`,
`
`E
`v
`2
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`39 a
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`40
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`41
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`43 K
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`45
`
`Fractional Accessibility
`KOL
`Position
`Residue Exposure
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`FIG. la
`
`Residues In Subgroup
`Ill
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`
`3 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Scp.28,2004
`
`Sheet 2 of 24
`
`US 6,797,492 B2
`
`Fractional Accessibility
`KOL
`Position
`Residue Exposure
`
`JS39
`Residue Exposure
`
`Residues In Subgroup
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`FIG. 1b
`
`4 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Scp.28,2004
`
`Sheet 3 of 24
`
`US 6,797,492 B2
`
`Fractional Accessibility
`KOL
`J539
`Position
`Residue Exposure Residue Exposure
`
`Residues In Subgroup
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`FIG. le
`
`5 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Sep.28, 2004
`
`Sheet 4 of 24
`
`US 6, 797 ,492 B2
`
`Position
`Res idue Exposure
`
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`0 .00 Bu
`0.92 Ex
`0.00 Bu
`0.62 mE
`i .00 Ex
`i .00 Ex
`
`0.34 mB
`0.71 mE
`1.00 Ex
`0.73 mE
`0.75 mE
`1 .00 Ex
`0.69 mE
`0.79 mE
`0.21 mB
`0.62 ME
`0.00 Bu
`0.92 Ex
`0.00 Bu
`0.00 Bu
`0.00 Bu
`0.46 pB
`0.00 Bu
`0.75 mE
`0.91 Ex
`1.00 EX
`0.74 mE
`0.62 mE
`0.00 Bu
`0.95 Ex
`0.23 mB
`0.15 Bu
`0.00 Bu
`0.39 mB
`1.00 Ex
`O.i4 Bu
`0.70 mE
`0.95 Ex
`0 .31 mB
`
`Residues In Subgroup
`Ill
`IV
`
`11
`
`SF
`Q
`s
`y
`E
`A
`L
`L
`TK
`T
`a
`Q
`p
`p
`p
`ARP
`s
`s
`v
`v
`s
`s
`VL
`G
`s
`SA
`p
`PA
`G
`G
`0
`Q
`s
`T
`IV
`A
`RM
`T
`I
`I
`s
`T
`c
`c
`w
`w
`y
`YF
`a
`a
`a
`OE
`H
`KR
`p
`PS
`G
`G
`K
`OR
`A
`A
`p
`p
`v
`K
`l
`MLP
`v
`MIL
`I
`IV
`y
`YF
`GE
`G
`VI
`IV
`p
`SP
`ON L EOA
`R
`R
`
`s
`E
`L
`T
`a
`0
`p
`A
`v
`s
`v
`A
`L
`G
`a
`T
`v
`A
`I
`T
`c
`w
`y
`a
`a
`K
`p
`G
`a
`A
`p
`L
`L
`v
`I
`y
`G
`I
`p
`0
`A
`
`v
`
`0
`s
`A
`L
`T
`Q
`p
`p
`s
`
`A
`s
`G
`s
`PL
`G
`0
`s
`v
`T
`I
`s
`c
`w
`y
`0
`a
`H
`PA
`G
`RK
`A
`p
`K
`LV
`VI
`I
`FY
`G
`v
`p
`0
`A
`
`VI
`
`ND
`F
`M
`L
`T
`Q
`p
`H
`s
`v
`s
`E
`s
`p
`G
`K
`T
`v
`T
`IFM
`s
`c
`w
`y
`a
`0
`R
`p
`G
`SRG
`A
`p
`T
`T
`v
`I
`y
`G
`v
`p
`D
`R
`
`Q
`s
`v
`L
`T
`Q
`p
`p
`s
`AV
`s
`GA
`TA
`p
`G
`a
`R
`v
`T
`I
`s
`c
`w
`y
`0
`OH
`LV
`p
`G
`T
`A
`p
`K
`l
`l
`I
`y
`G
`VI
`p
`0
`R
`
`FIG. 2a
`
`6 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Sep.28,2004
`
`Sheet 5 of 24
`
`US 6, 797 ,492 B2
`
`VI
`
`F
`s
`G
`s
`IF"
`s
`s
`N
`s
`A
`s
`L
`T
`I
`s
`G
`L
`KOT
`T
`E
`D
`E
`A
`D
`y
`y
