(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)

(22)

(65)

Appl. No.: 09/810,502

Filed: Mar. 16, 2001

Prior PubUcatlon Data

US 2002/0034765 Al Mar. 21, 2002

Related U.S. Application Data

(63) Cont inuation of applicat ion 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 conlinuation­io-part of application No. 07/702,217, filed on May 17, 1991, now abandoned.

(51) Int. C l.7 ................................................ C12N 15/00

(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 239400 9/1987 EP 255694 2/1988 EP 274394 7/ 1988 EP 328404 6/1989 EP 323806 7/1989 EP 327000 8/ t989 EP 332424 9/1989 EP 434257 6/1991 EP 438312 7/ t991 GB 2 188 638 10/1987 WO WO 87/02671 5/1987 WO WO 89/00999 2/ 1989 WO WO 91/08298 6/1991 WO WO 91/09967 7/1991 WO WO 9t/ !0742 7/ t99! WO WO 91/11198 8/1991 WO WO 91/11534 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.: US 6,797,492 B2 Sep.28,2004 (45) Date of Patent:

English, D. et al., Single Separation of Red Blood Cells: Granulocytes and Mononuclear Leukocytes on Discontinu­ous Density Gradcnts of Ficoll-Hypaque, 1974, J. Immu­nological Methods, 5, pp. 249-252 (cumulative).

Bernstein, F.C. el al., The Protein Data Bank: A Computer­based 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 Ribo­nucleic 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 Differen­tiation 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 Phago­cyte 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 cer­evisiae, 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 con­verts the antigenicity of a fi rst mammalian species to that of a second mammalian species. The method will simulta­neously change immuoogenici ty 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 Celltrion, Inc., Exhibit 1119

US 6, 797 ,492 B2 Page 2

ffnIER PUBLICATIONS

Bcrkner, K.L. et al., Effect of the tripartite leader on syn­thesis o f 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 (cumula­tive). 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 That 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 (cumu­lative). Lo, S.K. et al., Transient Adhesion of Neu1rophils To Endot­helium, 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 (cumula­tive). Padlan, E. et al., Modeling of Antibody Comining Sites, 1991, Methods in enzymology, 203, pp. 30-21 (cumula­tive). Padlan, E., A Possible Procedure for Reducing The Immu­nogenicity 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: Graft­ing 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~a­bindjog ... 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 Activi ties', 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-An­tibody 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 Discontinu­ous Density Graclicnrs of FicolJ-Hypaque" 1974 J. lmmu­oological 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 Celltrion, Inc., Exhibit 1119

U.S. Patent Scp.28,2004 Sheet 1 of 24 US 6,797,492 B2

Fractional Accessibility Residues In Subgroup

Position KOL J539 II Ill Residue Exposure Residue Exposure

, E 1.00 Ex E 1.00 Ex a a E 2 v 0.23 mB v 0.37 mB v v VM

3 a 0.82 Ex K 0.82 Ex a TO a 4 l 0.00 Bu L 0 .10 Bu L L L 5 v 0.87 Ex L 1.00 Ex v ROKT VL

6 a 0.00 Bu E 0.09 Bu a E E 7 s 0.94 Ex s 0.94 Ex s s s . 8 G 1.00 Ex G 1.00 Ex G G G 9 G 0.00 Bu G 0.00 Bu A p G 10 G 1 .00 Ex .G 1.00 Ex E AGT GA 1 1 v 0.90 Ex L 0.81 Ex v L LF 12 v 0.25 mB v 0.25 mB K v v 1 3 a 0.71 mE a 0.87 Ex K K a 14 p 0.59 PB p 0.64 mE p p p

15 G 1.00 Ex G 1.00 Ex G TS G 16 R 0.73 mE G 1 .00 Ex SA EQ G 17 s 0.66 mE s 0.75 mE s T s 18 L 0.28 mB L 0 .26m8 v L l 1 9 R 0.66 mE K 0.75 mE RK TS RK 20 l 0.00 Bu c 0.00 Bu v L L 2 1 s 0.71 mE s 0.82 Ex s T s 22 c 0.00 Bu c 0.00 Bu c c c 23 s 1 .00 Ex A 1 .00 Ex K T A 24 s 0.00 Bu A 0.00 Bu ATV F V A 25 s 0.87 Ex s 1 .00 Ex s s s 26 G 1 .00 Ex G 1 .00 Ex G G G 27 F 0.10 Bu F 0 .10 Bu GYO FLG F 28 I 0.85 Ex D 0 .72 mE T s TN 29 F 0.00 Bu F 0.00 Bu F LI F 30 s 0.74 mE s 0.83 Ex SNVI s s 36 w 0.00 Bu w 0.00 Bu w w w 37 v 0.00 Bu v 0.00 Bu v I v 38 R 0.10 Bu R 0 .31 me R R A 39 a 0.1 5 Bu a 0.28 mB a a a 40 A 0.95 Ex A 0.75 mE A p A 41 p 0.90 Ex p 0.73 mE p p PS 42 G 1.00 Ex G 1.00 Ex G G G 43 K 0.86 Ex K 0.86 Ex ORKH KR K 44 G 1.00 Ex G 1.00 Ex G AG GS 45 L 0.00 Bu L 0 .00 Bu L L L

FIG. la

3 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Scp.28,2004 Sheet 2 of 24 US 6,797,492 B2

Fractional Accessibility Residues In Subgroup

Position KOL JS39 I II Ill

Residue Exposure Residue Exposure

46 E 0.75 mE E 0.73 mE E E E

47 w 0.10 Bu w 0.04 Bu w w w 48 v 0.00 Bu I 0.00 Bu MV LI v 49 A 0.00 Bu G 0.00 Bu G AG GSA

66 A 0.36 mB K 0.51 pB R A A

67 F 0 .00 Bu F 0.00 Bu v LV F

68 T 0.87 Ex I 0.88 Ex T T T

69 I 0.00 Bu I 0.00 Bu VMI IV I

70 s 0.78 mE s 0.79 mE TS ST s 71 R 0. 11 Bu A 0.00 Bu ALA KV R 72 N 0.61 mE 0 0.55 pB OK 0 ON 73 0 0 .44 pB N 0 .43 pB PETAS T DN 74 s 0.85 Ex A 0.97 Ex s s s 75 K 0.88 Ex K 0.77 mE TF KR K 76 N 0.69 mE N 0.68 mE NST N N 77 T 0.41 pB s 0.33 mB TO a T 78 l 0 .00 Bu L 0.00 Bu AV VF LA 79 F 0.45 pB y 0.35 mB y vs YF 80 L 0.00 Bu L 0.00 Bu M L L 81 a 0.53 pB a 0.69 mE E TK SIN Q

82 M 0.00 Bu M 0.00 Bu L ML M 82a D 0.73 mE s 0.58 pB SVRT TSN I A ND 82b s 0.98 Ex K 0.96 Ex s NS s 82c L 0.00 Bu v 0.00 Bu L VM L 83 R 0.73 mE R 0.83 Ex RFI OT RE 84 p 0 .75 mE s 0.90 Ex s PA PA 85 E 0.82 Ex E 0.90 Ex E VA ED 86 D 0.00 Bu D 0. 11 Bu D 0 D 87 T 0.54 pB T 0.47 pB T T T 88 G 1.00 Ex A 0.00 Bu A A A 89 v 0.58 PB L 0.63 mE v TV VL 90 y 0.00 Bu y 0.00 Bu y y y 91 F 0.00 Bu y 0.08 Bu y y y 92 c 0.00 Bu c 0.00 Bu c c c 93 A 0.00 Bu A 0.00 Bu A A AT 94 A 0.17 Bu R 0.15 Bu A RH RP

JHI JH2 JH3 JH4 JH5 JH6 103 w 0.09 Bu w 0.07 Bu w w w w w w 104 G 0.00 Bu G 1.00 Ex G G G G G G

FIG. 1b

4 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Scp.28,2004 Sheet 3 of 24 US 6,797,492 B2

Fractional Accessibility Residues In Subgroup

Position KOL J539 II Ill Residue Exposure Residue Exposure

JHI JH2 JH3 JH4 JH5 JH6

105 a 0.93 Ex a 0.99 Ex a R a a a 0 106 G 0.00 Bu G 0.00 Bu G G G G G G io7 T 0.22 mB T 0.26 mB T T T T T T 108 p 0 .99 Ex L 0.67 mE L L M L L T , 09 v 0.00 Bu v 0.00 Bu v v v v v v 110 T 0.76 mE T 0.69 mE T T T T T T , , 1 v 0.00 Bu v 0.00 Bu v v v v v v , , 2 s 0.98 Ex s 0.74 mE s s s s s s , , 3 s 0.94 Ex A 0.84 Ex s s s s s s

FIG. le

5 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Sep.28, 2004 Sheet 4 of 24 US 6, 797 ,492 B2

Position Residues In Subgroup Res idue Exposure 11 Ill IV v VI

, 0 1 .00 Ex Q Q SF 0 ND 2 s 1.00 Ex s s y s s F 3 v 0 .77 mE v A E E A M 4 L 0 .00 Bu L L L L L L 5 T 0.92 Ex T T TK T T T 6 Q 0.00 Bu Q Q a a Q Q

7 p 0.62 mE p p p 0 p p 8 p i .00 Ex p ARP p p p H 9 s i .00 Ex s s s A s s 10 , 1 A 0.34 mB AV v v v A v i2 s 0.71 mE s s s s s s 13 G 1.00 Ex GA G VL v G E 14 T 0.73 mE TA s SA A s s is p 0.75 mE p p PA L PL p 16 G 1 .00 Ex G G G G G G 17 a 0.69 mE a Q 0 a 0 K 18 R 0.79 mE R s T T s T 19 v 0.21 mB v IV A v v v 20 T 0.62 ME T T RM A T T 21 I 0.00 Bu I I I I I IFM 22 s 0.92 Ex s s T T s s 23 c 0.00 Bu c c c c c c 35 w 0.00 Bu w w w w w w 36 y 0.00 Bu y YF y y y y 37 Q 0.46 pB 0 a a a 0 a 38 Q 0.00 Bu OH a OE a a 0 39 L 0.75 mE LV H KR K H R 40 p 0.91 Ex p p PS p PA p 4 1 G 1.00 EX G G G G G G 42 M 0.74 mE T K OR a RK SRG 43 A 0.62 mE A A A A A A 44 p 0.00 Bu p p p p p p 45 K 0.95 Ex K K v L K T 46 L 0.23 mB l l MLP L LV T 47 L 0.15 Bu l MIL v v VI v 48 I 0.00 Bu I I IV I I I 49 y 0.39 mB y YF y y FY y 57 G 1.00 Ex G G GE G G G 58 v O.i4 Bu VI VI IV I v v 59 p 0.70 mE p SP p p p p 60 0 0.95 Ex 0 ON L EOA 0 0 D 6 1 R 0 .31 mB R R R A A R

FIG. 2a

6 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Sep.28,2004 Sheet 5 of 24 US 6, 797 ,492 B2

Position Residues In Subgroup Residue Exposure II Ill IV v VI

62 F 0.12 Bu F F F F F F 63 s 0.85 Ex s $ s s s s 64 G 0.00 Bu GA G GS G G G 65 s 1 .00 Ex s s SY s s s 66 K 0 .4i pB K K TSN s K IF" 67 s 1.00 Ex s $ $ s s s 68 G 1.00 Ex G G G G OG s 69 A 0.71 mE T N TN H N N 70 s 1.00 Ex s T TKS T T s 71 A 0 .00 Bu A A AV A A A 72 s 1 .00 Ex ST s Tl s s s 73 L 0.00 Bu L L L L L L 74 A 0 .74 mE A T T T T T 75 I 0.00 Bu I I I I v I 76 G 1 .00 Ex ST s SN T s s 77 G 1 .00 Ex G G GR G G G 78 l 0.00 Bu l L VA A L L 79 0 0.76 mE OR 0 OE o RO KOT 80 s 1 .00 Ex ST A AV A A T 81 E 0.78 mE EG E EG E E E 82 D 0.09 Bu D D D D D D 83 E 0.64 mE E E E E E E 84 T 0.34 mB A A A A A A 85 D 0.30 mB D D D D D D 86 y 0.00 Bu y y y y y y 87 y 0.16 Bu y y YF y y y 88 c 0.00 Bu c c c c c c

JL-1 JL·2 JL·3 JL·4 JL·5 98 F 0.04 Bu F F F F F 99 G 0 .00 Bu G G G G G 100 T 0.59 pB T G G s s 101 G 1.00 Ex G G G G G 102 T 0 .00 Bu T T T T T 103 K 0.82 Ex K K K a a 104 v 0 .00 Bu v L L L L 105 T 0.86 Ex T T T T T 106 v 0 .19 Bu v v v v v 106a L 0 .70 mE L L l L L 107 G 1.00 Ex G G G s G

• additional residues after position 66: 66a D 66b SRO

FIG. 2b

7 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Scp.28, 2004 Sheet 6 of 24 US 6,797,492 B2

Position Residues In Subgroup

Residue Exposure II Ill IV

, E 0.99 Ex 0 0 E D

2 I 0.16 Bu ' I I I

3 v 0.87 Ex 0 v v v 4 l 0.00 Bu M M L M

5 T 0.80 mE T T T T

6 0 0.00 Bu 0 0 0 a 7 s 0.89 Ex s s s s 8 p 0.67 mE p p p p

9 A 1.00 Ex s L G ON

10 I 0.94 Ex s s T s , , T 0.30 mB L L l L

12 A 0.59 pB s p s A

13 A 0.00 Bu A v L v 14 s 0.78 mE s T s s 15 L 0.79 mE v p p L

16 G 1 .00 Ex G G G G

17 0 0.64 mE 0 E E E

18 K 0.74 mE R p R R

19 v 0.22 mB v A A A

20 T 0.65 mE T s T T

2i I 0.00 Bu I I L I

22 T 0.69 mE T s s N

23 c 0.00 Bu c c c c 35 w 0.00 Bu w w w w 36 y 0.00 Bu y y y y

37 0 0.14 Bu 0 L 0 0 38 a 0.24 mB Q Q Q 0

39 K 0.69 mE K K K K 40 s 1.00 Ex p p p p

41 G 1.00 Ex G G G G 42 T 0 .90 Ex K a a a 43 s 0.30 mB A s A p

44 p 0.00 Bu p p p p

45 K 0.90 Ex K OER R K

46 p 0.43 pB L L L l 47 w 0.16 Bu L L L l 48 I 0.00 Bu I I I I

49 y 0.42 pB y y y y

57 G 1.00 Ex G G G G 58 v 0.13 Bu v v I v 59 p 0.61 mE p p p p

60 A 1.00 Ex s 0 0 D 61 A 0.36 mB R R R R 62 F 0.00 Bu F F F F 63 s 0.94 Ex s s s s

FIG.3o

8 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Sep.28, 2004 Sheet 7 of 24 US 6, 797 ,492 B2

