US 20060039904A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0039904 A1 Wu et al. (43) Pub. Date: Feb. 23, 2006
(54) EPH RECEPTOR FC VARIANTS WITH Publication Classification ENHANCEDANTIBODY DEPENDENT CELL-MEDIATED CYTOTOXCITY (51) Int. Cl. ACTIVITY A61K 39/395 (2006.01) C07K 16/28 (2006.01) (75) Inventors: Herren Wu, Boyds, MD (US); (52) U.S. Cl...... 424/133.1; 424/143.1; 530/388.22 Changshou Gao, Potomac, MD (US) Correspondence Address: (57) ABSTRACT JOHNATHAN KLEIN-EVANS ONE MEDIMMUNE WAY The present invention relates to novel Fc variants that GAITHERSBURG, MD 20878 (US) immuno-specifically bind to an Eph receptor. The Fc vari (73) Assignee: MEDIMMUNE, INC., Gaithersburg, ants comprise a binding region that immunospecifically MD binds to an Eph receptor and an Fc region that further comprises at least one novel amino acid residue which may (21) Appl. No.: 11/203,251 provide for enhanced effector function. More Specifically, this invention provides Fc variants that have modified bind (22) Filed: Aug. 15, 2005 ing affinity to one or more Fc ligand (e.g., FcyR, C1q). Additionally, the Fc variants have altered antibody-depen Related U.S. Application Data dent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) activity. The invention fur (60) Provisional application No. 60/608,852, filed on Sep. ther provides methods and protocols for the application of 13, 2004. Provisional application No. 60/601,634, Said Fc variants that immunospecifically bind to an Eph filed on Aug. 16, 2004. receptor, particularly for therapeutic purposes.
T231 Cells (High EphA2 Expressors) 2 uglml 0.2 uglimt D0.02 uglml
2 O
1 O
A549 Cells (High EphA2 Expressors) 70
60 - E2 uglml 5 O m 0.2 uglml 40 - O0.02 uglim
: IgG Patent Application Publication Feb. 23, 2006 Sheet 1 of 22 US 2006/0039904 A1
A CAG. GTG CAG. CTG GTG. GAG TCT GGGGGA. GGC, GTT GTG CAGCCT GGAAGG. 48. Gln Wall Gln Leu Val Glu Ser Gly. Gly-Gly Wal Wall Gln Pro Gly Arg. 5 TCC CTG AGA CTC TCC TGT GCA GCC. TCT GGA TTC, ACC TTC AGT AGC TAT 96 Ser Leu Arg leu. Ser Cys Ala Ala Ser Giy Phe-Thr Phe, Ser Ser Tyr, 32 GAC ATG TCT TGG GTT, CGC CAG GCT CCG. GGC AAG. GGT CTG GAG. TGG GTC 144 Asp Met Ser Trp Wal Arg Gln Ala Pro Gly, Lys Gly: Leu . Glu. Trp Wall 48 GCA AAA GTT. AGT AGT GGTGGT GGT AGC ACC TAC TAT TTAGAC ACT GTG. 192. Ala Lys Val Ser Ser Gly-Gly Gly Ser. Thr. Tyr Tyr Leu Asp Thr Val- 54 CAGGGC. CGATTC ACC ATC. TCC AGA GAC AAT âGT AAG AAC ACC CTA TAC. 240 Gln Gly Arg Phe Thr. Ile. Ser. Arg Asp Asn. Ser-Lys Asn Thr. Leu Tyr- 80. CTG CAA ATGAAC TCT CTG AGA GCC:GAG. GAC ACA GCC GTG. TAT TAC TGT 288. Leu Gln Met Asn. Ser Leu Arg Ala Glu-Asp-Thr Ala Wal Tyr Tyr Cys 95 GCA AGA CAT CTG. CAT GGC.AGT TTT GCT TCTTGG GGC CAA. GGG.ACT ACA 336 Ala Arg His Leu. His Gly ser Phe Alaser Trp Gly-Gln Gly. Thr Thr l12 GTG. ACT GTT TCT AGT 35l Wall. Thr Wal Ser Ser. li