`c
`
`Position
`Residue Exposure
`
`Residues In Subgroup
`Ill
`IV
`II
`
`62
`63
`64
`65
`66
`67
`68
`69
`70
`71
`72
`73
`74
`75
`76
`77
`78
`79
`80
`8 1
`82
`83
`84
`85
`86
`87
`88
`
`F
`s
`G
`s
`K
`s
`G
`A
`s
`A
`s
`L
`A
`I
`G
`G
`l
`0
`s
`E
`D
`E
`T
`D
`y
`y
`c
`
`F
`98
`99
`G
`100
`T
`101 G
`102 T
`103
`K
`104 v
`105
`T
`106 v
`106a L
`107 G
`
`0.12 Bu
`0.85 Ex
`0.00 Bu
`1 .00 Ex
`0 .4i pB
`1.00 Ex
`1.00 Ex
`0.71 mE
`1.00 Ex
`0 .00 Bu
`1 .00 Ex
`0.00 Bu
`0 .74 mE
`0.00 Bu
`1 .00 Ex
`1 .00 Ex
`0.00 Bu
`0.76 mE
`1 .00 Ex
`0.78 mE
`0.09 Bu
`0.64 mE
`0.34 mB
`0.30 mB
`0.00 Bu
`0.16 Bu
`0.00 Bu
`
`0.04 Bu
`0 .00 Bu
`0.59 pB
`1.00 Ex
`0 .00 Bu
`0.82 Ex
`0 .00 Bu
`0.86 Ex
`0 .19 Bu
`0 .70 mE
`1.00 Ex
`
`F
`s
`GA
`s
`K
`s
`G
`T
`s
`A
`ST
`L
`A
`I
`ST
`G
`l
`OR
`ST
`EG
`D
`E
`A
`D
`y
`y
`c
`
`JL-1
`F
`G
`T
`G
`T
`K
`v
`T
`v
`L
`G
`
`F
`$
`G
`s
`K
`$
`G
`N
`T
`A
`s
`L
`T
`I
`s
`G
`L
`0
`A
`E
`D
`E
`A
`D
`y
`y
`c
`
`F
`s
`GS
`SY
`TSN
`$
`G
`TN
`TKS
`AV
`Tl
`L
`T
`I
`SN
`GR
`VA
`OE
`AV
`EG
`D
`E
`A
`D
`y
`YF
`c
`
`F
`s
`G
`s
`s
`s
`G
`H
`T
`A
`s
`L
`T
`I
`T
`G
`A
`o
`A
`E
`D
`E
`A
`D
`y
`y
`c
`
`JL·2
`F
`G
`G
`G
`T
`K
`L
`T
`v
`L
`G
`
`JL·3
`F
`G
`G
`G
`T
`K
`L
`T
`v
`l
`G
`
`JL·4
`F
`G
`s
`G
`T
`a
`L
`T
`v
`L
`s
`
`v
`
`F
`s
`G
`s
`K
`s
`OG
`N
`T
`A
`s
`L
`T
`v
`s
`G
`L
`RO
`A
`E
`D
`E
`A
`D
`y
`y
`c
`
`JL·5
`F
`G
`s
`G
`T
`a
`L
`T
`v
`L
`G
`
`• additional residues after position 66:
`66a D
`66b SRO
`
`FIG. 2b
`
`7 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Scp.28, 2004
`
`Sheet 6 of 24
`
`US 6,797,492 B2
`
`Position
`Residue Exposure
`
`,
`
`2
`3
`4
`5
`6
`7
`8
`9
`10
`
`, ,
`
`12
`13
`14
`15
`16
`17
`18
`19
`20
`2i
`22
`23
`35
`36
`37
`38
`39
`40
`41
`42
`43
`44
`45
`46
`47
`48
`49
`57
`58
`59
`60
`61
`62
`63
`
`Residues In Subgroup
`IV
`Ill
`II
`
`E
`0
`I
`I
`v
`v
`M
`L
`T
`T
`0
`0
`s
`s
`p
`p
`G
`L
`s
`T
`l
`L
`s
`p
`v
`L
`s
`T
`p
`p
`G
`G
`E
`E
`p
`R
`A
`A
`s
`T
`L
`I
`s
`s
`c
`c
`w
`w
`y
`y
`0
`L
`Q
`Q
`K
`K
`p
`p
`G
`G
`a
`a
`s
`A
`p
`p
`OER R
`L
`L
`L
`L
`I
`I
`y
`y
`G
`G
`v
`I
`p
`p
`0
`0
`R
`R
`F
`F
`s
`s
`
`D
`I
`v
`M
`T
`a
`s
`p
`ON
`s
`L
`A
`v
`s
`L
`G
`E
`R
`A
`T
`I
`N
`c
`w
`y
`0
`0
`K
`p
`G
`a
`p
`p
`K
`l
`l
`I
`y
`G
`v
`p
`D
`R
`F
`s
`
`0
`
`'
`
`0
`M
`T
`0
`s
`p
`s
`s
`L
`s
`A
`s
`v
`G
`0
`R
`v
`T
`I
`T
`c
`w
`y
`0
`Q
`K
`p
`G
`K
`A
`p
`K
`L
`L
`I
`y
`G
`v
`p
`s
`R
`F
`s
`
`E
`I
`v
`l
`T
`0
`s
`p
`A
`I
`T
`A
`A
`s
`L
`G
`0
`K
`v
`T
`I
`T
`c
`w
`y
`0
`a
`K
`s
`G
`T
`s
`p
`K
`p
`w
`I
`y
`G
`v
`p
`A
`A
`F
`s
`
`0.99 Ex
`0.16 Bu
`0.87 Ex
`0.00 Bu
`0.80 mE
`0.00 Bu
`0.89 Ex
`0.67 mE
`1.00 Ex
`0.94 Ex
`0.30 mB
`0.59 pB
`0.00 Bu
`0.78 mE
`0.79 mE
`1 .00 Ex
`0.64 mE
`0.74 mE
`0.22 mB
`0.65 mE
`0.00 Bu
`0.69 mE
`0.00 Bu
`0.00 Bu