Position Residues In Subgroup Residue Exposure II Ill IV

64 G 0.00 Bu G G G G 65 s 1.00 Ex s s $ $ 66 G 1.00 Ex G G G G 67 s 1.00 Ex s s s s 68 G 1.00 Ex G G G G 69 T 0.75 mE T T T T 70 s 0.98 Ex OEO D D D 71 y 0.09 Bu F F F F 72 s 0.70 mE T T T T 73 l 0.00 Bu l L L L 74 T 0.43 pB T K T T 75 I 0.00 Bu I I I I 76 N 0.83 Ex s s s s 77 ,. 0.83 Ex s R R s 78 M 0.00 Bu L v L L 79 E 0.63 mE a EO E a 80 A 0.96 Ex p A p A 81 E 0.91 Ex ED E E E 82 D 0. 13 Bu D D D D 83 A 0.55 pB F l v F v 84 A 0.00 Bu A G A A 85 I 0.58 pB T v v v 86 y 0.00 Bu y y y y 87 y 0. 11 Bu y y y y

88 c 0.00 Bu c c c c

JK·1 JK-2 JK·3 JL-4 JL·5 98 F 0.00 Bu F F F F F 99 G 1.00 Ex G G G G G 100 A 1 .00 Ex a a p G a 101 G 0.00 Bu G G G G G 102 T 0.00 Bu T T T T T 103 K 0.79 mE K K K K A 104 L 0.00 Bu v L v v L 105 E 0.89 Ex E E D E E 106 L 0.44 pB I I I I I 106a , 07 K 0.77 mE K K K K K

FIG. 3b

9 of 45 Celltrion, Inc., 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· (SEQ ID NO: 6)

BamHI Xbol 5·. AA! AAi GCGGC.cOC ATCG AT GAGCTCAAGT ATGTAGACGGGGTACG -3

Not I Clo I Soc I <SEO 1D NO: 7)

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· (SEQ ID NO: 9)

Eco RI

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'

FIG. 4

(SEO ID NO: 8)

(SEQ ID NO: 10)

(SEQ ID NO: 11 )

(SEQ ID NO: 12)

(SEO ID NO: 13)

10 of 45 Celltrion, Inc., 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 1 S'· CAT TCG CTI NX, AGA TCT MG CTI~ AGT ~ATC ACA GTI CTC TCT AC .3·

V 9 S · TGG CTC l'GC AGC TG.t. TGG TG -3' (SEQ ID NO: ) 5)

V 10 S'· CAC CAT CAG CTG CAG AGC CA-3' (SEQ ID NO: 16)

V 1 1 5'· CTG TCT GGG ATC CCA G.t.T TC ·3' (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)

13 S'· GA.A TGT GCCTAC m CTAGAG GAT CCAACT G.t.GG.MGCAAAG ·3' (SEQ ID NO: 21 )

A 1 5·. CAT TCG CTI ACC AGA TCT .3· (SEO ID NO: 22)

A 2 5-. GM TGT GCC TAC TTT CT A G .3· (SEQ ID NO: 23)

Oligodeoxynucleotides for PC~ Ampl ificat ion 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 5" TTA GTG MG CCT GGA OOG TCC CTG AAA CTC .3· (SEO ID NO: 25)

V 3 S'· GC.c CCT TCC CAG GAG CTI 00C GAA CCC MG ACA TG ·3' (SEQ ID NO: 26)

V 4 S'· AAG CTC CTG ~ AOO OOC TGG AGT TGG TCG CAG CC -3 {SEO ID NO: 27)

V 5 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)

V 6 5'· TGT ADC TAC AAA TGA ACA GTC TGA GGG CTG AGG ACA CAG CCT TGT ATI ·3' V 7 5'· CTG TGA GM GGG TGC CTI OGC CCC AGT N3 .~ (SEO ID NO: 29)

• (SEO ID NO: 30)

V 8 5'· AA(!, GOA CCC TTC TCA CAG TCT CCT CAG GTG -3' (SEO ID NO: 31)

I 2 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 Celltrion, Inc., Exhibit 1119

U.S. Patent Sep.28,2004

-(.0

f . <..!)

M

Li:

;f ! M

)( - II(

> II( )(

zr ; - )( ..... >

a: {.) a..

==>

Sheet 10 of 24

+ N <

)( )I( )( )(

US 6, 797 ,492 B2

"' ~ x

-:c E

"' co

a: {.) a..

==>

---"O c :f

)(

II( )( )(

)(

c e -c:

M a: Cl (..)

N a: Cl (.)

..... a: Cl (..)

12 of 45 Celltrion, Inc., Exhibit 1119

MCS

Sspl

(201

2)

FIG. ?

a

Xho

l(4)

Hum

an C

-kap

pa b

y PC

R H

ind

111(

16)

$Ph

1(26

) P

stl(

32)

Soll(

34)

Bom

HI \.

l Kho I

Ba

ri

pSP7

2 Xba

1(40

) B

amH

l(46)

S

mal

(53)

K

pnl (

59)

Soc

I (65

) E

coR

I (67

) C

lol(

74)

Eco

RV

(81)

B

gl 1

1(85

)

(2.5

kb)

/

..,.,.

_Col

EI O

ri Amp

R

(CON

T. O

N FI

G.7

8) I

Bom

HI

( 0.9

kb)

Xho

l(4)

Hin

dill(

16)

Sph

l(26)

Ps

t I (3

2)

Sol

l(34

) X

bol(

40)

Bom

Hl(4

6)

Xbo

l (54

) S

ocl(

60l

Soc

l(569

)

( p89

4 3)

Bom

Hl(9

77)

pSP7

2/H

UM

AN C

-S

mol

{983

) Ko

ppa

(3.4

kb)

Kp

n 1(

989)

IC

So

c I

(995

) T

l E

coR

l(9

97

) C

lo I(

1004

) Ec

oRV(

101

1)

8g

lll{1

01

5)

d • 00

• ""C

~

......

(D =

......

00

~

"!='

N

.,.~

N

Q ~

00

=­ ~ ~ .... .... .... 0 -N ~ e VJ

~~

-....J "° -....J ~ "° N t::d

N

13 of 45 Celltrion, Inc., Exhibit 1119

Xho

l(4)

(CO

NT

FROM

FI

G. ?

A)

Hin

d01(

16)

Spe

l(22 l

V18

4VK

Hni

dl+

Xbo

l IH

nidl

+Xbo

I VE

M±R

ED 1

84 V

-Kop

po b

y PC

R

(p8

95

2)

pSP7

2/R

ei1B

4 Ko

ppo(

4.0k

b)

Bom

HI(5

87)

Xbo

l(593

) ~

Soc

l(599

)

Hum

an C

-Kap

pa

Soc 1

(110

8)

Bom

Hl(1

516)

S

mol

(152

2)

Kpn

1(15

28)

Soc

l(153

4)

EcoR

H15

36)

Clo

1(15

43)

EcoR

V(15

50)

Bgl

II (1

554)

Spe I

+ C

lo!

( 1.5

kb

Kopp

a Ch

o in)

FI

G. 7

b {C

ONT

ON

FfG

. 7C

) 1 S

pel

+ C

lal

Hin

dll

Spel

X

bol

'I

I (0

.6 k

b)

Bom

H1(

6)

Pvu

l(552

3) _

_ _...

. ----­

Pst

l(539

3)

Amp

~

(p89

42)

p05

I lgH

-Enh

once

r/Hyg

B (6

.1 kb

}

Bom

HI

(199

5}

Sol

l SV

40

L(A

)n

I . ~

(200

8)

~

"K

pnl{2

347)

Ad

2 M

LP

Not

l(342

9)

Nde

l(342

3)

Clo

l (34

17)

Nhe

l(341

1}

Pvu 1

1(29

93)

Soc

[{340

5)

Spe

l{339

9)

Bam

Hl(3

393

d • 00

• ""C

~

......

(D =

......

00

~

"!='

N

.,.~

N

Q ~

00

=­ ~ ~ .... .... N

0 -N ~ e VJ

~~

-..-l

\C

-..-l ~

\C

N t::d

N

14 of 45 Celltrion, Inc., Exhibit 1119

(Fro

m F

IG.

78)

i

Pvu

1(70

10)

Pst

l(68

80)

Bom

H1(

6)

( p89

53)

p05

/ lgH

-Enh

once

r I H

ygB

/ H

uman

ized

18

4-K

oppa

v1

B4VK

l7.

7kb)

Not

l(491

6}

\ Hu

man

C-K

oppo

N

del (

4910

) C

lol(

4904

) ~

~'

v1B4

VK

Eco

R1{

4901

) S

ocl{4

897)

K

pnll4

891)

B

omH

1(48

89)

tk-H

ygB

Bom

H[(1

995)

S

ol 1

(200

8)

lgH

-E nh

once

r

Kpn

l(234

7)

V40

Enh

ance

r Ad

2 M

LP

Pvu I

I (29

93)

Ad2

Lead

er

Bom

H 1

(339

3)

Spel

(339

9)

Kpn

l(371

7)

Kpn

l(381

5}

FIG.

7c

d • 00

• ""C

~

......

(D =

......

00

~

"!='

N

.,.~

N

Q ~

00

=­ ~ ~ .... .... ~ 0 -N ~ e VJ

~el'.

-..-l

\C

-..-l ~

\C

N t::d

N

15 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Sep.28,2004 Sheet 14 of 24 US 6,797,492 B2

-• N !

t N < x -a:>

. - c:

(..!) :r: e E -LL "' .E

CD

l··· :: ::1 !;: )(

~ .... M llC )(

C"')

a: 0 <..>

!e L: M )(

.. :::1 ~ M M )(

a: a: (.)

)( (.)

c.. c.. N

> ==> a: C> (.)

~ i···· .. ··l ~ ~ ~

a )( )( .. )(

-a: C> <..>

N l··· ···: :>- -··· •• ••• > )(

-!. (ij c: - en .2> -- en _J

t "C en s~ - c: < :f --'5> co

16 of 45 Celltrion, Inc., Exhibit 1119

VENE

ERED

184

VH

BY P

CR

Hin

dm

Bom

HI

Bg

ll*

Sp

el

..

I

Kpn

l(862

3)

Eco

RH

8617

l\tk

( 0.5

kb)

Pvu

l(798

6}

· ~ ~tk-Neo

Pst1

1785

6) ~P ~

SV40

UA

)n

p05/

lgH

-Enh

once

r I

/ORI

N

eo/1

84 V

H-S

hort

Hum

an C

-Gom

mo

4 (8

. 7 k

b)

Sol

l(195

7l

lgH

-Enh

once

r K

pnl(2

296)

Not

1(5

893)

~

Ad2

Lead

er, fJ

SV

40 E

nhan

cer

Ad2

MLP

P

vull (

2942

) X

hol(3

097)

C

lol(5

887)

S

ocl(5

881)

Sh

ort

Hum

an C

-G<m

no 4

P

stl(4

887)

Bg

l 11 (

4856

) Chi

mer

ic 18

4 vu

.~

Bgl 1

1(32

68)

Hind

111

(334

7)

Spe

H33

53l

Nco

l(340

1)

Hin

d 11

1(35

33)

Smo I

(404

2)

Bom

Hl(4

102)

FI

G.9

o

Cj

• rJ'J

• '"'C

~ ~

~ =

~

\J')

('

>

'!='

N

:0

N g ~

\J')

=­ ~ ('> ..... .... Ul

0 ~

N ~

~

rJJ

~Cl'

, -..

..J

\0

-....J

~ "° N ~

N

17 of 45 Celltrion, Inc., Exhibit 1119

Kpn

l{862

3}

EcoR

I (86

17)\

tk

Pvul(7986}~ ~tk-Neo

Pstl(7856)~ ~

p05/

lgH

-Enh

once

r I

/ORI

N

eo/1

84 V

H-S

hort

Soll(

1957

l lg

H-E

nhon

cer

Kpn

l(229

6)

SV40

L(A

ln

Hum

an C

-Gom

mo

4

Not

1(5

893)

---w

-..

Clo

U58

87)

Soc

l(588

1 l

Sho

rt Hu

man

C-G

omno

4

Pst

1(4

887)

Bg

l II (

4856

)

(8. 7

kb)

Ad

2 Le

oder

i" v

1B

4--

h

SV 4

0 En

hanc

er

Ad2

MLP

Pv

ull (

2942

) X

hol(3

097)

FIG.

9b

Bgl

11(

3268

) Hi

nd Il

l ( 33

4 7)

Spe

ll335

3l

Nco

1(34

01)

Hind

111(

3533

) S

mol

(404

2}

Bom

Hl(4

102)

d • 00

• ""C

~

......

(D =

......

00

~

"!='

N

.,.~

N

Q ~

00

=­ ~ ~ .... .... 0\

0 -N ~ e VJ

~~

-..-l "° -..-l ~ "° N t::d

N

18 of 45 Celltrion, Inc., Exhibit 1119

Pvu

l(353

6)

Pst

I(34

06l

p05(

MC

S)

(4.2

Kb)

SV

40 E

nhan

cer

Hin

d 11

1(72

6)

Ad2

MLP

Pvu

II( 1

006)

Ad2

Lead

er

r-.1

-B

omH

l(140

6)

Spel

(141

3)

Soc

l(141

9)

Nhe

I(142

5)

Clo

l(143

1)

Nde

l(143

7)

Not

I (1

443)

Kpn

l +

EcoR

1(3.