GAGATT GTG CTA ACT CAG TCT. CCA, GCC. ACC CTG TCT CTC AGC CCA GGA 48 Giu. Ike Wall Leu. Thri Gln Ser. Pro Ala. Thr. Leu Ser Leu Ser Pro Gly 16 GAA AGGGCG ACT CTTTCCTGCCAGGCC AGCCAA AGTATT AGC AAC TTC 96 Glu Arg Ala Thr. Leu. Ser. CysGlin Alaser Glin ser, Ile ser Asn. Phe 32 CTA CAC TcG TAT CAA CAA. AGGCCTGGT CAA GCC CCA AGG CTT CTC ATC 144 Lieu. His Trp Tyr Glin Gin Arg-Pro Gly, Glin. A la Pro Arg Lea Leu Ile 48 CGCTAT, CGT TCC CAG TCC ATC. TCT GGG ATC. GCC GEC GGTTC AGT GGC 192 Arg Tyr Arg Ser Gln-Ser Ile iser Gly. Ile. Pro Ala Arg Phe Ser Gly 52 AGT GGA TCA GGG ACA GAT TTC, ACC CTC-ACT, ATC TCC. AGT CTG GAG CCT 240 Ser Gly Ser Gly. Thr: Asp Phe-Thr Lau Thr Ile Ser. Ser. Leu Glu Pro 80 GAA GAT TTT GCA GTC TATTAC TGT. CAA CAG. AGT GGC AGC TGG CCT CTG 288. Glu Asp Phe Ala Wall. Tyr Tyr Cys Gln: Gln Ser Gly Ser. Trp Pro Leu, 96 ACGTTCGGA GGGGGG ACC. AAG GTG GAA ATT. AAG 32. Thr Phe Gly Gly Gly. The Lys Val Glu Ile Lys 107 FIG. 1 Patent Application Publication Feb. 23, 2006 Sheet 2 of 22 US 2006/0039904 A1
A CAAA T G CAGCT GGT GCA GT CT GGGCCT GA. GGTGA A GAAGCCTGGGA CCT CA GT GAA GGT C 60 Gln Met Gin Leu Wai Gln Ser Gly Pro Glu Wall Lys lys Pro Gly Thr Ser Wall Lys Wol
T CCT GCAAGGCT T C T G GATTCACC TTT GA, CGATT ACT CCA GAACT GGG T G CGACA GGCT 12O Ser Cys Lys Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ser Met Asn Trp Val Arg Gin Ala
CGT GGA CAA, CGC CT GA GTGGA TAGGATT TAT TAGAAA CAAA GCTAATGACT ACA CAA CA 18O Arg Gly Gin Arg Leu Gu Trip Ile Gly Phe Ile Arg Asn Lys Ala Asn Asp Tyr Thr Thr
GA GTA CGCT GA CTCT G T GAA. GGG TAGA GT CACCAT TA CCAGGGA CAT GT CCA CGA, GCACA 24 O Glu Tyr Ala Asp Ser Wol Lys Gly Arg Wal Thr Ile Thr Arg Asp Met Ser Thr Ser Thr
GCCTA CAT GGA, GCT GAGCAGCCTGA, GATCCGAGGA, CA, CGGCCGT GTATTACT G T GCGAGA 3OO Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Wol Tyr Tyr Cys Ala Arg
TACCC TAGGCAT CAT GCTA TGGA CTCCTGGGGCCAAG GAA CCT CGGT CACCGT C T CCT CA 360 Tyr Pro Arg His His Ala Met Asp Ser Trp Gly Gin Gly Thr Ser Wol Thr Wol Ser Ser
B
GA CAT CCAGA T G ACCCA GT CTCCAT CCTCCCTGT CT GCA TCT G TAGGAGA CAGA GT CACC 8O Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Wol Gly Asp Arg Vol Thr
A TCA CT TGCA. GGGCCAGCCAAA GTAT TA GCA ACA ACC TACA CTGGTA, TCAGCAGAAA CCA 12O Ile Thr Cys Arg Ala Ser G.In Ser Ile Ser Asn Asn Leu His Trp Tyr Gln Gin Lys Pro