`0.00 Bu
`0.14 Bu
`0.24 mB
`0.69 mE
`1.00 Ex
`1.00 Ex
`0 .90 Ex
`0.30 mB
`0.00 Bu
`0.90 Ex
`0.43 pB
`0.16 Bu
`0.00 Bu
`0.42 pB
`1.00 Ex
`0.13 Bu
`0.61 mE
`1.00 Ex
`0.36 mB
`0.00 Bu
`0.94 Ex
`
`FIG.3o
`
`8 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Sep.28, 2004
`
`Sheet 7 of 24
`
`US 6, 797 ,492 B2
`
`Position
`Residue Exposure
`
`Residues In Subgroup
`Ill
`IV
`II
`
`64
`65
`66
`67
`68
`69
`70
`71
`72
`73
`74
`75
`76
`77
`78
`79
`80
`8 1
`82
`83
`84
`85
`86
`87
`88
`
`98
`99
`100
`101
`102
`103
`104
`105
`106
`106a
`, 07
`
`G
`s
`G
`s
`G
`T
`s
`y
`s
`l
`T
`I
`N
`
`,.
`
`M
`E
`A
`E
`D
`A
`A
`I
`y
`y
`c
`
`F
`G
`A
`G
`T
`K
`L
`E
`L
`
`K
`
`0.00 Bu
`1.00 Ex
`1.00 Ex
`1.00 Ex
`1.00 Ex
`0.75 mE
`0.98 Ex
`0.09 Bu
`0.70 mE
`0.00 Bu
`0.43 pB
`0.00 Bu
`0.83 Ex
`0.83 Ex
`0.00 Bu
`0.63 mE
`0.96 Ex
`0.91 Ex
`0. 13 Bu
`0.55 pB
`0.00 Bu
`0.58 pB
`0.00 Bu
`0. 11 Bu
`0.00 Bu
`
`0.00 Bu
`1.00 Ex
`1 .00 Ex
`0.00 Bu
`0.00 Bu
`0.79 mE
`0.00 Bu
`0.89 Ex
`0.44 pB
`
`0.77 mE
`
`G
`s
`G
`s
`G
`T
`OEO
`F
`T
`l
`T
`I
`s
`s
`L
`a
`p
`ED
`D
`F l
`A
`T
`y
`y
`c
`
`JK·1
`F
`G
`a
`G
`T
`K
`v
`E
`I
`
`K
`
`G
`s
`G
`s
`G
`T
`D
`F
`T
`L
`K
`I
`s
`R
`v
`EO
`A
`E
`D
`v
`G
`v
`y
`y
`c
`
`JK-2
`F
`G
`a
`G
`T
`K
`L
`E
`I
`
`G
`$
`G
`s
`G
`T
`D
`F
`T
`L
`T
`I
`s
`R
`L
`E
`p
`E
`D
`F
`A
`v
`y
`y
`c
`
`G
`$
`G
`s
`G
`T
`D
`F
`T
`L
`T
`I
`s
`s
`L
`a
`A
`E
`D
`v
`A
`v
`y
`y
`c
`
`JK·3
`F
`G
`p
`G
`T
`K
`v
`D
`I
`
`JL-4
`F
`G
`G
`G
`T
`K
`v
`E
`I
`
`JL·5
`F
`G
`a
`G
`T
`A
`L
`E
`I
`
`K
`
`K
`
`K
`
`K
`
`FIG. 3b
`
`9 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Sep.28, 2004
`
`Sheet 8 of 24
`
`US 6, 797 ,492 B2
`
`Mouse Light Chain Variable Region
`s· upstream primer • FR 1 of variable region
`5·. TCT CGG ATC CGA (CT)AT (TC)GT G(AC)T {GC)AC CCA (GA) .3·
`BatnHI
`(SEQ ID NO: 1)
`
`3' downstream primer • kappa constant region
`5·. TCT CM GCJ TTG GTG GCA AGA T(GA)G AT A CAG TTG GTG CAG C ·3'
`Hind 111
`(SEO 1D NO: 2)
`
`Mouse Heavy Chain Variable Region
`s· upstream primer • FRi of variable region
`i) s·. TTC TGG AJC C(CG)A GGT (GCT)CA (AG)CT G(AC)A G(GC)A GTC (T A)GG ·3'
`BamHI
`<SEO ID NO: 3)
`ii) 5" nc TGG ATC C(CG)A GGT (GCT)AA GCT GGT G(GC)A GTC (i A)GG .3·
`Som HI
`csEa 10 NO: 4)
`
`3· downstream primer • lgG2a CH1 reg ion
`5·. TCT CM GCT TAC CGA TGG (GA)GC TGT TGT m GGC .3· (SEO ID NO: 5)
`Hind Ill
`SHORTEN VERSIO'J OF THE 9G4 HEAVY CKA.IN CO'-lST ANT REGO>J
`
`5" ATI TGG ATC C TC TAG A CA TCG CGGATA GAC AAG AAC .3·
`BamHI Xbol
`5·. AA! AAi GCGGC.cOC ATCG AT GAGCTCAAGT ATGTAGACGGGGTACG -3
`Not I
`Clo I
`Soc I
`<SEO 1D NO: 7)
`
`(SEQ ID NO: 6)
`
`TKPROMOTERFRAGMENT
`5'- TAT AGA ATi C GG TAC CCT TCA TCC CCG TOO CCC G .3·
`Eco RI
`Kpn I
`5·. TGC GTG TTC GM TTC GCC .3·