9 kb

)

CM

VIE

Prom

oter

EcoR

I(1)

Clo

l(23)

H

indl

0(29

) B

omH

l(375

}

7 A

p

Ori pC

MVI

E-AK

1-0H

FR

(9.2

kb)

tk-N

eo(A

KI)

. '

BGH(

A)n

.,

-Bom

Hl(2

766)

Bgl

ll(55

70}

Kpn

l(534

0)

EcoR

I(504

1)

lgH

enh

ance

r by

PCR

B

glll

S

oll

Kpn

l

Eco

Rl(4

565)

\Q

om

Hl(4

766)

(0

.3kb

)

FIG.

fOa

(CO

NT

ON

FIG

. !Cl>

)

d • 00

• ""C

~

......

(D =

......

00

~

"!='

N

.,.~

N

Q ~

00

=­ ~ ~ .... .... -....J

0 -N ~ e VJ

~~

-....J

\C

-....J ~

\C

N t::d

N

19 of 45 Celltrion, Inc., 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)

SV40 L(A)n

Notl(3379) Ndel(3373) Clo1{3367) Nhell3361) Soc1{3355) Spel(3349)

BomHl(3342)

Pvul(5472) Pst 1(5342)

Soll(1957) lgH -Enhancer

KpnI(2296) V40 Enhancer

Ad2 MLP Pvul1(2942)

f'Amp ~tk-Neo

(p8941) ·-H.1--' p05/lg H-Enhoncer /Neo (6 .2 kb)

t ke Promoter (Octamer and CAT Region by PCR)

EcoRI EcoRI ld

(0.1kb)

Ad2 Leader SV40 L(A)n

Notl(3379) Ndel(3373) CLol(3367)

"

Ad2 MLP Sol!(1957) lgH- Enhancer

Kpnl(2296) SV40 Enhancer

(Hind III minus)(2662)

Nhe1{3361) Sac1(3355) Spel(3349) BamH1(3342)

Pvu II {294 2)

FIG.10b

20 of 45 Celltrion, Inc., Exhibit 1119

EcoR

l (1)

H

ind

Ill (

29)

Bom

Hl(3

75) tk

-Hyg

B pA

L2

( 5.6

kb)

Bom

Hll2

275)

So

ll(25

51}

FJG.

I la

Bo

mH

l {1

.9kb

tk

-Hyg

B}

Sspl

(201

2)

Xho 1

(4)

Hin

d 11

1(16

) Sp

h 1(

26)

Pstl(

32)

Soll

(34)

X

bol (

40)

Bom

H1(

46)

Sm

ol(5

3)

Kpn

l(59)

So

c I (6

5)

Eco

Ril6

7)

Clo

1(7

4)

EcoR

V(81

) ~

Bgl

ll(8

5)

pSP7

2 (2

.5 kb

)

Col

EIO

ri

Bam

HI

(CO

NT O

N FI

G. 1

1 b)

d • 00

• ""C

~

......

(D =

......

00

~

"!='

N

.,.~

N

Q ~

00

=­ ~ ~ .... .....

\0

0 -N ~ e VJ

~el'.

-..-l "° -..-l ~ "° N t::d

N

21 of 45 Celltrion, Inc., Exhibit 1119

Xho

l(4)

Hin

dlll(

16)

Sph 1

(26)

Ps

t 1(3

2)

Sol

l(34)

X

bol(4

0)

Bom

H1(

46} tk

-Hyg

B

pSP7

2 /t k

-Hyg

B (4

.4 k

b)

FJG.

11b

Bom

Hl(1

946)

S

mol

( 195

3)

Kpn

l (19

59)

Socl

(196

5) \

Ec

oRl(1

967l

C

lol(

1974

) Ec

oRV(

1981

l Bg

l 11(

1985

)

r (CO

NT

FROM

FIG

. lla

)

Kpn

l(610

9)

tk

EcoR

l(610

3) \

{ ,E

coR

l(1)

Pvul

(547

2) ~

Pstl(

5342

)

Not

1(3

379)

N

del(3

373)

C

lol(3

367)

N

hel(3

361)

So

cl(3

355)

S

pel(3

349)

B

omH

l(334

2)'

Sol

l(195

7)

IgH

-Enh

ance

r //V

Kpn I

(229

6)

SV 40

Enh

ance

r (H

ind I

ll m

inus

)(266

2)

Pvu l

l(294

2)

/ (C

ONT

ON

FIG

. 11c

)

0 • 00

• ~

~ .....

(D =

.....

(J) ~

"P

N

~Ot:i

N ~ (J) =­ ('!) ~ .....

N

Q

0 -N .&:>. L:

r:J') °' ~

\C

-.....l ~

\C

N

~

N

22 of 45 Celltrion, Inc., Exhibit 1119

FfG.

I lc

(CON

T. F

ROM

FIG

.1th

)

Sol I

+ S

mol

( 1

.9kb

tk-H

ygB)

Ec

oRl

ond

fill in

end

s

Pvu

l(552

3)

Pst

l(539

3) ~p

p05/

lgH

-Enh

once

r/Hyg

8 (6

.1 k

b)

tk-H

ygB

Bom

Hl(1

995)

A

d2

w.

MP

-S

olH

2008

) Ad

2 le

ader

) (

,_

IgH

-Enh

once

r '-

-K

pnl (

2347

)

Not

l(342

9J

Nde

l(342

3)

Clo

ll341

7)

Nhe

l(341

1)

Socl

(340

5)

Spe

l(339

9)

Bom

HI(3

393)

SV 4

0 En

hanc

er

(Hin

dIII

min

us)(2

713)

Pv

u I [

(299

3)

d • 00

• ""C

~

......

(D =

......

00

~

"!='

N

.,.~

N

Q ~

00

=­ ~ ~ .... N

""""'

0 -N ~ e VJ

~el'.

-..-l "° -..-l ~ "° N t::d

N

23 of 45 Celltrion, Inc., Exhibit 1119

U.S. Patent Sep.28,2004 Sheet 22 of 24 US 6,797,492 B2

HEAVY CHAIN

vlB4: DVKLVESGGDLVKPGGSLKLSCAASGFTFS (DY'YMS) WVRQAP mlB~: DVKLVESGGDLVKLGGSLKLSCAASGFTFS [DYYM.S ) WVRQTP

Gal: EVQLVESGGDLVQPGRSLRLSCAASGFTFS (BLGMT ) WVRQAP

GKGLELVA [AIDNDGGSISYPDTVKG) RFTISRDNSKNTLYLQM EKRLELVA (AIONDGGSISYPOTVKG ) RFT!SRDNAKNTLYLQ~ GKG!...EV.'T\lA 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 .. .

LIGHTCHAJN

(SEQ ID NO: 33)

(SEQ ID NO: 34)

(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 QQKPGQPPKLLIY (RASNI.ES ) GIPARFSGSGSRTDFTLTINPV QQKPGQPPKLLlY [10.STRES ) GVPDRFSGSGSGTDFTLTISSL

E.AEDVATYYC ( QQSNEDPLT ) FGQGTKLE! KR . .. (SEO ID NO: 36)

EADDVATYYC ( QQSNEDPLT ) FGAGTKLELKR ... (SEO ID NO: 37)

QAEDVAVYYC lQQYYSTPYS) FGQGTKLEIKR ... (SEQ ID NO: 38)

FIG. 12

24 of 45 Celltrion, Inc., Exhibit 1119

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

IO I 0

t2 ~ ~

~ . <D

~ (.!)

~ <D ~ Ci:

N II I"") ~ ,...

0 I If) II 0 '"O u ~

(1) 0

I"') I/) '"O ~

u '"O ~

'-- <: ~

Q.) Q.)

a:l c: co c - Cl> I 0 E > 0 ·-I - .....

o• • I 0 ~ J . ~ .....

I c • (1)

~ Ol u •' I c

/ 0 0 -, u ,>' , ~ ,

/ 0 ~

/ 0 0 ..... ~ ,~ I

I 0 -I I

~ ... I

0 0 0 0 o~ 0 0 0 0 0 0 0 0 ~

,,., N -(z=u)sN~d sOlXl/puno9 ~d:> uoe~

25 of 45 Celltrion, Inc., Exhibit 1119

• u c 0 0 cu E

Cl)

cu

- O.· E

::J

E ·-)( cu

:E

~

0

Tim

e C

ou

rse

of

Pla

sma

184

Lev

els

Det

erm

ined

15

min

. A

fter

Eac

h W

eekl

y l.V

. D

ose

10

0

10

•

.x ..

.. :-

-: ·<>

· :-

=-:-

-.. ~:

. . .

. .

. o ~ h

lgG

4 --

.. (

) .

.

• • •

. • · .. ~~

...

. fi

. /

··A···

···~l84

He

mlc

hlm

erl

c ••

•

hlB

4

-x

-

f 'X V

enee

r

\ 'A

. G

al/R

el

hlB

4

. •

0)

. • 0 -'

1

0 5

10

1

5 2

0

Da

ys

. • . • . • . • • '

I •

' '

' I

I I

• '

"'P

••

I '

I

25

3

0 3

5 4

0

j ~

L!

• 00

• ""C

~ .....

~ =

.....

00

~

'?

N

.,.oo

N

Q

Q ~

00

=­ ('> ~ .... N ~

0 ....,

N ~ e \J)

.5"

-...J

\C

-...J ~

\C

N co N

26 of 45 Celltrion, Inc., Exhibit 1119

us 6,797,492 132 1

METHOD FOR REDUCING THE IMMUNOGENIClTY OF ANTIBODY

VARIABLE DOMAINS

RELAfED APPLICATIONS

This applica1ion is a con1inua1ion of applica1ion Ser. No. 08/905,280, filed Aug. I, 1997, now abandoned, which is a con1inua1ion of applica1ion Ser. No. 08/609,218, filed Mar. l, 1996, now abandoned, which is a cominuation of appli­cation Ser. No. 08/109,187, filed Aug. 19, 1993, now abando11ed, which is a conlinuation-in-part of application Ser. No. 07/702,217, fi led May 17, 1991, now abandoned.

13RIEF DESCRHYrION OF THE DRAWINGS

FIGS. lA- l C. Solvent exposure of sidechains of frame­work residues in KOL and J539 Fvs and the residues which occur most frequently at these positions in 1be various human VI I subgroups.

FIGS. 2A and 213. Solveol exposure of sidecbaios of framework residues in KOL VL and the residues which occur mosl frequently al 1hesc positions in tbe various huma11 V-lambda subgroups.

FIGS. 3A and 313. Solven1 exposure of sidechains of framework residues in J539 VL and 1he residues which occur mosl frequemly al these positions in the various human V-kappa subgroups.

FIG. 4. Primers used 10 isolate ONA encoding murine kappa light chain variable region and murine IgG2a heavy chain variable region using PCR. Oligodcoxynucleotides used as PCR primers to genera1e a shortened IgG4 heavy chain. Oligocleoxynucleo1ides used in PCR to re-engineer 1he thymidine kinase (TI<) promo1er to facilitate the expres­sion of the neomycin resistance gene. Oljgodeoxynucleotide primers used in PCR 10 clone the lgH enhancer sequence. Oligocleoxynucleotides used as PCR primers to generate a human kappa light chain constant region.

FIG. 5 . Oligodeoxynucleotides used in 1he cons1ruction of 1he "veneered" I 134 heavy and ligh1 chain variable regions plus 1bosc necessary 10 fuse 1he human signal and intronic sequenccds onto these variable regions.

FIG. 6. PCR-recombina1ion s1ra1egy used in the veneering of lhe l 134 kappa light chain variable region.

FIGS. 7 A- 7C. Outline of the inserlion of the "veneered" kappa ligh1 chain variable region and kappa constant region inlo the lighl chain expression vec1or.

FIG. 8. PCR-recombina1ion stra1egy used in 1he veneering of the 1134 heavy chain variable region.

FIGS. 9A an 9B . Outline of the insertion of the "veneered" heavy chain variable region into the heavy chain expression vector.

2 BACKGROUND OF TI fE INVENTION

The identification and production of murine monoclonal antibodies has lead to numerous 1herapcutic applications of these exquisi1ely specific molecules in human disease. The 5 technologies of molecular biology have further expanded the utility of many anlibodies by allowing for the creation of class switched molecules whose fu nctionality has been improved by the acquisition or loss of complement fixation. 'J11e size of 1he bioactive molecule may also be reduced so

10

JS

20

25

as to increase the tissue 1arget availability of the antibody by ei ther changing the clas.<; from an lgM to an lgG, removing most of the heavy chain constant region in the creation of a F(ab)2 or both heavy and light chain constant regions maybe dispensed wilh in the formation of a Fv antibody. Common to all of 1hese potentia lly 1herapeutic forms of antibody are the requisite CDRs (complemea1ary determining regions) which guide the molecule to its ligand and the framework residues (FRs) which support these latter structures and dictate the disposition of the CORs relative to one another, Wiater European Patent Application, Publication No. 239, 400; Riechmann et al., Na/Lire 332: 323-327 (1988). Crys­tallographic analyses of numerous antibody structures reveal that 1he combining sile is composed almost entirely of the CDR residues arranged in a limited number of loop motifs, Pad Ian and heriff, L 990. llle necessity of the CD Rs to form these structures combined with the appreciated hypervari­ablity of their primary sequence leads to a great diversity in the antigen combining site, but one which has a finite number of possibili1ics. Thus, hypermutability and a limited

30 primary sequence repetoire for each CDR would suggest 1hat the CDRs derived for a given antigen from one species of animal would be the same derived from another species. Hence, 1bey should be poorly immunogenic, if at all, when

35 presented to a recipient organism in a non-foreign context.

Monoclonal antibody producing bybridomas have been mosl readily obtained from immunized rodents. Develop­ment of similar reagents from human sources has been frust rated by the current inabil ily to maintain long lerm

40 cultures of cells which produce sufficient quanlities of antibody. Additional problems arise from the regulatory s tandpoint when cells of huma11 origin are employed for the production of agents to be used i.n man. These considerations have led to the widespread use of rodent mono-clonal

45 antibodies for the imaging and trea1ment of malignancy, prophyllactic administration to guard agaios1 1oxic shock, modification of graft rejection episodes, and to temper acute inflammatory reac1ions. In all scenarios where completely rodenl or par1ially rodent (ie, rodent-human chimeras)

50 amibodies have been used for therapy the recipients have oflen illicited an immune response directed 1oward the antibody. "lltesc reactions have limited the duration and effectiveness of the therapy.