GGGAAA GCCCCTAAGCT CCTGATCA AG TAT GCCTT CCA GT C CAT CTCTGGGGTCCCA TCA 18O Gly Lys Ala Pro Lys Leu Lleu Ile Lys Tyr Ala Phe Gin Ser Ile Ser Gly Wol Pro Ser
A GGTTCA GT G GAA GTGGA, TCT GGGACA GATTTT ACT TTCAC CAT CAGCA GCCTGCAGCCT 2 O Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gin Pro
GAAG AT TTT GCA ACA, TATT ACT GT CA ACA GGCCAA CAGCT GGCCGCT CAC GT TCGGCGGA, 3OO Glu Asp Fhe Ala Thr Tyr Tyr Cys Gin Gin Ala Asn Ser Trp Pro Leu Thr Phe Gly Gly
GGGACCAAG G T G GAGAT CA AA 32 Gly Thr Lys Wal Glu Ile Lys
FIG. 2 Patent Application Publication Feb. 23, 2006 Sheet 3 of 22 US 2006/0039904A1
A GAG.Glu wal',GTG. CAG.Gln. LauCTG wal.GTG. GluGAG TCT.Ser GGGGly. Gly-Gly-WallGGAGGT:GTG: wal.GTA: CGG,Arg. CCTPro GGGGly. GGGGly 48.16 TCC. CTG XGA CTCTCC GGGTTC Acc. GTCAGF GATTAC. 96. Sér, Léu. Arg Leu Ser. C. Gly. Phe Thr, Wal Serasp Tyr 32 TCCser. ATGAACMet. Asn. TGGTrp valGTCCGC Arg CAG,Gin GCTAla: CoA.Pro. GGGGly. AAGLys GGEGiy CTGLieu. GAGTGGGlu Trp ATTIle 14448. GGGTTT-ATT,...... W. 1 AGAAACAAA. . . . GCT. AAT, GCCTACv. . . CA. ACAGAG. TACAGT:GCA h92 Glyphe. Ile Arg Asn. Lys. Ala Asn. A Thr Thr-Glu Tyr Ser Ala 64 Serfict. wallGTG AAG.Lys GGTGly. AGATTCACCArg Phe Thr...I ATC. re. TCASer Arg.AGAGAT Asp GATTCA.Asp. Ser AAA-AACLys Asn ThrACG 24080 - - - - ... ." Y. AG. GAC ACA GCC GTG TAT. 288. CTGLieu TATTyr: CTGLieu. CAAATGAACAGCCTG.A.A.A.A.Glin Met Asn Ser Leu Lys. Th ilu-Asp Thr Ala Val Tyr - 96.. TAC TGT- ACC ACA. TAC CCT AGG TAT CAT GCT. ATG GAC TCC TGG GGC CAG 336 Tyr. Cys. Thr. Thr Tyr Pro Arg T. r. His Ala. tet Asp set Trp Gly-Gln ll 2 GGC ACC ATG GTC ACC GTCTCC, TCA 350 Gly. Thr Met Wall Thr Wal Ser Ser. l2O
B GCC ATC CAG. TTG ACT CAG. TCTCCA TCC, CC CTG TCT, GCA TCT, GTA GGA 48. Aka Ile Gln Leu Thr Gln Ser: Pro, ser Leu Ser Ala Serval. Gly lé GiCAGA...... GTC. . . ACC. . . . . ATC,. " ACT TGC. A T. ATT.Ile AGCSer' AAC,Asn. AsnAAC 96.32 Asp Arg Wall. Thr Ile CTA CAC TGG TAC CTG CAG &AGCCAGGG cag. TCTCCA CAG CTC CTG ATC 144 Leu. His Trp. Tyr Leu Gin Lys-Pro Gly-Gln-Sér Pro Glin Leu Leu. Ile 48 TTC AGT GGC 192 Phe Ser Gly 64. 'AT TyrTAT GGCGly. TTCPhe CAGGlin TCC ATC :AGT.GGA, 'Glyser TCTGGG Gly. ACAThr GATAsp-Phe. TTC. Thriéu.ACT Thr fie Ser SerAGT CTGLeu CAAGln CCTPro 24080 Ser. - GAAGAT TTT GCA ACT TAC. TACTGT. CAA CAG, GCC. AAC AGC TGGCCGCTC 288. Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Gln, Ala-Asn Ser Trp Pro Leu .96 ACG TTC GGC. GGA. GGG Acc. AAG. CTG GAG ATC AAA 32l Thr. Phe Gly. Gly-Gly. Thr. Lys. Levi Giu. Ile Iys lO?