`Eco RI
`
`(SEQ ID NO: 9)
`
`(SEO ID NO: 8)
`
`lg H ENHANCER
`s·. TIT TAG ATC l G! CGA. CAG ATG GCC GAT CAG MC CAG .3·
`Boll I Sal I
`S'· TTG GIC GAC GGIACC MT ACA m TAG MG TCG AT .3·
`Sal I
`Kpn I
`HUw.N KAPPA CCNS'TANT REGIC)-J
`5'· TCT COO ATC CTC TAG MG AA T GGC TGC AAA GAG C ·3'
`s·. TCT CGC TAG CGG ATC CTi GCA Bt\G GA T GAT AGG G ·3'
`
`(SEQ ID NO: 10)
`
`(SEQ ID NO: 11 )
`
`(SEQ ID NO: 12)
`
`(SEO ID NO: 13)
`
`FIG. 4
`
`10 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Scp.28, 2004
`
`Sheet 9 of 24
`
`US 6,797,492 B2
`
`Oligodeoxynucleotides for PCR Amplification of the LEN light Chain
`Variable Reg ion
`
`(SEQ ID NO: 14)
`S'· CAT TCG CTI NX, AGA TCT MG CTI~ AGT ~ATC ACA GTI CTC TCT AC .3·
`S · TGG CTC l'GC AGC TG.t. TGG TG -3'
`(SEQ ID NO: ) 5)
`
`S 1
`V 9
`
`V 10 S'· CAC CAT CAG CTG CAG AGC CA-3'
`V 1 1 5'· CTG TCT GGG ATC CCA G.t.T TC ·3'
`
`(SEQ ID NO: 16)
`(SEQ ID NO: 17)
`
`V 12 S· GAA TCT GGGATCCCAG.t.CAG .3·
`(SEQ ID NO: 18)
`V 1 3 S'· GTI GCA ACA TCT Tc.A GCC TCC ~ CTG CTG ATG ·3' (SEQ ID NO: 19)
`
`V 14 5·. GTGGAGGCT GAAGAT GTIGCAACTTATTACTG ·3· (SEQ ID NO: 20)
`S'· GA.A TGT GCCTAC m CTAGAG GAT CCAACT G.t.GG.MGCAAAG ·3' (SEQ ID NO: 21 )
`13
`
`A 1
`A 2
`
`5·. CAT TCG CTI ACC AGA TCT .3·
`(SEO ID NO: 22)
`5-. GM TGT GCC TAC TTT CT A G .3· (SEQ ID NO: 23)
`
`Oligodeoxynucleotides for PC~ Ampl ification of the mi 84
`Heavy Chain Variable Region
`
`V 1
`
`s·. CCC TCC AGG CTI CAC TAA GTC TCC CCC .3· (SEO ID NO: 24)
`
`V 2
`V 3
`
`V 4
`V 5
`
`V 6
`V 7
`
`V 8
`I 2
`
`5" TTA GTG MG CCT GGA OOG TCC CTG AAA CTC .3· (SEO ID NO: 25)
`S'· GC.c CCT TCC CAG GAG CTI 00C GAA CCC MG ACA TG ·3' (SEQ ID NO: 26)
`
`S'· AAG CTC CTG ~ AOO OOC TGG AGT TGG TCG CAG CC -3 {SEO ID NO: 27)
`5·. TGT TCA m GT A GGT Ac.A OOG TGT TCT TGG MT TGi CTC TGG AGA TOO TG .3·
`(SEO ID NO: 28)
`5'· TGT ADC TAC AAA TGA ACA GTC TGA GGG CTG AGG ACA CAG CCT TGT ATI ·3'
`5'· CTG TGA GM GGG TGC CTI OGC CCC AGT N3 .~
`(SEO ID NO: 29)
`• (SEO ID NO: 30)
`5'· AA(!, GOA CCC TTC TCA CAG TCT CCT CAG GTG -3'
`(SEO ID NO: 3 1)
`S'· GM TGT GCC TAC TTAAGC m CTA ~ GAT CCT ATA MT CTC TGG CCA TG .3·
`(SEO ID NO: 32)
`
`Si, A1, and A2. as above
`
`FIG. 5
`
`11 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`US. Patent
`
`Sep.28,2004
`Sep. 28, 2004
`
`Sheet 10 of 24
`Sheet 10 0f 24
`
`US 6, 797 ,492 B2
`US 6,797,492 32
`
`(.0
`
`.
`<..!)
`Li:
`
`M
`
`-
`
`f
`
`>
`
`;f ! M
`-
`; -
`zr
`
`.....
`>
`
`)(
`)I(
`)(
`)(
`
`)(
`II(
`II(
`)(
`
`)(
`
`a:
`{.) a..
`==>
`
`
`
`+ N <
`
`[1'-
`
`m4
`
`"' ~ x
`-:c
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`
`.....
`a:
`Cl
`(..)
`
` :25$50#50E8.93
`wmnwmmg
`
`12 of45
`
`BI Exhibit 1119
`
`12 of 45
`
`BI Exhibit 1119
`
`