FIGS. lOA and 1013. Outline of the insertion of neomycin selectable expression vec1or.

FIGS. llA- lOC. Ou1line of the insertion of hygromycin 55

selectable expression vector.

Various attempls have been made to minimize or elimi­nate the immunogenicity of non-human amibodies while preserving their an1igen-binding properties. Initially, chi­meric amibodies were constructed containing the rodent variable regions and lheir as.sociated CDRs fused to human constant domains. The following references generally

FIG. 12. Amino acid sequence compleition of 1he ·•veneered"-1134, murine 1134 and human Gal heavy chain variable regions and the ··veneered" 1B4, murine 1B4 and 60 human Len kappa light chain variable regions. Check marks idicate 1he individual amino acid residues converted.

describe chimeric an1ibody technology: Lobuglio et al., Proc. Natl. Acad. Sci. USA 86: 4220-4224 (1989); U.S. Pat. No. 4,816,567; PCT lllternational Publication No. WO 87/02671, published May 7, 1987; European Patent Publi­cation No. 255,694, published Feb. 10, 1988; European

FIG. 13. Competitive binding assay of native murine 1B4 (opeo diamonds) and recombina11t •·veneered" lB4 (closed diamonds).

rl G. 14. lnmunogenicity of a veneered murine an tibody in Rhesus monkeys is shown.

65 Patent Publication No. 274,394, published Jul. 13, 1988; European Patent Publication No. 323,806, published Jul. 12, L989; PCT ln1crnational Publication No. W0/89/00999,

27 of 45 Celltrion, Inc., Exhibit 1119

US 6,797,492 B2 3

published Feb. 9, 1989; European Patent Publication No. 327,000, published Aug. 9, 1989; European Patent Publica­tion No. 328,404, published Aug. 16, 1989; and European Patent Publication No. 332,424, published Sep. 13, 1989. Tbese proved to be Jess immunogenic bu.I still approxi- 5

mately half o( tbe recipients mounted an immune response to the rodent variable region framework residues. Further reduction of the "foreign" nature of the chimeric antibodies bas been achieved by g rafting only the CORs Crom the rodent monoclonal into a human supporting framewo rk prior 10

to its subsequent fusion with an appropriate constant domain, Wiater European Patent Application, Publication No. 239,400; Riechmann et al. , Nature 332: 323-327 (1988). The procedures employed to accomplish COR­grafting often resuJt in imperfectly "humanized" antibodies. JS

That is to say, the resultant an tibody has either lost avidity (usually 2-3 fold, at best) or in an attempt to retain its original avidity a significant number of the murioe frame­work residues have replaced the corresponding ones of the cbosea human fromewo rk. In this later case, the immuno- 20 geoicity of the modified " humanized" antibody is difficult to anticipate a priori.

4 DETAILED DESCRIPTION OF THE

INVEN110N

The present invemion relates to a "'humanization" proce­dure which simultaneously reduces the iromunogeoicity of the rodent monoclonal antibody while preserving its ligand biadiag properties in their entirety. Since the antigenicity of a protein is primarily dependent on the nature of its surface, tbe immunogenicity of an xeoogeoic or allogenic antibody could be reduced by replac ing the exposed residues which differ from those usually fouad in aaother mammalian species amibodies. This judicious replacement of exterior residues shou Id have little, or no, effect on the interior domains, or on the ioterdomain contacts. Thus, ligand bind­ing properties should be unaffected as a consequence of altera tions which are limited 10 the variable regioa frame­work residues. The process is refered lo as ''veneeriag" siace only the outer surface or skin of the antibody is altered, the s upporting residues remain undisturbed.

The procedure for "veneering" makes use of the available sequence data for human antibody variable domains com­piled by Kabat et al., "Sequences of Proteins of Immuno­logical Interest'', 4th ed., Bethesda, Md.: National Ins titutes of Health, 1987, updates to this database, and other acces­sible U.S. and foreign databases (both nucleic acid aad protein). The subgroups into which the various sequences have been combined are presented in FIGS. 1- 3, indicating the most frequently occurring amino acid at each framework position. Also presented are tbe sequences of the various J-minigeaes. The solvent accessibilities of the amino acids, as deduced from the kaowa lbree-dimensioaal structure for human aad mouse antibody fragments, are included in these figures .

High resolution X-ray crystallography of the variable domains of the antibodies KOL and 1539 have been sub-

The ligand binding characteristics of an antibody com­bining s ite are determined primarily by the structme and relative disposition of the CD Rs, although some neighboring 25

residues also have been found to be involved in antigen binding (Davies et al., Ann. Rel~ Biocliem. 59: 439-473 [1990]). Fine specificity can be perserved in a "humanized" antibody only if the CDR structures, their interaction with each other, and their interaction with the rest of the variable 30 domains are strictly maintained. One may anticipate tha t the key residues represem .. interior" and interdomain contact residues, hence those surface exposed residues which are immediately available for immune surveillaace should be non-inclusive of the structural residues. 35 jected to extensive refinement beginning witb the structures

available from the Protein Data Bank (Bernstein e t al., J. Mo/. Biol. 112: 535-542 1977; fi le 2FB4 for KOL and file 2FBJ fo r J529). The solvent accessibilities were computed as described by Padlan Proteins: Struct. Funcl. Genet. 7:

OBJECTS OF THE INVEN110N

It is, accordingly, an objective of the present invention to provide a means of converting a monoclonal amibody of one mammalian species to a monoclonal antibody of another mammalian species. Another object is lo identify the amino acid residues respoasible (or species specificity or immuao­genicity on the exterior of the moaoclonal antibody. Another object is judiciously replace o r veneer the exterior amino 45 acid residues of one species witb those of a second species

40 (1990).

so that the antibodies of the first species will not be immu­nogeaic in the second species. A further object is to make replacements only in framework regions of tbe heavy and light chains of the antibody molecule and not in tbe

50 complementarity-determining regions . Another object of the inveation is to provide novel ONA sequences iacorporatiag the replacement amino acid residues. Another object is to provide a vector containing the DNA sequences for the altered antibody. Another object is to provide a eukaryotic or

55 procaryotic host transformed with a vector containing the ONA sequence for the veneered antibody.

SUMMARY OF THE INVEN110N

Tbere are two steps in tbe process of "veneering" . First, the framework of a first animal species, i.e. the mouse, variable domains are compared with those corresponding fra meworks of a second animal species, i.e. hurnaa. It is iatendecl that this invention will allow the antigenic alter­ation of aay animal species antibody. The preseat iaventioa is illust rates !be conversion of murine antibody to human antibody, but tbis is for illustraiive purposes oo]y. The most homologous human variable regions are then compared residue for residue io the corresponding murine regions. This wil l also defiae the humaa subgroup to which each mouse sequeace most closely resembles. Second, those residues in the mouse framework which differ from its human counterpart are replaced by the residues present in the human counterpart. This switching occurrs only with those residues which are at least partially exposed (mE and Ex; FIGS. 1-3). One retains in the "veneered" mouse antibody: its CDRs, the residues neighboring tbe CDRs, those residues defined as buried or mostly buried (mB and

A unique method is disclosed for identifying and replac­ing immuooglobulio surface amino acid residues which converts the antigenici ty of a lirs t mammalian species to that of a second mammalian species. The method will simulta­neously change immuoogeoici ty and strictly preserve ligiod binding properties. The judicious replacement of exterior amino acid residues has ao effect on the ligaad bindiag properties but greatly alters imrnu nogeaic ity.

60 Bu; FIGS. 1- 3), aad those residues believed to be involved with iolerdornain contacts (boldface, FIGS. 1- 3).

Human and murine sequences frequently differ at the N-terminus of both heavy and light chains. The N-termioi are contiguous with the CDR surface and are in position to

65 be involved in ligand binding. Thus, wisdom would dicta te that these murine termini be retained ia its .. veaeered" vers ion.

28 of 45 Celltrion, Inc., Exhibit 1119

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Finally, replacement of some amioo acid lypes could bave a sigoificant effecl on the tertiary structure or electrostatic inleractioos of tbe variable region domains. Heoce, care sbould be exercised io tbe replacemenl of prolioe, glycine, and charged amno acids.

These criteria aod the following procedures are used to prepare recombinant DNA sequeoces wbich incorporate the CDRs of a first mammalian species, animal, mMAb, both light and heavy chains, in10 a second mammalian species, human, appearing frameworks tbal can be used lo 1raosfec1 mammaliao cells for the expression of recombinaot bumao antibody with the antigen specificity of the animal mooo­clonal antibody. The preseot ioventioo further comprises a method fo r constructing and expressing the altered antibody comprising: (i) mutagenesis and assembly of variable region domains iocluding CDRs aod mutageoesis aod assembly of variable regioo domaios including CDRs and FRs regions; (ii) preparatioo of an expression vector including at least one variable region which upon transfection into cells results in tbe secretion of protein sufficieot for avidity and specifici ty determinations; and (iii) co-amplification of beavy and light cbain expression vectors in appropriate cell lines. The present invention provides recombinant methods for incor­porating CDRs from animal monoclonal antibodies into framewo rks which appear to be human immunoglobulin in nalure so tbat tbe resulting recombinant antibody will be either weakly immunogenic or non-immunogeuic when administered lo bumans. Preferably tbe recombioaot immu­noglobulios will be recogoized as self proteins wbco admin­istered for tbreapeutic purposes. This method of "veoeering" wi ll render the recombinant antibodies useful as therapeutic agents because they wiU be either weakly immunogenic or non-immunogenic when administered to humans. The ioveotion is further cootemplated lo ioclude the recombinant conversioo of aoy aoimal moooclooal aotibody ioto a recombioaot '·human-appearing" moooclonal ao tibody pro­viding that a suitable framework regioo can be ideotified (as described below). The anima l monoclonals may include, but are not limited to, those murine monoclonal antibodies described by Yao Voorhis et al.,J. Exp. Med. 158: 126-145 (1983) which bind lo human leukocytes and tbe appropriate mMAbs produced by bybridomas deposited io the Hybri­doma Cell Bank maintaioed by the Americao Type Culture Collection (ATCC) and described in the ATCC Catalog of Cell Lines 8 Hybridomas, No. 6, 1988.

·rne CDR sequeoces from the animal monoclonal ao ti­body are derived as fo llows. Total RNA is extracted from the murine bybridornas, for example the 1B4 myeloma cells described by Wrigbl el al. , Proc. Nari. Acad. Sci. USA 80: 5699-5703 (1983), the 60.3 cells described by Beauy el al., J. l mmunol. 131:2913-2918 (1983), the TSl/ 18 cells described by Sanchez-Madrid et al., J. Exp. Med. 158: 1785-1803 (1983), and other anti-CD18 or CDll mono­clonal antibodies and hybridomas as described in Leukocyte Typing 111, Springer-Verlag, New York ( 1988), using stan­dard methods involving cellular solubilizalion with guaoi­dinium isothiocyaoate (Chirgwin et al., Bioc/1e111. 18: 5294-5299 [1979]). The murinc 1B4 mMAb will be used as the primary example of animal MAb that can be "veneered" by the unique process being disclosed. The invention is in tended to include 1be conversion of any animal immuno­globulin to a "human-appearing" imrnunoglobulin. ll is further inte nded lbat " buman-appearing" immunoglobulin (lg) cao cootain either kappa or lambda light chaios or be ooe of any of the following heavy chain isotypes (alpha, delta, epislon, gamma and mu).

Pa.irs of degenerate oligodeoxynucleotide primers (FIG. 4) representing sequences w ithin framework 1 of the murine

6 kappa ligbt chain variable region aod ligbt c bain coustaot domain, or those within framework 1 of the murine lgG2a heavy chain variable region aod heavy chain constant CH! domain are syotbesized oo an Applied Biosystem 381A

5 DNA synthesizer, removed from the resin by treatment with concentrated NH40H and desalted on a NAP-5 column eluted with H~O. Total RNA, about 2 µg, is reverse trao­scribed for 30-min at 42° C. using Moloney MLV reverse transcriptase, about 200 units (BRL), and about 10 pmoles

10 of the constaot region complemeolary strand primers for either the heavy or light chain. The reverse transcriptase is beat inactivated, about 95° C. for about 5 min, and the reactions are made to contain in about 100 pl of PCR buffer about 50 pmoles of each of the paired primers aod aod 2.5

JS units of Taq polymerase. About 45 cycles of amplification (2', 94° C.; 2', 55° C.; 2' 72° C.) are followed by gel purification of the anticipated 400+base pair (bp) DNA fragmeolS. Prior 10 subclooing those DNAs ioto a blunt­ended intermediate plasmid such as pSP72 (Promega) they

20 are terminally pbospborylated using T4 polynucleotide kinase. Multiple clones represeotiog tbese PCR amplified sequences are grown and submitted to DNA sequence deter­minations using Sequenase® and T7 and SP6 specific sequencing primers. A unique DNA sequence representing a

25 murioe lgG2a heavy chaio variable region is obtained by analysis of tbe derived amino acid sequences. Replacement of the "murine-appearing" framework residues with those residues compatible with a human variable regioo is accom­plished utiliziog tbe following unique processes. An appro-

30 priate human framework is determined utilizing the criteria discussed below. The light c hain variable region framework with sufficient homology to tbc the mlB4 framework was delermined to be the burnan LEN framework (FR). The Len FR s hows a similarity of 90% and an ideo1i1y of 81 % when

35 compared to murioe 1B4. This sequeoce, with its leader, 3' in trouic sequences and engrafted mlB4 CDRs had been subcloned into the intermediate vector pGEM3Z (Promcga), as described in Daugherty e t al. Nucleic Acids !?es. 19: (1991). About eight oligocleoxyoucleo1ide primers (FIG. 5)