FIG. 3
Patent Application Publication Feb. 23, 2006 Sheet 5 of 22 US 2006/0039904 A1
1 2
Fc-mutants ... h
s. I||
|11 13 15 17 19 21 23 25 27 29 31 33 35 37 9 4 43| Clone Number
Clone 1: RI-1 (L234E) Clone 23: RIV-49 (I332E) Clone 2: RI-2 (L235R) Clone 24: RIV-50 (A330V) Clone 3: RI-13 (L235A) Clone 25: RIV-59 (A330G) Clone 4: RI-16 (L235W) Clone 26: RIV-65 (A330Y) Clone 5: RI-61 (L235P) Clone 27: RIV-84 (332S) Clone 6: RI-63 (L235P) Clone 28: RIV-90 (A330G) Clone 7: RI-69 (L235V) Clone 29: RIV-100 (P329H) Clone 8: RI-83 (G236E) Clone 30: RIV-112 (1332W) Clone 9: RI-102 (L235Y) Clone 31: RIV-116 (L328V) Clone 10: RII-III-19 (D265L) Clone 32: RIV-122 (A330T) Clone 11: RII-III-81 (S298.I) Clone 33: RIV-125 (1332F) Clone 12: RII-III-121 (S298T) Clone 34: RIV-135 (I332Y) Clone 13: RII-III-123 (S298F) Come 35: RIV-141 (A330L) Clone 14: RII-III-145 (E269S) Come 36: RIV-150 (A327N) Clone 15: RII-III-20-4-F5 (E269G) Clone 37: RIV-151 (A330D Clone 16: RIV-2 (P329Q) Clone 38: RIV-159 (I332Y) Clone 17: RIV-3 (332E) Clone 39: RIV-161 (332Y) Clone 18: RIV-21 (L328S) Clone 40: RIV-165 (A327G) Clone 19: RIV-22 (A330K) Clone 41: RIV-168 (L328V) Clone 20: RIV-23 (332E) Clone 42: RIV-173 (L328S) Clone 21: RIV-43 (A327W) Clone 43: RIV-189 (A330R) Clone 22: RIV-47 (332H) Clone 44: RIV-203 (A330C)
FIG. 5 Patent Application Publication Feb. 23, 2006 Sheet 6 of 22 US 2006/0039904A1
25
15
10
5
O
& Š
FIG. 6 Patent Application Publication Feb. 23, 2006 Sheet 7 of 22 US 2006/0039904 A1
uu Ogye OO AloxonoMo Aueo-led Patent Application Publication Feb. 23, 2006 Sheet 8 of 22 US 2006/0039904A1 A 2. d Vitaxin (NSO) witaxin (293) O A Fo-332e (293)
VIn 1'' ?u ?a O O.
Antibody concentration
Vitaxin (NSO) Vitaxin (293) E Fo-332 (293) O ? V d f ?a O
Antibody concentration FIG. 8 Patent Application Publication Feb. 23, 2006 Sheet 9 of 22 US 2006/0039904 A1
O) eg 9 o L C O U D O O 2 E C
up SR p Sp AloxooAOueled Patent Application Publication Feb. 23, 2006 Sheet 10 of 22 US 2006/0039904A1
i
%,
AuSixo.o.ouesied US 2006/0039904A1
MoxooAO % AloxooAO % Patent Application Publication Feb. 23, 2006 Sheet 12 of 22 US 2006/0039904A1
A
Log Conc. (ng/ml) 4.