`

`~ "° N
`-....J "° -....J
`
`
`~~
`
`N
`t::d
`
`e
`
`VJ
`
`~
`
`0 -N
`
`....
`~ .... ....
`00 =(cid:173)~
`
`~
`Q
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`.,.~
`N
`"!='
`~
`00
`
`(D = ......
`~ ......
`""C
`•
`00
`d •
`
`8glll{1015)
`EcoRV( 1011)
`Clo I( 1004)
`EcoRl(997)
`Soc I (995)
`Kpn 1(989)
`Smol{983)
`BomHl(977)
`
`Socl(569)
`
`Tl
`IC
`Koppa (3.4 kb)
`pSP72/HUMAN C-
`
`( p8943)
`
`(CONT. ON FIG.78) I
`
`Amp R
`
`/ ..,.,._ColEI Ori
`
`(2.5 kb)
`pSP72
`
`FIG. ?a
`
`Socl(60l
`Xbol (54)
`BomHl(46)
`Xbol(40)
`
`Soll(34)
`Pst I (32)
`
`Sphl(26)
`Hindill(16)
`Xhol(4)
`
`( 0.9kb)
`
`Bari
`
`Human C -kappa by PCR
`
`BomHI \.l Kho I
`
`BomHI
`
`Bgl 11(85)
`EcoRV(81)
`
`Clol(74)
`EcoRI (67)
`Soc I (65)
`
`Sspl(2012)
`
`MCS
`
`Kpnl (59)
`Smal(53)
`BamHl(46)
`Xba 1(40)
`
`Soll(34)
`Pstl(32)
`$Ph 1(26)
`Hind 111(16)
`Xhol(4)
`
`13 of 45
`
`BI Exhibit 1119
`
`

`

`N
`t::d
`N
`\C
`~
`-..-l
`\C
`-..-l
`
`
`~~
`
`e
`
`VJ
`
`~
`
`0 -N
`
`~ .... ....
`00 =(cid:173)~
`
`N
`
`~
`Q
`N
`.,.~
`N
`"!='
`~
`00
`
`(D = ......
`~ ......
`""C
`•
`00
`d •
`
`BamHl(3393
`Spel{3399)
`Soc[{3405)
`
`Pvu 11(2993)
`Ad2 MLP
`~ "Kpnl{2347)
`. ~ (2008)
`Soll
`
`I
`
`(1995}
`BomHI
`
`Nhel(3411}
`Clol (3417)
`Ndel(3423)
`Notl(3429)
`
`SV 40 L(A)n
`
`(6.1 kb}
`
`p05 I lgH-Enhoncer/HygB
`~ (p8942)
`
`Amp
`
`Pstl(5393)
`
`Pvul(5523) __ _.... ----(cid:173)
`
`I
`
`BomH1(6)
`
`(0.6 kb)
`
`Xbol
`VEM±RED 184 V-Koppo by PCR
`
`Hindll Spel
`
`'I
`
`Xbol IHnidl+XboI
`Hnidl+
`
`(CONT FROM FIG. ?A)
`
`( 1.5 kb Koppa Cho in)
`Spe I+ Clo!
`
`{CONT ON FfG. 7C) 1 Spel + Clal
`
`FIG. 7b
`
`Bgl II (1554)
`EcoRV(1550)
`Clo1(1543)
`EcoRH1536)
`Socl(1534)
`Kpn 1(1528)
`Smol(1522)
`BomHl(1516)
`
`Soc 1(1108)
`
`Human C-Kappa
`
`Koppo(4.0kb)
`pSP72/Rei1B4
`
`(p8952)
`
`~ Socl(599)
`Xbol(593)
`BomHI(587)
`
`Spel(22l V184VK
`Hind01(16)
`
`Xhol(4)
`
`14 of 45
`
`BI Exhibit 1119
`
`