40 are synthesized representing the primers necessary to gen­erate by polymerase chain reaction (PCR) amplification four DNA fragments. Incorporated into all but the terminal oligodeoxyoucleotide primers were those sequeoces corre­sponding to the veneered MAb lB4 ligbt chain, with its

45 unaltered CD Rs, and at least 15 bases of 5'-lerminal comple­mentarity to al low for the subsequent PCR-directed recom­bination of these four fragments. For the purposes of exem­plifying the "veneering,. process the LEN light chain variable region already cootainiog ao eografted set of CD Rs

so represeoting those within the light cbaio of murioe 1B4 was used as the template into which mutations were placed so as to easily create the "veneered" framework sequeoce. Tbe appropriate primer pair (Sl & V9, VlO & Yll, etc.), about 50 pmole each, was combined with about 10 og of plasmid

ss DNA representing tbe LEN CDR-grafted framework, about 2.5 uoits of Taq DNA polymerase and about twenty-five (25) cycles of PCR amplification ensued (cycle periods: l ', 94° C.; 1', 55° C.; 2' 72° C.) . The products o[ the four reactions, purified by agarose gel electrophoresis, are combined, about

60 10 ng of each DNA fragment, aloog with lermioal oligode­oxynucleotide primers (Al & A2, FIG. 6) and Taq DNA polymerase. The combined fragments were PCR amplified (25 cycles of: 2', 94° C.; 2', 55° C.; 2' 72° C.). Followiog restrictioo eodoouclease digestioo with Hind 111 and Xba I

65 the amplified DNA is purified by agarose gel electrophoresis and subcloned into compatible sites of an intermediate vector pSP72 (Promega) which contains the human kappa

29 of 45 Celltrion, Inc., Exhibit 1119

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light chaio coostaoi rcgioo (sec FIG. 7). Gcoomic DNA, ioitiate RNA syothcsis. A stroog promoter is ooc which about l 11g, purified from a human B cell line (GM0108A: causes mRNAs to be ioitiated at high frequency. Expression NIGMS Hurnao Genetic Mutant Cell Repository, Institute vectors may include, but are not limited to, cloniog vectors, for Medical Research, Camden, NJ.) is used as a template modified clooiog vectors, specifically designed plasmids or for PCR amplification (FIG. 4) of about a 920 base pair 5 viruses. Tbe heavy cbain immunoglobulin molecule is trao-fragmen t containiog the splice acceptor for the kappa light scribed from a plasmid carrying the neomycin (0418) cbaio coostant domaio, the exon and a portion of its resistaocc marker while tbe light cbain irorouooglobulin is 3'-uotranslatcd region. The PCR produci is purified by transcribed from a plasmid carrying the hygromycio B agarose gel e lectrophoresis, d igested with Barn Hl resistance marker. With the exception of the drug resistance endonuclease, and subclooed into pSP72 previously linear- 1o portioo of these plasmids they are identical. The preferred ized with Barn I-11. The individual clones representing tbe progenitor of tbe immuooglobulin expression vectors is tbe pSP72 intermediate vector containing both tbe 1B4 pD5 (Berkner and S harp, Nucl. Acids Res. 13: 841- 857 "veneered" light chaio variable region aod the human kappa [1985]) eukaryotic expression vector which contains the constant region derived by PCR amplification of human origin of adenovirus replication, the SV40 eohanccr domaio, DNA arc used to determine the DNA scqueoce of the JS the adcoovirus major late promoter, the adenovirus 2 tripar-"veneered" light chain variable region. tite leader, a 5' splice donor from the adenovirus third leader

The "veneered" heavy chain portion of the recombinant and a 3' splice acceptor derived from an immunoglobulin antibody is derived from the mutated version of the murioe locus, a multiple cloning site placed in the Bam 1-11 si te 1B4 heavy chain variable region fused to the human con- subsequent to receipt of the vector, and the SV40 late stant region of a gamma 4 subtype obtained from a lambda 20 polyadenylation signal (FIG. 10). The origin of replication is library constructed by Flanagao and Rabbits, Nature 300: removed by digestion with Eco Rl and Kpn I and replaced 709-713 (1982). The variable region of tbe "veneered" by two fragments represcotiog the neo selectable marker heavy chain is constructed from five DNA fragments rep- gene (derived (rom plasmid pCMVIE-AKl-DHFR as an resenting a signal sequence, portions of tbe mutated murioe Eco Rl/Bam Hl about L.8 Kb fragment) and the lg beavy heavy chaio variable region, and an intronic sequence (FIG. 2s cha in enhancer(obtaincd as a PCR amplified fragmen t usiog 8). Oligodeoxynucleotide primer pairs (FIG. 5) are synt be- bumao DNA as the template, aod the oligodeoxynuc)eotides sized representing the primers necessary lo generate by PCR listed in FIG. 4 as the primer pair, following its digestion amplilication these five DNA fragments from about 10 ng of with Bgl 11 aod Kpn 1). The resultant expression vector is plasmid DNA template obtaioed from a pSP72 intermediate fouod to lack a small portico of tbe TK promoter responsible vector containing lhe heavy chain variable region previously 30 for the transcription of the neomycio gene. This is replaced used to determine the murine 1B4 CDR sequence. Ampli- by insertion into the Eco RI site about a 0.14 Kb PCR l:lcation of 1be signal fragmen1, variable region fragments, ampli6ed fragment derived from Lbe CMVIE-A.Kl-DHFR and intron-coniainiog fragmen t was as described above. The DNA using the primer pair listed io FIG. 4. The resultant agarose gel purified products are combined, about 10 ng of heavy chain expression vector (p894l) is modified by each product, with terminal oligodeoxyoucleotide primer 35 removal of the iodicated Hind 111 and Xba I sites using pairs (FIG. 8) and the PCR-generated in vitro recombined standard procedures. To convert this vector ioto ooe express-template is amplified using the s tandard procedures ing the bygromycin B selectable marker the neomycin-described above. Prior to subcloning into a Hind 111 and resistance cassette is removed by digestion first with Eco Rl Barn 1-11 digested expression vector containing ibe human followed by DNA polymerase-directed fill io of the 5' heavy chain gamma 4 constaot region (FIG. 9), this recom- 40 overhand, then subsequeot Sal I digestion. Tbe about 1.9 Kb bioed product is similarly digested aod agarose gel purified. bygromycio B expressioo cassette, TK promoter aod TK Individual cloocs are submitted to DNA sequence determi- polyadenylatioo signal flanking the hygromycin B gene, nation usiog Scqucoase® and T7 aod SP6 specific scquenc- (obtaioed as a 1.8 kb Bam Hl fragment io plasmid pL690, ing primers and one is chosen fo r subsequeot expression. Gritz and Davies, Gene 25: 179-188 [1981]) is removed The gamma 4 heavy chain constant region is subcloned as 45 from the plasmid pAL-2 by Barn 111 digestion and sub-about a 6.7 Kb Hind 111 fragment derived from the plasmid cloned into the Barn Hl site of the intermediate vector pAT84 into the Hind 111 site of the intermediate vector pSP72. The bygromycin B cassette is removed from tbis pSP72. This plasmid is then used as the template DNA from vector by d igestion wi.th Sma I and Sall aod cloned ioto the which a s hortened version of tbe gamma 4 constant regioo expression vector linearized as described above to c reate a is subclooed usiog PCR amplification aod the primer pairs so blunt end and Sal I end DNA fragment (FIG. 11). indicated in FIG. 4. Eukaryotic expressioo vectors are Expression of the 1B4 "veneered" kappa light chain is coostructed as described below. accomplished by transferring this cistron from the pSP72-

Expression vectors are defined hereio as DNA sequences based intermediate cloning vector (p8952), cootaining the that are required for the transcription of cloned copies of human kappa constant region, to the hygromycio B select-geoes and the translation of their mRNAs in ao appropriate 55 able eukaryotic expression vector (FIG. 7). Ao about 1.5 kb host. Such vectors can be used to express eukaryotic geoes DNA fragment resultiog from the endonuclease digestion of io a variety of hosts such as bacteria, blue-green algae, plaot p8952 with Spe I aod Cla I is purified by agarose gel cells, yeast cells, insect cells and animal cells. The iromu- electrophoresis and ligated ioto the expression vector which noglobulins may also be expressed in a oumber of virus has previously been linearized, following digestion with the systems. Specifically designed vectors allow the shuttling of 60 same two restriction enzymes, and agarose gel purified. Tbe DNA between bost sucb as bacteria-yeast or bacteria-aoimal heavy chain eukaryotic expression vector is constructed in cells. Ao appropriately constructed expression vector should two steps. First, the p8950 vector contairriug the modified contain: ao origio of replication for autooomous replication heavy chain variable region of murioe 1B4 fragment is in host cells, selectable markers, a limited oumber of useful digested with Bgl 11 aod Barn Hl. The agarose gel purified restriction enzyme sites, a potential for bigh copy number, 65 0.75 kb fragment is ligated ioto the Barn Hl site of the p8941 and strong promoters. A promoter is defined as a DNA vector and recombinant clones containing this fragment in sequence that directs RNA polymerase to bind to DNA and the proper orientation are identified. Plasmid DNA from one

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US 6,797,492 B2 9

such clone is linearized by Barn Hl digestion and ligated with a 1.78 Kb Barn 11 1 fragment representing a short version of Lhe human gamma 4 constant region, derived from plasmid pAT84 by PCR am11lifica1ion. Following the identification of clones containing these inserts in the appro­priate orientation, plasmid DNAs (one which is referred to as p8953) are grown and purified for transfcction into recipient mammalian cells.

Equal amounts, about I 011g, of the plasmids encoding the 184 "veneered" lgG4 heavy chain and the 1B4 "veneered,. kappa light chain are transfccted by standard calcium phos­phate precipitation procedures into the monkey kidney cell line CV-l P or the human embryonic lddney cell line 293. The culture supcroants, assayed by a trapping ELISA {described below), were fou nd to coa1ain a human kappa light chain/human lgG4 immuno-globulin. Immulon-2 (Dynalech Labs.) 96-wcll plates are coated overnight with about a 5 11g!ml solution of mouse anti-human kappa chain cons tant domain monoclonal antibody (cat. #MC009, The Binding Site, Inc., San Diego, Calif.) in about 0.J M NaHC03 buffer (pH 8.2) at about 4° C., and blocked with about l % bovine scrum (BSA) in about 0.1 M NaJ-IC03 for about 1 hour at about 25° C. After this and all subsequent steps, washing was performed with phosphate buffered saline (PBS). The wells arc then challenged with condi­tioned medium coataining recombinant anti-CD18 antibody, or with predetermined quanti ties of human lgG4/kappa purified by protein AScpharose (Pharmacia Fine Chemicals) chromatography from human lgG4 mycloma serum (cat.# 8P026, The Binding Site, Inc.). All samples are diluted in PBS containing about 0.05% Tween-20. About 100 pl ali­quots arc incubated for about 1 bour at about 37° C. in triplicate, and standard calibration curves are constructed using lgG4 concentrations ranging from about 10 ng/ml to about 100 ng/ml. Bound and fully assembled human IgG4 (either native or the recombinaot 1B4 human .. veneered" lgG4 constructs) arc detected with about 100 id aliquots of a 1:500 dilution of mouse anti-human IgG4 Fe monoclonal antibody conjugated to alkaline phosphatase (cat #05-3822, Zymed Laboratories, Inc.) in phosphate buffered saline {PBS) containing aboul I% 13SA. Af1er incubation for about 1 hour al about 37° C. and subsequent washing, 1he quan­lilics of bound conjugate are detected by incubating all samples with a l mg/ml solution of p-nitrophenyl phosphate in 0.1 M 2,2' amino methyl-propanediol buffer, p11 10.3, for about 30 minutes at about 25° C. The aclsorbance of the wells is de1ermincd with a UV Max ELISA plate reader (Molecular Devices) set at 405 nm. The antibody secreted by the Lransfected human 293 cells or monkey kidney CVl P cells, either following transient expression or subsequent to stable clone isolation, is isolated by protein A chromatography, 1he concentration of recombinant human anli-CD18 antibodies determined by the trapping Elisa described above, and used to compete with the binding of radiolabeled murinc LB410 the CD18 ligand on 1he surface

10 Fullerton, Calif.) and total protein measured at OD280 with a Kratos Spec1roflow 757 detector (Kratos, Mawah, NJ.). A s ingle 125I-1B4 peak composed of coincident 00280 and radioactivity tracings characterist ically elutes at about 6

5 minutes, 30 seconds following sample injeciion. Specific ac1ivi1y of 1he product is generally about JO ,uCi/pg protein, and 97-99% of the counts arc precipitable with 10% trichlo­roacetic acid. The binding of this radiolabelcd antibody is assessed on human PMNs purified on a discontinuous Ficoll/

10 1 lypaque gradient (English, D. and Anderson, B. R., J. lmmunol. Methods 5: 249- 255, 1974) and activated with about 100 ng/ml phorbol myristate acetate for about 20 minutes at about 37° C. (Lo ct al., J. Exp. Med. 169: 1779-1793, 1989). To determine the avidity of antibodies

JS for CD18 molecules on the PMN surface, about lxl05

activated PMNs are incubated in a buJTer such as I lanks balanced salt solution containing about 20 mM Hepes (pl I 7.2), about 0.14 uniis apro1inin (Sigma Chemical Co.) and abou t 2% human serum albumin {binding buffer) containing

20 about 1.3 ng 1251-lll4 (2.8xJO-J I M) in the presence of increasing concentrations of unlabeled 1B4 antibody (about 10-7 to 10-15M) in about a 300 Jll reaction volume for aboul 1 hour at about 4° C. with constant agitation. Cell bound 1B4 is separa1ed from tbe unbound antibody by

25 ccnlrifugation through a 0.5 M sucrose cushion (4,800xg, 3 minutes); the tubes arc frozen on dry ice, and lhe tips cut o[ and counted wilh an LKB gamma counter. The IC50 of 1he anti-CD18 antibody for the inhibition of 125l-1B4 antibody binding is calculated us ing a four parameter filler program

JO (Rodbard el al., In, "Radioimmunoassay and Related Pro­cedures in Medicine", lnlc rnational Atomic Energy Agency, Vienna, vol 1, 469- 504, 1978). The affinity of the "'veneered" r-an1i-CDl8 antibody for 1he CD18 ligand is determined in a similar manner using murine 1251-1B4