B 3 5ug/ml 1.25 ug/ml sS 2
1 0 -8- idea 3F2-wt 3F2-1M 3F2-3M 9. 4. C D 3F2-Wt 3 - A 3F2-1M a?: () 3F2-3M S O O
log ConC. FIG. 12
Patent Application Publication Feb. 23, 2006 Sheet 14 of 22 US 2006/0039904 A1
A A549 Cells (High Epha2 Expressors) 50 -0- 12 G3 FC-Var is2 40 -- 12G3 wT x 30 s CD 20 3 10 21-1 O O 4 ng 40 ng 400 ng Antibody Conc (in ng Iwell)
B A549 Cells (High EphA2 Expressors)
4. O 50 to 1
3 O 25 to 1
20
12G3H11 12G3H1 1 (I332E) 12G3H11 IgG No Ab 400 ng 40 ng 4 ng 400 ng 40 ng 4 ng 400 ng 40 ng 4 ng 400ng
FIG. 14 Patent Application Publication Feb. 23, 2006 Sheet 15 of 22 US 2006/0039904 A1 T231 Cells (High EphA2 Expressors) 32 ug/ml 0.2 uglml D 0.02 uglml
A549 Cells (High EphA2 Expressors) 70
60 - s2 uglml as 50 - 0.2.Zugim uC/ml s 40 - O0.02 uglml und O is 30 - O SS 20 - 10 -
O va V - eS s L en Patent Application Publication Feb. 23, 2006 Sheet 16 of 22 US 2006/0039904A1
Hey8 Cells (High Eph A2 Expressors) 60 s2 ug/ml 0.2 uglml 50 O0.02 ug/ml 3040
2O
10 S
100:1 50:1 25:1 100:1 50:1 25:1 1 00:1 50:1 25:1 100:1 50:1 25:1 3F2-3M 3F2-1M 3F2WT R347 ET SKOV3 Cells (Moderate EphA2 Expressors)
x 2 uglml 0.2 uglml 0.02 uglml Patent Application Publication Feb. 23, 2006 Sheet 17 of 22 US 2006/0039904A1 A498 Cells (Low EphA2 Expressors) 70 2 uglml 60 0.2 uglml 50 D 0.02 uglml 40
30 : s 20 | | |& | | U : : s 10 O : & & 8 2: ES g s g s g s s S s wo- N w N v CN v- L N 3F2-3M 3F2-1M 3F2WT ld
SKMEL28 Cells (Express integrin oVB3 and Low Eph A2) 20 18 16 S2 uglml Ca 14 0.2 uglml 12 O 0.02 uglml X 810 U 8 & 6 4 s
O s
Vitaxi Vitaxin 3M Parental
FIG. 17 Patent Application Publication Feb. 23, 2006 Sheet 18 of 22 US 2006/0039904 A1
e i e P G A hike . . . . A :
Bhal 3 is s sh v. Ephs . 53 RhAge E. g Enid E. s Ephia, l sh Ash r. . Ephia EE has of Ephs st Ephra , & 8. Bh82h 8 BhB . Ehsa . . . 8 this is tr. L. 8
C: s 3 s 3 : 3 s B 8 s 8 s 8 i, s 38 8 8 8 ... I s A a chase B s f : e 8 s k s 8 Ephs 4 f. El 8 3. Ephs is s . r
Ephs. A g c s ------, A. . . . . Eshk E H ------f . crat is Eyn A3 3: DRI ------s (, shashi AI ------A. Eghk a RI ------C. At y phasa to ------A. Eph Ashery ------8th Eik ------A sphat eth ------i. C, f his cca. ------xs Eshkzar V------shkeh C. ------as a as a s Ephs ------Ensa A. E. A Er ------a -a-. a- a a rphis s s s A A or A it c e c is c q x. A c I. orju K. sec s
Bhal Age A s 3. Eph A RF Bhasa s Ephigh f sh A& Aoss 1 vs f G Eamas ?: r Bhash EE W K s
Ehk? is s 8th As sphs 1. s sha s Enkh . Eshk: Le e Ezr g Bhish he e s - nks s 8 s FIG. 18A Patent Application Publication Feb. 23, 2006 Sheet 19 of 22 US 2006/0039904 A1
ph A. c. sc. 8 g e 3. sha A 3. Enasa R 3. pha2a as 3 & 3. Ephia s Ephasa H 3 s 56 ph Ash g na s 9 s has h s 33 pha23 . hah A. 33 Ephs 3 8 A. 3. Enk & g s A. phs 36
ph Al $ As cro sen A ha. v c as Ga KE Ephiarp is s pharp B s Ephs War sh Asapf v if s shishu s ph A rif Eph A. swif w sphs IV w 3. Ephs a sv s g pha2 sv A. 3S she pr v if 3 Eph is a Lac C - - - 3. phas H 4 acc 3.