`

`N
`t::d
`N
`\C
`~
`-..-l
`\C
`-..-l
`l'.
`VJ
`
`~e
`
`e
`
`~
`
`~
`
`....
`~ ....
`00 =(cid:173)~
`
`0 -N
`
`FIG. 7c
`
`Kpnl(3815}
`
`Kpnl(3717)
`
`Pvu II (2993)
`Ad2 MLP
`
`Spel(3399)
`BomH 1(3393)
`Ad2 Leader
`
`V40 Enhancer
`Kpnl(2347)
`lgH-E nhoncer
`Sol 1(2008)
`BomH[(1995)
`
`v1B4VK
`
`\ Human C-Koppo
`
`BomH1(4889)
`Kpnll4891)
`Socl{4897)
`EcoR1{4901)
`Clol(4904) ~ ~'
`Ndel (4910)
`Notl(4916}
`
`p05 / lgH-Enhoncer I HygB/
`
`Humanized 184-Koppa
`
`v1B4VK l7.7kb)
`
`~
`Q
`N
`.,.~
`N
`"!='
`~
`00
`
`(D = ......
`~ ......
`""C
`•
`00
`d •
`
`( p8953)
`
`tk-HygB
`
`BomH1(6)
`
`Pstl(6880)
`
`Pvu 1(7010)
`
`(From FIG. 78) i
`
`15 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`US. Patent
`
`Sep.28,2004
`Sep. 28, 2004
`
`Sheet 14 of 24
`Sheet 14 0f 24
`
`US 6,797,492 B2
`US 6,797,492 B2
`
`!e L:
`.. :::1 ~
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`l··· :: ::1 !;:
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`16 of45
`
`BI Exhibit 1119
`
`“mammal!IIIi§§
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`.mmmmmuunwQE9;m53,~>Ag
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`_J
`
`-··· •• ••• >
`
`16 of 45
`
`BI Exhibit 1119
`
`
`
`

`

`~
`-....J
`\0
`-....J
`l',
`rJJ
`~
`
`~C
`
`N
`~
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`"° N
`
`~
`N
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`....
`.....
`\J') =(cid:173)~
`
`('>
`
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`:0
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`('>
`\J')
`
`~ = ~
`
`~
`~
`'"'C
`•
`rJ'J
`•
`Cj
`
`FIG.9o
`
`Hind 111(3533)
`Ncol(3401)
`SpeH3353l
`Hind 111(3347)
`Bgl 11(3268)
`Xhol(3097)
`Pvull (2942)
`Ad2 MLP
`SV40 Enhancer
`Kpnl(2296)
`lgH-Enhoncer
`Soll(1957l
`
`BomHl(4102)
`Smo I (4042)
`
`Chimeric 184 vu.~
`Ad2 Leader, fJ
`
`Bgl 11 (4856)
`Pstl(4887)
`Short Human C-G<mno 4
`
`Socl(5881)
`Clol(5887)
`Not 1(5893) ~
`
`Human C-Gommo 4
`/ORI Neo/184 VH-Short
`p05/lgH-Enhoncer I
`
`(8. 7 kb)
`
`SV40UA)n
`
`Pst117856) ~P ~
`· ~ ~tk-Neo
`Pvul(7986}
`
`EcoRH8617l\tk
`Kpnl(8623)
`
`( 0.5 kb)
`
`I
`BomHI
`VENEERED 184 VH BY PCR
`
`Bgll*Spel
`
`Hindm
`
`..
`
`17 of 45
`
`BI Exhibit 1119
`
`

`

`~ "° N
`-..-l "° -..-l
`
`N
`t::d
`
`~~
`VJ
`
`e
`
`~
`
`0 -N
`
`0\
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`00 =(cid:173)~
`
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`Q
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`"!='
`~
`00
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`(D = ......
`~ ......
`""C
`•
`00
`d •
`
`Hind 111(3533)
`Nco1(3401)
`Spell3353l
`Hind Ill ( 334 7)
`Bgl 11(3268)
`Xhol(3097)
`Pvull (2942)
`Ad2 MLP
`SV 40 Enhancer
`Kpnl(2296)
`lgH-Enhoncer
`Soll(1957l
`
`Human C-Gommo 4
`/ORI Neo/184 VH-Short
`p05/lgH-Enhoncer I
`
`(8. 7 kb)
`
`Bgl II (4856)
`Pst 1(4887)
`Short Human C-Gomno 4
`
`Socl(5881 l
`CloU5887)
`Not 1(5893)---w-..
`
`SV40L(Aln
`
`FIG. 9b
`
`BomHl(4102)
`Smol(4042}
`
`v1B4--h
`Ad2 Leoderi"
`
`Pstl(7856)~ ~
`Pvul(7986}~ ~tk-Neo
`
`EcoRI (8617)\tk
`Kpnl{8623}
`
`18 of 45
`
`BI Exhibit 1119
`
`