35 antibody and increasing quan1i1ics, as determined by tbe trapping Elisa, of unlabeled r-an1i-CDl8. The results of the binding assays are showa in FIG. l3 and indicate that the avidity of tbe "veneered" recombinant 104 antibody is equa I to that of the muriae 184 monoclonal antibody. This result

40 shows that an antibody with presumptive human iso1ypc may be recombinaotly constructed from the murine parcnl antibody by the introduction o( numerous point mutations in its framework residues and expressed fused to human kappa and gamma 4 constanl domains without loss in avidity for

4S lhe antigen. It can be inferred from this result that Lhe point mutations within the framework regions do not alter 1he presentation of 1he murinc I 84 light chain and heavy chain CDRs. Many of the examples of c.-onstruction of recombi­nant human antibodies coataining complementarity regions

so replaced by those found within murinc monoclonal antibod­ies have resulted in loss of avidity for the ligand or antigen. Thus, although these lauer transmutations are possible, 1he successful maintenance of avidity is not assured. This pro­cedure described above dcmonslratcs that when s iricl allcn-

ss tion is payed 10 the framework regions, and the nature of !he amino acids within each framework, ''humanization" may potentially be achieved without the loss of avidity which accompanies the transfer of CDRs to the "generic" human frameworks {"humanization") employed by Winter, Euro-

of activated human PMNs. Affinities of r-anli-CD18 anti­body constructs arc determined using a competitive 1251-1 B4 soluble binding assay with stimulated human polymorpho-nuclear leukocytes (PMNs). Purified murine anti-CD18 monoclonal antibody (50 ug) is iodina1ed using chloramine-T (I-luntcr, W. M. and Greenwood, F. C., Nature 194: 495-496, 1962), and the radiolabcled antibody purified using a Bio-Sil TSK250 (Biorad, Richmond, Calif.) gel filtralion HPLC column {which fractionates proteins in 1hc range of 1-300xl03 daltons) equilibrated in 0.1 M phos- 65

phate buffer, pH 7.0. Efllucnl radioactivity is monitored wilh

60 pean Patent Publication No. 239,400, published Sep. 30, 1987.

To identify human framework sequences compatible wi1b the CDRs of, say, murinc lB4, human frameworks with a high degree of sequence sim ilarity to those of murfoe 1B4 arc idcntificcl. Sequence similarity is measured using iden­tica l residues as well as evolutionarily conservative amino acid substitutions. Similarity searches arc performed using an in-line detector (Beckman Model 170; Beckman,

31 of 45 Celltrion, Inc., Exhibit 1119

US 6,797,492 B2 11

the murine 1B4 framework sequence from which the CDR sequences bad been removed. T his sequence is used to query a database of human immunoglobulin sequences Lhat bad been derived from multiple sources. Sequences with a high degree of sequence similarity are examined individually for 5

tbeir potential as humanizing framework sequences. In this way, the human homologue providing the murine CDRs with the structure most similar to their native murine frame­work is selected as the template for the construction of the "veneered" variable regions (FIG. 12). ShouJd human 10

frameworks of sufficient similarity noi be identifiable from compi led sequences, it is possible to isolate from human genomic ONA a group of closely related variable regions using recombinant technology. Thus, a degenerate 5' upstream oligodeoxynucleotide primer may be designed JS from the conserved sequences within the amino-terminus of each of the various human FRl regions and paired with a degenerate 3' downstream oligodeoxynucleotide primers fashioned from the FR sequence detem1ined from the murine monoclonal whose CD Rs o ne wishes to transfer into 20 a human context. These primer pairs are then used to PCR amplify from a human genomic template those DNA sequences which are flanked by the primer pair. T he result­ing DNAs may then be cloned and the DNA sequence derived from individual members will describe various 25

murine-related human variable regions. Tbe pauci ty of somatic mutations in framewo rk residues and the conserva­tion of amino acid sequence between mouse and man make this approach possible.

12 variable region, beginning with the signal peptide encoding region and ending wi th the 3' int ronic sequence which resides between the variable region coding domain and the IgG4 constant region sequence, wi th the desired poiot muta­tions to create a "veneered" variable region framework. T hese live fragmen ts are purified by agarose gel electrophoresis, combined, about 10 ng of each DNA fragmen t, along with terminal oligodeoxynucleotidc primers (AJ & A2, FIG. 5) and Taq DNA polymerase. The combined fragments were PCR amplified (25 cycles of: 2', 94° C.; 2', 55° C.; 2' 72° C.). By virtue of the complementary ends of the five fragme nts, the polymerizatioo/ -deoatu.ra tion/ polymerization cycles of the polymerase chain reaction result in the formation, and subsequent amplification, of the combined sequences. Following 25 cycles of amplification the combined 0.8 Kb fragment is electrophoretically purified from an agarose gel and was digested with restriction enzymes Spe I and Barn Hl. Following agarose gel electrophoresis, the purified DNA fragment is ligated into tbe heavy chain expression vector, p8958 (see PIG. 9), in place of the chineric variable region existing in this vector. Each "veneered" variable region, with its associated human cons tant region, residing wi thin a pD5-based ex')Jression vector plasmid was co-transfected into 293 cells and CV! P cells aod recombinant bumao antibody is fouod to be present in tbe conditioned medium 48 hours post transfection. T he "veneered" recombinant antibody is isolated by protein A chromatography. The avidity of !his antibody for the CD18 ligand displayed on the surface of activated human PMNs is

30 compared with that of the murine 1B4 monoclonal antibody parent. FIG. 13 shows that although each antibody contains the same set of six CDRs within different framework domains, they exhibit identical avidity for tbe ligaocl. Thus, the avidity of an antibody molecule does oot rely upon the

The construction of a complete recombinant human !gG4 antibody, whose heavy and light chain variable domains contain the CDR residues of the murine monoclonal antibody, wi th complete retention of the specifici ty and avidity of tbe parent murioe monoclo nal antibody is dis­closed. The construction of tbe ·'veneered" light chain framework derived from the human sequence of LEN fused with a human kappa light chain constant region is described above. The murine variable region framework sequence, devoid of CDR sequences, is used 10 query a database of complete human variable region sequences. Tbe human sequences tha t are most similar to the murine framework region are then analyzed individually to determine both their sequence identity and similarity to the murine framework region. ln the case of murine 1B4 these sequences include, but are not limited to, "Gal", chosen because of its high degree of both similarity and identity with the 1B4 heavy chain sequence. The Gal FR bas been found to be 85% similar and 79% identical-to murine 1B4. These values are based upon the Dayhoff similarity matrix of evolutionarily conserved amino acid substitutions (R. M. Schwartz, M. 0. Dayhoff, in Atlas of Protein sequence and structure M. 0. Dayhoff, Eds. (National Biomedical Research Foundation, Washington, DC [1979]) (FIG. 12). To prepare a recombi­nant DNA encoding the murine beavy chain CDRs in the context of a human-appearing framework the following procedures are performed. A set of ten short oligodeoxy­nucleotides are synthesized. Each pair is combined in a separate PCR reaction with the DNA template representing the murine 1B4 heavy chain variable region, amplified and isolated following PCR of the RNA of the murine hybri- 60 doma 1B4 as described above. Thus, about 50 pmole of each primer pair was combined wi th abou t 10 ng of plasmid DNA representing the murine 1B4 heavy chain variable region, about 2.5 units of Taq DNA polymerase and about twenty­five (25) cycles of PCR amplification ensued (cycle periods: 65

l ', 94° C.; l', 55° C.; 2' 72° C.). The products of the five reactions (FIG. 8) encoded portions of the lB4 heavy chain

35 variable region framework residues which are surface exposed, rather the proper structure in which the CDRs arc presented must be significantly influenced by the buried and inter/intra active residues. The parent murine monoclonal aotibody demonstrates ao IC50 of about LO to about 0.7 nM,

40 the "veneered" molecule has a similar IC50 .

This invention further relates to a method of inhibiting the influx or migration of leukocytes capable of expressing CD18 antigen (leukocyte integrin, beta subunit) on their surface in10 a site of inflammation or a tissue area or organ

45 that will become inflamed following an influx of the cells. The inflammation which is the target of the method of the preseot ioven1ion may result from an iofectioo with patho­genic microorganisms such as gram-positive and gram­negalive bacteria, parasites and fungi. The response may

so also be induced by viruses and non-infectious means such as trauma or reprefusion following myocardial infarction or stroke, immune responses to foreign antigen and autoim­mune responses. The recombinant human anti-CD18 anti­bodies are useful in the treatment of inflammation in lung,

55 central nervous system, kidney, joints, endocardium, eyes, ears, skin, gastrointestinal tract and urogenital system. Dis­ease s lates in which the recombinant human anti-CD18 antibodies are useful as therapeutic agents include, but arc not limited 10: infectious diseases where active infection exists at any body site, such as meningitis; conditio ns such as chronic or acute secondary inllammations caused by aotigen deposition; and o ther conditions such as, encepha­litis; arthritis; uveitis; colitis; glomerulonephri1is; dermatitis; psoriasis; and respiratory dist ress syndrome associated with sepsis and/or trama. Other inllammatory diseases which may be responsive Lo recombinant human anti-CD18 antibody include, but are not limited to, immune disorders and

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Jun. 6, 1989 and the hybridoma was designated HB 10164. Previous experiments had de termined this antibody to be an IgG 2a with a kappa light chain (Wright et a l., Proc. Natl. Aca. Sci. USA 80: 5699-5703(1983)).

Total RNA was extracted from tbe 1B4 myeloma cells using standard methods involving cellular solubilization with guanidinium isotbiocyaoatc (Chirgwi.n et al., Biochem. 18:5294-5299 (1979]). Sets of degenerate oligodeoxynuclc­otide primers (FIG. 4) representing sequences wi thin Crame-

conditions involving T-ccll and/or macropbagc auacbrncnl/ recognition, such as acute and delayed hypersensit ivity, graft vs. bost disease; primary autoimmune conditions sucb as pernicious anemia; infection related autoimmune conditions such as Type I diabetes meUitis; flares during rheumatoid 5

ar1hritis; diseases that involve leukocyte diapedesis, such as multiple sclerosis; antigen-antibody complex mediated dis­eases including certain of the secondary infection states listed above; immunosuppression; and transplan t rejection. InOammatory conditions due to toxic sbock or trallma such 10 work 1 of the murine kappa ligbt chain variable region and

kappa ligbt chain constant domain, or those within frame­work 1 of tbe murine !gG2a heavy c hain variable region and heavy chain constant CHl domain were synthesized by standard phosphoramidite procedures on an Applied Bio-

as adu ll respiratory distress syndrome and re perfusion injury; aod disease states due to leukocyte dyscrasias and metastasis, are included within the scope of Ibis inven1ion. The present invention is also applicable to the inhibition of leukocyte-endothelia l allachment for diagnostic and thera­peutic pllrposes; such as the ia1rogenic opening or the endotbelium to prevent the ingress of leukocytes during the ingress of a therapeutic drug in the instance of chemo­therapy; or to enhance the harvesting of leukocytes from patients.

Recombinanl human anti-CD18 antibodies o r an active fragmen t thereof can be used to treat the above mentioned diseases. An active fragmen t will include the F(ab')2, the Fab and any other fragment tbat can bind to the CD18 antigen. Recombinant human aoti-CD18 antibodies can be administered alone for non-infectious disease states or com­bined wi th antibiotics or other an ti-in(ective agems for the treatment of infectious diseases for reasons discussed above. Administration will generally include the antibodies and possibly other subs1anccs in a physiologically acceptable medium o r pbarmaceutical carrier. Such physiologically accepiable media or pharmaceutical carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose, buffered saline and the like. The antibodies and any anti-infective agent will be administered by parenteral routes which include intravenous, intramuscular, subcutaneous and in traperito­neal injection or delivery. The amount of 1he antibodies and the mix ture in the dosage form is dependent upon the particular disease state being treated. The amount of tbe recombinant human anti-CD18 antibody utilized in a dosage Corm can range from about 1 10 about l,000 mg, wi th a range of from about 10 mg to about 100 mg being preferred. The antibodies can be administered dai ly or less tbao daily as de1ermined by the treating physician. The fo llowing examples illustrate the present invention without, however, limiting the same thereto.

EXAMPLE 1 Preparation of a "Veneered" Recombinant Antibody

An anti.body was produced in wbich the variable domain of the light chain comprises the framework region of a murine light chain modified to contain surface exposed amino acids of human derivation. The variable domain of tbe heavy chain is similarly de rived from tbe murine heavy chain with point mutations wbich replace muri.ne exposed residues with human-appearing residues. Tbe light chain human framework region was derived from human myeloma protein LEN. Tbe CDR and framework sequences f:rom the murine monoclonal antibody lB4 wbicb binds 10 CD18 (the beta subunit of the leukocyte integrin B-2 family which [ncludes: LFA-1, Mac-1, and p150.95) were derived as follows. The hybridoma designated 1B4 which produces 1B4 monoclonal antibody was deposited under the Budapest Treaty at the International Depository Authority: American Type Cultur e Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. Viability was determined on

JS system 381A DNA synthesizer. Removal of the oligodeoxy­nucleotides (oligos) from the resin was accomplished by treatment w ith concentrated NH40H followed by desalting on a NAP-5 column (Pharmacia) with f-120 elu tion (when the o ligos were<45 bases in length), or by use of an OPC

20 columo (Applied Biosystems Inc) with 20% acetonitrile elution (wben the oligos were>45 bases in length), as recommended by the manufacturers. Total RNA (2 .ug) was reversed transcribed for 30' at 42° C. using Mo loney MLV reverse transcriptase (200 units, BRL) and 10 pmoles of the

25 constant region complementary strand primers representing ei ther heavy or lighl chain in a buffer (fin a I volume of 20 pl) containing 50 mM Tris HCI, pH 8.3,75 mM KCI, 3 mM MgC~, 10 mM DTT, and 20 units of RNAsin (Pharmacia). The reverse traoscriptase was heat inactivated (95° C., 5')

30 and the reactions were made to contain in 100 pl of PCR buffer (10 mM Tris HCI, pH 8.3, 50 mM KC!, 1.5 mM MgC12 , 0.01% gelatin, 200 p M eacb dNTP), SO pmoles of each of the pa ired primers, and 2.5 units ofTaq polymerase (Perkin Elmer/Cetus). Polymerase chain reaction (PCR)

35 amplification was carried out essentially as described by Saiki c t al., Science 230: 1350-1354 (1985) and others (Mullis el al. , Cold Spring Harbor Symp. Quant. Biol. 51: 263-273 [1986], Dawasaki and Wang, PCR Technology, Princples and Applications fo r DNA Amplification, Erlich,

40 Ed , Stockton Press, NY, pp. 89- 97 (1989), Tung et al., ibid. pp. 99- 104 (1989)). Forty five cycles of amplifica tion by a DNA Thermal Cycler (Perkin Elmer Cetus lnstruments)(2', 94° C.; 2', 55° C.; 2' 72° C.) were followed by gel purifi­cation of the anticipated 4-00+base pair (bp) DNA fragments.