`

`N
`t::d
`N
`\C
`~
`-....J
`\C
`-....J
`
`
`~~
`
`e
`
`VJ
`
`~
`
`0 -N
`
`....
`~ ....
`00 =(cid:173)~
`
`-....J
`
`~
`Q
`N
`.,.~
`N
`"!='
`~
`00
`
`(D = ......
`~ ......
`""C
`•
`00
`d •
`
`pCMVIE-AK1-0HFR
`
`(9.2 kb)
`
`Ori
`
`Ap
`
`7
`
`BomHl(375}
`
`Hindl0(29)
`Clol(23)
`EcoRI(1)
`
`EcoR1(3.9 kb)
`Kpnl +
`
`-BomHl(1406)
`Ad2 Leader
`
`Not I (1443)
`Ndel(1437)
`Clol(1431)
`NheI(1425)
`Socl(1419)
`Spel(1413)
`
`r-.1
`
`Pvu II( 1006)
`Ad2 MLP
`Hind 111(726)
`SV40 Enhancer
`
`(4.2Kb)
`p05(MCS)
`
`PstI(3406l
`Pvul(3536)
`
`(CONT ON FIG. !Cl>)
`
`FIG. fOa
`
`(0.3kb)
`\Q
`Bglll Soll Kpnl
`lgH enhancer by PCR
`
`EcoRl(4565)
`
`omHl(4766)
`
`EcoRI(5041)
`
`Kpnl(5340)
`Bglll(5570}
`.,
`
`Promoter
`CMVIE
`
`-BomHl(2766)
`
`'
`tk-Neo(AKI)
`
`.
`
`BGH(A)n
`
`19 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Sep.28, 2004
`
`Sheet 18 of 24
`
`US 6, 797 ,492 B2
`
`1 (CONT. FROM FIG. IOo)
`
`Pvu1(54 72)
`Pstl(5342) ~/
`/Amp
`
`(p8928)
`p05/IgH-Enhoncer/Neo
`(6.1kb)
`
`Soll(1957)
`lgH -Enhancer
`KpnI(2296)
`V40 Enhancer
`Ad2 MLP
`Pvul1(2942)
`
`t ke Promoter
`(Octamer and CAT
`Region by PCR)
`EcoRI
`EcoRI
`ld
`(0.1kb)
`
`SV40 L(A)n
`
`Notl(3379)
`Ndel(3373)
`Clo1{3367)
`Nhell3361)
`Soc1{3355)
`Spel(3349)
`BomHl(3342)
`
`Pvul(5472)
`Pst 1(5342)
`
`f'Amp
`
`~tk-Neo
`
`·-H.1--'
`(p8941)
`p05/lg H-Enhoncer /Neo
`(6 .2 kb)
`
`SV40 L(A)n
`
`Ad2 Leader
`"
`
`Notl(3379)
`Ndel(3373)
`CLol(3367)
`Nhe1{3361)
`Sac1(3355)
`Spel(3349)
`BamH1(3342)
`
`Ad2 MLP
`
`Sol!(1957)
`lgH- Enhancer
`Kpnl(2296)
`SV40 Enhancer
`(Hind III minus)(2662)
`Pvu II {294 2)
`
`FIG.10b
`
`20 of 45
`
`BI Exhibit 1119
`
`

`

`e
`
`~
`
`0 -N
`
`\0
`.....
`~ ....
`00 =(cid:173)~
`
`~
`Q
`N
`.,.~
`N
`"!='
`~
`00
`
`(D = ......
`~ ......
`""C
`•
`00
`d •
`
`l'.
`VJ
`
`~e
`
`~ "° N
`-..-l "° -..-l
`
`N
`t::d
`
`(CONT ON FIG. 11 b)
`
`BamHI
`
`{1.9kb tk-HygB}
`BomHl
`Soll(2551}
`
`BomHll2275)
`
`FJG. I la
`
`(2.5 kb)
`pSP72
`
`ColEIOri
`
`( 5.6 kb)
`pAL2
`
`tk-HygB
`
`Sspl(2012)
`
`~ Bglll(85)
`
`EcoRV(81)
`
`Clo 1(74)
`EcoRil67)
`Soc I (65)
`
`Kpnl(59)
`Smol(53)
`BomH1(46)
`Xbol (40)
`
`Soll (34)
`Pstl(32)
`Sph 1(26)
`Hind 111(16)
`Xho 1(4)
`
`BomHl(375)
`
`Hind Ill (29)
`EcoRl (1)
`
`21 of 45
`
`BI Exhibit 1119
`
`