45 Prior to subcloning the DNAs into a blunt-ended interme­diate plasmid (pSP72, Promega) they were terminally phos­phoryla1ed using T4 polynucleotide kinase (Boehringer Mannheim). Multiple clones representing these PCR ampli­fied sequences were isolated form OHS transformed E.coli

so plated on LB agar plates containing 50 .uglml ampicillin, grown by described procedures (Maniatis ct al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), plasmid DNAs were extracted from the bacteria using the DNA

55 preparation procedures of Bimboin and Doly, Nucleic Acid Res. 7: 1515 (1979), and lhe double-stranded plasmid DNAs were submitted to DNA sequence determinations using Sequenase® (United States Biochemicals) and 17 and SP6 specific sequencing primers (Boehringer Mannheim) using

60 the protocols recommended by Lhe manufacturer. A unique DNA sequence representing a murine lgG2a heavy chain variable region was obtained, as was a kappa light chain variable region sequence.

To give lbe final appearance of a "'veneered" murine light 65 chain, several residues within a template composed of the

human LEN framework, into which bad been grafted the CDRs described for 1B4, were replaced by corresponding

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residues found in tbe murine 1B4 ligbt cbain framework. Replacement of the buman LEN variable region residues with tbose unique to MAb 1B4 took place as follows. Eigbl oligodeoxynucleotides (FIG. S) were synthesized represent­ing tbe primers necessary to generate by PCR amplification four DNA fragments. Incorporated into all but the te rminal oligodeoxynucleotides were those sequences corresponding to the MAb 1B4 light chain variable region framework residues to be point mutated and at least 15 bases of 5'-terminal complementarity (see FIG. 6). Tbe appropriate primer pair (50 pmole each) was combined with 10 ng of a 1B4 CDR-grafted LEN framework-containing plasmid DNA, 2.5 units of Taq DNA polymerase, PCR reaction components and buffer, and twenty-five (25) cycles of PCR amplification ensued (cycle periods: l ', 94° C.; 1', 55° C.; 2' 72° C.). The products of tbe four reactions, purified by agarose gel electropboresis, were combined (10 ng of eacb DNA fragmen t) along witb a terminal oligodeoxynucleotide primer pair (amplifier) (FIGS. S & 6), Taq DNA polymerase, PCR reaction components and buffer, and tbe subsequent recombined fragments were amplified, as described above, for twenty-five (see FIG. 6). Following restriction endonu­clease digestion with Hindlll and Xbal the amplified DNA was purified from an agarose gel and subcloned into these same sites of an intermediate vector pSP72 (Promega) which contained the human kappa light chain constant region, obtained as follows. DNA (l pg) purified from a buman B cell line (GM01018A; NIGMS Human Genetic Mutant Cell Repository, Institute for Medical Research, Camden, NJ. 08103) was used as a template for the oligodeoxynucleotide primers described in FIG. 4 to PCR amplify a 920 base pair fragment containing the splice acceptor for the bum an kappa light chain constant domain, the exon and a portion of its 3'-untranslated region (PCR primer pair cboice was selected based on tbe kappa constant region sequence described by Hieter et al., Ce!l 22: 197-207 (1980). The PCR product was purified by agarose gel electropboresis, digested witb Baro Hl endonuclease, and subcloned into pSP72 (Promega) previously linearized with Barn Hl.

lbe individual clones representing tbe pSP72 intermedi­ate vector containing both the 1B4 "veneered" light chain variable region derived as described above, and the human kappa constant region, derived by PCR amplification of human DNA, were used to verify the DNA sequence of tbe "veneered" light chain variable region. The " veneered" heavy chain portion of the recombinant antibody was derived from a point muta ted murine 1B4 beavy chain variable region fused to tbe human constant region of gamma 4 subtype obtained from a lambda library con­structed by Flanagan and Rabbitls, Nature 300: 709-713 (1982).

16 fragmen ts comprising the "veneered" ligbt chain variable region. Prior to subcloning into a Hind Ill and Barn HI digested expression vector this recombined product was similarly digested and agarose gel purified. DNA was

5 obtained following growtb of individual bacteria I clones and submined to DNA sequence determination using Seque­nase® and T7 and SP6 specific sequencing primers in order to verify the sequence of the reconstructed variable region and its flanking domains.

10 Tbe gamma 4 heavy cbai11 constant region bad been

subcloned as a 6.7 Kb Hind rII fragment derived from tbe plasmid pA.f84 (Fla.nagan and Rabbit ts, supra) into the Hind III site of the intermediate vector pSP72 (Promega). Tbis plasmid was then used as the template DNA from which a shortened version of the gamma 4 constant region was

JS obtained using the standard PCR amplification procedures described above and the primer pairs indicated in FIG. 4. Eukaryotic expression vectors were constructed as described below sucb that the heavy cbain immunoglobu lin molecule was transcribed from a plasmid carrying the neomycin

20 (0418) (Rothstein and Rezoikoff, Cell 23: 191- 199 [1981]) resistance marker, wbile the ligbt cbain immunoglobulin was transcribed from a plasmid carrying the hygromycin B resistance marker (Gritz and Davies, Gene 25: 179-188 [1983]). With the exception of the drug resistance portion of

2s these plasmids they are identical. Tbe progeni tor of the imrnunogJobulin expression vectors

was the pD5 eukaryotic expression vector (Berkner and Sbarp, Nucl. Acids Res. 13: 841-857 [1985]) which con­tained the origin of adenovirus replication, the SV40

30 enhancer domain, the adenovirus major late promoter, the adenovirus 2 tripartite leader, a 5' splice donor from the adenovirus third leader and a 3' splice acceptor derived from an immunoglobulin locus, a multiple cloning s ite, and the SV40 late polyadenylation signal (FIG. 10). T be origin of

35 replication was removed by digestion with Eco Rl and Kpn I and replaced by two fragments representing the neo selectable marker gene (derived from plasmid pCMV1E­A.Kl-DHFR (Silberklang et al, Modern Approaches to Arli-1110/ Cell Tec/Jno/ogy, Ed. Spier et al., Bullerworth, U.K.,

40 [1987]) as an Eco Rl/Bam Hl 1.8 Kb fragment) and tbe lg beavy cbain enhancer (obtained as a PCR amplified frag­ment using standard procedures described above and human DNA as the template; the oligodeoxynucleotide primer pair is listed in FIG. 4) following its digestion witb Bgl II and

45 Kpn I. The resultant expression vector was found to Jack a small portion of the TK promoter responsible for the tran­scription of the neomycin gene. This was replaced by insertion into the Eco RI site of a 0.14 kb PCR amplified fragment derived from the CMVIE-A.Kl-DHFR DNA using

so the primer pair also listed in FIG. 4. The resultant heavy chain expression vector was subsequently modified by removal of the .indicated Hind lll and Xba I sites. To convert this neomycin selectable vector in to one expressing the

The variable region of the "veneered" beavy chain was constructed from five DNA fragments representing a signal sequence, mutated portions of tbe murine 1B4 heavy cbain variable region, and an in tronic sequence (FIG. 8). 55

Oligodeoxy-nucleotide primer pairs (FIG. S) were synthe­sized representing the primers necessary to generate by PCR amplification these five DNA fragments from 10 ng of plasmid ONA template containing the murine 1B4 heavy chain variable region previously used to determine the 60 murine 1B4 CDR and framework sequences. Amplification

bygromycin B selectable marker (FIG. 11) the neomycin­resistance casseue was removed by digestion first with Eco Rl followed by DNA polymerase-directed fill in of tbe 5' overhang, then subsequent Sal I digestion. The 1.9 kb bygromycin B expression casselle [TK promoter and TK polyadenylation signal flanking the hygromycin B gene obtained from Gritz and Davies, Gene 25: 179-188 (1983), as tbe 1.9 kb Baro Hl fragment in plasmid (pLG90)] was

of tbe five fragments was performed as described above for the four light chain variable region fragments. The agarose gel purified products were combined (10 ng of each product) with terminal primer pairs (FIG. S) and the PCR-generated 65

in vitro recombined template was amplified using the stan­dard procedure also described above for recombining the

removed from tbe plasmid pAL-2 by Barn Hl digestion and subcloned into the Baro Hl site of the intermediate vector pSP72 (Promega). The bygromycin B cassette was removed from tbis vector by digestion with Sma I and Sal I and cloned into the expression vector linearized as described above to create a blunt end and Sal I end DNA fragment.

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Expression of the 1B4 ·'veneered" kappa light chain was accomplished by transferring this cistron from its position within the pSP72 intermediate vector 10 the hygromycin B selectable eukaryotic expression vector (FIG. 7). A 1.5 kb .DNA fragmen t resulting from the endonuclease digestion of vlB4 VK/pSP72 intermediate vector with Spe 1 and Clal was purified by agarose gel clectro-phoresis and ligated into the expression vector which bad previously been linearized, by digestion with the same two restriction enzymes and agarose gel purified.

The 1B4 "veneered" heavy c hain eukaryotic expression vector was constructed in one step (FIG. 9) from an existing vector previously constructed to express a chimaeric form of the 1B4 heavy chain. The "veneered" heavy chain variable region created by PCR amplifcation (FIG. 8) was digested with Hind III and Barn Ill. The agarose gel purified 0.8 kb fragment was l.igated into the Hind Ill and Barn Hl sites of the pDS/IgH -Enhancer/Neo/ 1B4 VH-Short Human C-Gamma 4 expression vector following its endonuclease digestion wi th these two enzymes and subsequent purifica­tion by agarose gel electrophoresesis (FIG. 9). Transfor­mants containing both variable and constant regions were identified. Plasmid DNAs were grown (Maniatis et al., supra) and purified for transfection into recipient mamma­lian cells (Maniatis et al., supra; Birbion and Doty, supra.).

Equal amounts (10 pg) of the plasmids encoding the "veneered" Ig04 beavy cbain and the "veneered" kappa light chain were transfected by s tandard calcium phosphate precipitation procedures into human 293 cells and african green monkey kidney CV-lP cells. The culture supernatant fluids were assayed by a trapping Elisa (described below) for the secretion of a bum1tn kappa light chain containing !gG4 immunoglobulin.

An Elisa wa5 developed for the quantitatioo of tbe amounts of a 1B4 recombinant antibody expressed in con­ditioned mammalian cell growth medium. Immulon-2 (Dynatecb Labs.) 96-well plates arc coated overnight witb a S p g/ml solution o( mouse anti-buman k chain constant domain monoclonal antibody (cat. #MC009, The Binding Sile, toe., San Diego, Calif.) in O.lM Nal!C03 buffer (pH 8.2) al 4° C., and blocked with J % bovine serum (BSA) in O.lM NaHC03 for 1 hat 25° C. After this and all subsequent steps, washing was performed witb phosphate buffered saline (PBS). The wells are tben challenged witb condi­tioned medium containing recombinant anti-CD18 antibody, or with predetermined quantities of human IgG4 purified by protein A Sepbarose (Pharmacia Fine Chemicals) chroma­tography from hurnao lgG4 rnyeloma serum (cat.# BP026, The Binding Site, Inc.). All samples are diluted in PBS containing 0.05% Tween-20. 100 pl aliquots are incubated for lh at 37° C. in triplicate, and standard calibration curves are constructed using lgG4 concentralions ranging from 10 ng/ml 10 100 ng/ml. Bound and fully assembled human IgG4 (either native or recombinant "veneered"1B4 human IgG4 constructs) are detected witb 100 ,ul aliquots of a 1:500 dilution of mouse anti-buman IgG4 Fe monoclonal antibody conjugated to alkaline phosphatase (cat #05-3822, Zymed Laboratories, Inc.) in phosphate buffered saline (PBS) con­taining 1 % BSA After incubation for 1 h at 37° C. and subsequent washing, the quantities of bound conjugate are detected by incubating all samples with a 1 mg/ml solution of p-nitrophenyl phosphate io O.lM 2,2'amino-metbyl­propanediol buffer, pH 10.3, for 30 min at 25° C. The adsorbance of the wells is determined with a UV Max ELISA plate reader (Molecular Devices) set at 405 nm. All supernatant fluids contain this immunoglobulin, though in various amounts. The antibody secreted by the lransfected

18 293 cells is concentrated by protein A chromatography and the concentrations of the recombinant buman "veneered" anti-CD 18 antibody determined by the trapping El.isa described above, is used to compete with the binding of

5 radiolabeled murine 1B4 to tbe CD18 ligand on tbe surface of activated humao PMNs. Affin.ities of various anti-CD18 antibody constructs arc determined using a competitive 1251-m1B4 soluble binding assay with stimulated buman polymorphonuclear leukocytes (PMNs). Purified murine

10 anti-CD18 monoclonal antibody (SO ug; m1B4) is iodinated using chloramine-T (Hunter, W. M. and Greenwood, F. C., Nature 194: 495-496, 1962), aod tbe radiolabeled antibody purified using a Bio-Sil TSK250 (Biorad, Richmond, Calif.) gel filtration HPLC column (which fractionates proteins in

JS the range of l-300x103 daltons) equilibrated in O.lM phos­phate buffer, pll 7.0. Effiuent radioactivity is monitored wi tb an in-l ine detector (Beckman Model 170; Beckman, Fu.llerton, Calif.) and total protein measured at OD280 wi tb a Kratos Spectroflow 757 detector (Kratos, Mawah, NJ.). A

20 single 125J-mlB4 peak composed of coincident OD280 and radioactivity tracings characteristically elutes 6 minutes, 30 seconds following sample injection. Specific activity of the product is generally about 101tCif.ug protein, and 97-99% of the counts are precipitable with 10% trichloroacetic acid.

2s The binding of this radiolabeled antibody is assessed on bumao PMNs purified on a discontinuous Ficoll/Hypaque gradient (English, D. and Anderson, B. R., J. Immunol. Methods S: 249-255, 1974) and activated with 100 ng/ml pborbol myristate for 20 minutes at 37° C. (Lo cl al., J. Exp.

30 Med. l69: l779-1793, 1989). To determine the avidity of antibodies for CD18 molecules on the PMN surface, about lxl05 activated PMNs arc incubated in a buffer such as Hanks balanced salt solution containing 20 mM Ilepes (pH 7.2), 0.14 units aprotinio (Sigma Chemical Co.) and 2%

35 human scrum albumin (binding buffer) containing 1.3 ng 1251-mlB4 (2.8x10-11M) in the presence of increasing concentrations of unlabeled m1B4 antibody (10-7 to 10-lSM) in a 300 ul reaction volume for about 1 b al about 4° C. with constant agitation. Cell bound 1B4 is separated

40 from the unbound antibody by centrifugatioo through a 0.5M sucrose cushion (4,800xg, 3 minutes); the tubes are frozen on dry ice, and the tips cut off and counted with an LKB gamma counter. The 1C50 of the anti-CD18 antibody for the inhibition of 125I-mlB4 antibody binding is calcu-

45 lated using a four parameter filter program (Rodbard, D, Munson, P. J., and DeLean, In, "'Radioimmunoassay and Related Procedures in Medicine", International Atomic Eaergy Agency, Vienna, vol I, 469-504, 1978). T he affinity of the '·veneered" anti-CD18 antibody for the CD18 ligand

so is determined in a similar manner using cnurine 1251-m1B4 antibody and increasing quantities, as determined by the trapping Elisa, of unlabeled "veneered" anti-CD18 antibody. The results of the binding assays arc shown in FIG. 13 and indicate that the avidity of the "veneered" heavy cbain aod

ss light cbaio recombinant 1B4 antibody is equivalent to Lb at of the murine 1B4 monoclonal antibody.

The ·'veneered" beavy and light cbain expression vectors were co-transfected into CVlP monkey kidney cells using 20 pg of each plasmid 10 prepare 2 mL of the calcium

60 pbospbate precipitated solution. One mL was placed in the medium overlaying each 100 mm dish of CVlP cells. After 4 hr at 37° C. the medium was replaced wilb 1mLof15% glycerol in lxl-IBS (Hepes buffered salt). Following the 3 min glycerol sbock, 10 mL of PBS as added, the cell

65 monolayers were aspirated, washed once with 10 mL of PBS, and re-fed with fresb medium (DMEM+10% beat inactivated new born calf serum) containing 200 ,ug of

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hygromycin B and 800 11g of G418 per mL. Cloning cylinders (Fishney, In, Culture of Animal Cells, Alan R. Liss, Inc. New York, 1983) were used to isolate individual colonies prior to their expansion and subsequent assay for productivity. 1wo clones, #11 and #48, were found to express suJ'ficient amounts of v1B4 to warrant their expan­sion and ultimate accessioning.

EXAMPLE 2 Immunogenicity of a Veneered Murine Antibody MolecuJe

Human MAbs have been found io have the same phar­macokjnetics in Rhesus monkeys as they do in humans. After repeated dosing of human MAbs into these mo nkeys they were well tolerated and rarely resulted in immune recognition. Groups of three Rhesus monkeys were injected, at weekly intervals, for five weeks with one milligram of MAb (either murine, CDR-grafted, 1-lemi-chimeric, or Veneered) per kjlogram body weight. At various times follov•ing each injection the level of circulating MAb and the development of anti-MAb antibodies were assayed by ELISAs. The IB4 MAbs all bound their CD18 target on Rhesus PMNs and serum half-lives were initially about three

SEQUENCE LISTING

( 1) GENERAL INFORMATION:

(iii) NUMBER OF SEQUENCES: 38

(2) INFORMATION FOR SEQ ID NO : l :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO: 1 :

TCTCGGATCC GAYATYGTGM TSACCCAR

(2) INFORMATION FOR SEQ ID NO: 2 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 40 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

TCTCAAGCTT TGGTGGCAAG ATRGATACAG TTGGTGCAGC

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 33 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3:

TTCTGGATCC SAGGTBCARC TGMAGSAGTC WGG

20 to four hours. Trus value predominantly reflects the normal rapid turnover rate of the PMN population. Differences between the humanized versions of IB4 and its murine predecessor were appreciated by the trurd dose of antibody

5 when two of the three monkeys receiving the murine anti­body displayed moderate anaphylactic sympto ms. This response was never seen in animals treated wi th the other forms of the IB4 MAb during the six weeks of this study. Distinguishing differences between the recombinant IB4

JO MAbs were most evident following their fourth dose at which time peak plasma levels for the CDR-grafled MAbs (Hemi-chimeric and fully CDR-grafted) were significantly reduced relative to the Veneered MAb. This trend continued

15 for the remainder of the observation period (FIG. 14) and could be attributed to the progressively higher levels of anti-1B4 antibodies in these animals. These findings show that a veneering approach to humanization may not only result in recombinant an tibodies which retain all of their

20 affinity and potency, but these antibodies may also be less immunogeoic than those humanized by CDR-grafting and reshaping procedures.

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(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 33 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4:

TTCTGGATCC SAGGTBAAGC TGGTGSAGTC WGG

(2) INFORMATION FOR SEQ ID NO: S :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 33 base pairs {B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

{xi) SEQUENCE DESCRIPTION: SEQ ID NO : S :

TCTCAAGCTT ACCGATGGRG CTGTTGTTTT GGC

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS: {A) LENGTH : 36 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single {D) TOPOLOGY: linear

{ii) MOLECULE TYPE: cDNA

{xi) SEQUENCE DESCRIPTION : SEQ ID NO : 6 :

ATTTGGATCC TCTAGACATC GCGGATAGAC AAGAAC

(2) INFORMATION FOR SEQ ID NO: 7 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 46 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

{ii) MOLECULE TYPE : cDNA

{xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7:

-continued

AATAATGCGG CCGCATCGAT GAGCTCAAGT ATGTAGACGG GGTACG

(2) INFORMATION FOR SEQ ID NO : 8 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 34 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 8 :

TATAGAATTC GGTACCCTTC ATCCCCGTGG CCCG

(2) INFORMATION FOR SEQ ID NO : 9 :

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(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

TGCGTGTTCG AATTCGCC

(2) INFORMATION FOR SEQ ID NO : 10 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 36 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 10 :

TTTTAGATCT GTCGACAGAT GGCCGATCAG AACCAG

(2) INFORMATION FOR SEQ ID NO: 11 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 35 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

TTGGTCGACG GTACCAATAC ATTTTAGAAG TCGAT

(2) INFORMATION FOR SEQ ID NO: 12 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 34 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :

TCTCGGATCC TCTAGAAGAA TGGCTGCAAA GAGC

(2) INFORMATION FOR SEQ ID NO : 13 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 34 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 13 :

TCTCGCTAGC GGATCCTTGC AGAGGATGAT AGGG

(2) INFORMATION FOR SEQ ID NO: 14 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 50 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single

-continued

24

18

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-continued

(D) TOPOLOGY : linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 :

CATTCGCTTA CCAGATCTAA GCTTACTAGT GAGATCACAG TTCTCTCTAC

(2) INFORMATION FOR SEQ ID NO: 15 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 20 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 15 :

TGGCTCTGCA GCTGATGGTG

(2) INFORMATION FOR SEQ ID NO: 16 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 16 :

CACCATCAGC TGCAGAGCCA

(2) INFORMATION FOR SEQ ID NO : 17 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 20 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 17 :

CTGTCTGGGA TCCCAGATTC

(2) INFORMATION FOR SEQ ID NO : 18 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 20 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 18 :

GAATCTGGGA TCCCAGACAG

(2) INFORMATION FOR SEQ ID NO: 19 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 33 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

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50

20

20

20

20

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 19:

GTTGCAACAT CTTCAGCCTC CACGCTGCTG ATG

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 32 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GTGGAGGCTG AAGATGTTGC AACTTATTAC TG

(2) INFORMATION FOR SEQ ID NO : 21 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 42 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

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

GAATGTGCCT ACTTTCTAGA GGATCCAACT GAGGAAGCAA AG

(2) INFORMATION FOR SEQ ID NO: 22 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 22 :

CATTCGCTTA CCAGATCT

(2) INFORMATION FOR SEQ ID NO: 23 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 19 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 23 :

GAATGTGCCT ACTTTCTAG

(2) INFORMATION FOR SEQ ID NO : 24 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 27 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

CCCTCCAGGC TTCACTAAGT CTCCCCC

-continued

28

33

32

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18

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27

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(2) INFORMATION FOR SEQ ID NO : 25 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (DJ TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 25 :

TTAGTGAAGC CTGGAGGGTC CCTGAAACTC

(2) INFORMATION FOR SEQ ID NO: 26 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 35 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 26 :

GCCCCTTCCC AGGAGCTTGG CGAACCCAAG ACATG

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 35 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

AAGCTCCTGG GAAGGGGCTG GAGTTGGTCG CAGCC

(2) INFORMATION FOR SEQ ID NO : 28 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

-continued

TGTTCATTTG TAGGTACAGG GTGTTCTTGG AATTGTCTCT GGAGATGGTG

(2) INFORMATION FOR SEQ ID NO: 29 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 48 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 29 :

TGTACCTACA AATGAACAGT CTGAGGGCTG AGGACACAGC CTTGTATT

(2) INFORMATION FOR SEQ ID NO: 30 :

(i) SEQUENCE CHARACTERISTICS :

30

30

35

35

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(A) LENGTH: 29 base pairs (B) TYPE : nucleic acid (C} STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 30 :

CTGTGAGAAG GGTGCCTTGG CCCCAGTAG

(2) INFORMATION FOR SEQ ID NO: 31 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 30 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

AAGGCACCCT TCTCACAGTC TCCTCAGGTG

(2) INFORMATION FOR SEQ ID NO: 32 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : cDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

-continued

GAATGTGCCT ACTTAAGCTT TCTAGAGGAT CCTATAAATC TCTGGCCATG

(2) INFORMATION FOR SEQ ID NO : 33 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 120 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33 :

Asp Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45

Ala Ala Ile Asp Asn Asp Gly Gly Ser Ile Ser Tyr Pro Asp Thr Val so 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95

Ala Arg Gln Gly Arg Leu Arg Arg Asp Tyr Phe Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Leu Leu Thr Val Ser Ser 115 120

32

29

30

so

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(2) INFORMATION FOR SEQ ID NO: 34 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 120 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE : peptide

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 34 :

-continued

Asp Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Leu Gly Gly l 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30

Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45

Ala Ala Ile Asp Asn Asp Gly Gly Ser Ile Ser Tyr Pro Asp Thr Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95

Ala Arg Gln Gly Arq Leu Arg Arq Asp Tyr Phe Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Thr Leu Thr Val Ser Ser 115 120

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 115 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 35:

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arq 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asx Leu 20 25 30

Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Al a Asn Ile Lys Gl x Asx Gly Ser Glx Glx Asx Tyr Val Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95

Al a Arg Gly Trp Gly Gly Gly Asp Trp Gly Gln Gly Thr Leu Val Thr

Val Ser Thr 115

100 105 110

(2) INFORMATION FOR SEQ ID NO : 36 :

{i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 112 amino acids (B) TYPE : amino acid

34

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(C) STRANDEDNESS: single (D) TOPOLOGY : linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

-continued

Asp Ile Val Met Thr Gln Ser Ser Asn Ser Leu Ala Val Ser Leu Gly 1 s 10 lS

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp SO SS 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 7S 80

Ser Val Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110

(2J INFORMATION FOR SEQ ID NO : 37 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 112 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (DJ TOPOLOGY : linear

(ii) MOLECULE TYPE : peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 37 :

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly l 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile 65 70

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr 85 90

Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr 100 105

(2) INFORMATION FOR SEQ ID NO: 38 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 114 amino acids (BJ TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : peptide

75

Tyr Cys Gln Gln

Lys Leu Glu Leu 110

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 38:

Ser 95

Lys

Asn 80

Asn

Arg

Asp Ile Val Met Thr Gln Ser Ser Asn Ser Leu Ala Val Ser Leu Gly l 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

36

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-continued

20 25 30

Ser Asn Ser Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95

Tyr Tyr Ser Thr Pro Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110

Lys Arg

What is claimed is: 20

1. A me thod of producing a veneered immunoglobulin having the ligand binding properties of an immunoglobulin from a first mammalian species and having the immuno­geoic properties of reduced imrnunogenicity in a second

25 mammalian species, the process comprising:

a) comparing a variable domain of the first species immunoglobulio with variable domains of immunoglo­bulins from the second species at corresponding frame­work amino acid sequences; 30

acid residues at the corresponding pos1tton of the selected variable domain, these differing amino acid residues being Jjmited to those determined to be exposed to solvent, which are not directly adjacent to a complemeotarity determining region; and

d) producing an immunoglobulin having the lirs1 species variable domain w ith o nly the identified amino acid residues identified in step c) being replaced with the corresponding residues present in the selected va riable domain.

2 . The method of claim 1 wherein the first mammalian species is mouse.

3 . T be method of claim 1 wherein the second mammalian

b) selecting from the variable domains of the second species immunoglobulins the variable domain which is most similar lo the variable domain of the first species immunoglobulio at corresponding framework amino acid sequences; 35 species is human.

c) identifying framework amino acid residues of the first species variable domain which differ from the amino * * * * *

45 of 45 Celltrion, Inc., Exhibit 1119