`

`N
`~
`N
`\C
`~
`-.....l
`\C
`°' ~
`r:J')
`L:
`
`0 -N
`
`.&:>.
`
`Q
`N
`~ .....
`(J) =(cid:173)
`
`('!)
`
`~
`
`N
`t:i
`N
`"P
`~
`(J)
`
`~O
`
`(D = .....
`~ .....
`~
`•
`00
`0 •
`
`Pvu ll(2942)
`
`IgH-Enhancer
`Soll(1957)
`
`(Hind Ill minus)(2662)
`
`SV 40 Enhancer
`//V Kpn I (2296)
`
`(CONT ON FIG. 11c)
`
`/
`BomHl(3342)'
`Spel(3349)
`Socl(3355)
`Nhel(3361)
`Clol(3367)
`Ndel(3373)
`Not 1(3379)
`
`Pstl(5342)
`Pvul(5472) ~
`
`EcoRl(6103) \ { ,EcoRl(1)
`Kpnl(6109) tk
`
`r (CONT FROM FIG. lla)
`
`Bgl 11(1985)
`EcoRV(1981l
`Clol(1974)
`EcoRl(1967l
`Socl(1965) \
`Kpnl (1959)
`Smol( 1953)
`BomHl(1946)
`
`FJG. 11b
`
`pSP72 /t k-HygB
`
`(4.4 kb)
`
`tk-HygB
`
`BomH1(46}
`Xbol(40)
`
`Soll(34)
`Pst 1(32)
`Sph 1(26)
`Hindlll(16)
`Xhol(4)
`
`22 of 45
`
`BI Exhibit 1119
`
`

`

`e
`
`~
`
`0 -N
`
`~ ....
`00 =(cid:173)~
`
`""""'
`N
`
`~
`Q
`N
`.,.~
`N
`"!='
`~
`00
`
`(D = ......
`~ ......
`""C
`•
`00
`d •
`
`l'.
`VJ
`
`~e
`
`~ "° N
`-..-l "° -..-l
`
`N
`t::d
`
`IgH-Enhoncer
`-SolH2008)
`
`Ad2w.
`
`,_
`MP
`
`-
`
`'-
`
`Ad2 leader) (
`
`SV 40 Enhancer
`Kpnl (2347)
`
`(HindIII minus)(2713)
`
`Pvu I [ (2993)
`
`BomHI(3393)
`Spel(3399)
`Socl (3405)
`Nhel(3411)
`Cloll3417)
`Ndel(3423)
`Notl(3429J
`
`FfG. I lc
`
`BomHl(1995)
`
`p05/lgH-Enhoncer/Hyg8
`
`(6.1 kb)
`
`tk-HygB
`
`Pstl(5393) ~p
`
`Pvul(5523)
`
`fill in ends
`EcoRl ond
`
`( 1.9kb tk-HygB)
`Sol I+ Smol
`
`(CONT. FROM FIG.1th)
`
`23 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Sep.28,2004
`
`Sheet 22 of 24
`
`US 6,797,492 B2
`
`HEAVY CHAIN
`
`vlB4: DVKLVESGGDLVKPGGSLKLSCAASGFTFS
`mlB~: DVKLVESGGDLVKLGGSLKLSCAASGFTFS
`Gal: EVQLVESGGDLVQPGRSLRLSCAASGFTFS
`
`(DY'YMS) WVRQAP
`[DYYM.S ) WVRQTP
`(BLGMT ) WVRQAP
`
`GKGLELVA
`EKRLELVA
`GKG!...EV.'T\lA
`
`[AIDNDGGSISYPDTVKG) RFTISRDNSKNTLYLQM
`(AIONDGGSISYPOTVKG ) RFT!SRDNAKNTLYLQ~
`I NIJ<tBGSZZBY'VDSVKG ) RFTl SRDNAKNSLYLQt~
`
`..J
`..J
`.J
`NSLR.AE~TALYYCAR {-QGPJ.JUWrFOY) WGQGTLLTVSS .. .
`SSLRSEDTALY'iCA.R {-QGIU»..D'fi'OY ) WGQGTTLTVSS .. .
`NS~R \SDTALY'iCA.R {-----GWGGGD- ) WGQGTLVTVST .. .
`(SEQ ID NO: 33)
`(SEQ ID NO: 34)
`
`LIGHTCHAJN
`
`(SEQ ID NO: 35)
`
`v 1?~ : DJ V11TQSSNSLAV SLGERJ.. Tl SC [AASESVDSYGNSFMH- - ) W't
`miB4 : DlVLTQSPASLAVSLGQRAT lSC [AASESVDSYGNSF'MH-- ) WY
`Le~ : D l V1~!QSSNSLAVSLGERATlNC [JtSSQSVI..YSSNSl<NYLA ) WY
`
`..J.J
`..J
`..J
`QQKPGQPPKLLl'i [RASNIZS ) GlPDRFSGSGSGTDFTLTISSV
`(RASNI.ES ) GIPARFSGSGSRTDFTLTINPV
`QQKPGQPPKLLIY
`QQKPGQPPKLLlY [10.STRES ) GVPDRFSGSGSGTDFTLTISSL
`
`E.AEDVATYYC ( QQSNEDPLT ) FGQGTKLE! KR . .. (SEO ID NO: 36)
`EADDVATYYC ( QQSNEDPLT ) FGAGTKLELKR ... (SEO ID NO: 37)
`lQQYYSTPYS) FGQGTKLEIKR ... (SEQ ID NO: 38)
`QAEDVAVYYC
`
`FIG. 12
`
`24 of 45
`
`BI Exhibit 1119
`
`

`

`U.S. Patent
`
`Sep.28,2004
`
`Sheet 23 of 24
`
`US 6,797,492 B2
`
`~
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