ph A. C. s c s LLJ, K is or a Ena E. E. K e spinase I, SS hase . . 6 Bhak h . . f has . Eph Ashb L. L. s Eph A. D. L. L. Eph J. F. s Enki 3. Ehsaas Ehsaac g s sins s ent & 3. sphes cav cw
sphal rty as a - - - sts Eph A. v exy B ...... - - - s spara A Y 3. RhAh y : y s Rph Ad A y s: phasis fy - 6. Eph A. is 8. Ephia y wry A s& As Y sy a As E 3. Ephri - s ?ex a s s Bhra 8 a Y - - 3. sh 3 R A - as a 3. Ephs y c I As-w y A- - S&6 speed v A sec - - Ephs re- A a cer c civ Y - - SS3
Eph Ali - - - - - sh c L cc I v A vir at Ligh A. L. iii. ------R. R. R. A. A 58 Ephia ------E: A ------a - H a r at 3 sphaia r - - - - sis c s s sov v as - Is AAF A it wix vic ------: R is sphah ------St. sphad I co c is ------v L. AAFI s ------s:
phashphasa r - - - - A asss s D : ::::::::se - - - a - as as s a -A - - - a aA - -A - - - ra - stss pha as r is A R A v s s v - e as we c e ch ...... m - - 4. s Ephia - - c. a R Y D . . . a s a a s - w w - - a K K ------a s - a s ephen D Y x s is - I - - - he ol. - a ------s pha2a is 8 r ------s ------, a : Ephsah E. Y. g. s r - ros V ------A : s Eph 3 c s CA o O L - - - E QL - - - - as ------a s - . . . . e. s phi - - - s is car - - - a . ------, is a s sphe 6 E L S 3 g L - - - - EA y L. A r------A v v r on Knac - - 3.
FIG. 18B Patent Application Publication Feb. 23, 2006 Sheet 20 of 22 US 2006/0039904 A1
Eph Al on Age v ------a a x L. S3 Ephlia os P------phas abc A or KA Le c arc H. s: phaz ------V X sha - f ------y Eph A53D H Y Eph A50 s f sh A. Eph O. a Y s 6. shal AV has a y s Eph S. s y sh;3 s. a s a 3 s Beha & I ------Y B 6. sphs - y A. s
sha as on c sha S. Bhasa s RhA. 3. Ephia A A has a as A se ph As Rys a lad is as sax E. k a lar c. As phas L. Kac Y. g. a n is r i. shal L. KACY to a no e L. 8 A . . Ephra . a ex a e a s Ephs A 3 Ephs A sphed A 63 Ephes A 3
8 ...... S Eph A3 ------a ------a a - as a a S3 Eph Aa E . S. ph Asas , A .2 phashers R b c L. r A :E:::::::::::::: s spha srs R F GL AF A ...... Eph AE is is c. are x D . A. An vi. vos r. v c x v 8 Dec , s a st ephal grass t c ber 1. A A A ...... g . . . . . R. S. Epse r is E c s , a s r. A ...... Ehrhe A ...... a . . . . Eph 3 E A ...... Ephras 3 6 Ephesis 3.
Eph Al Loa- - 33 EEEph A3K 83
Eph 3 ------s Ephia F L E on P A 3. Bhasav L. B A 3 3. Eph A5by L. E Go e A A a ass Eph A7 was o De a 8 A. A. or as s spha v L. B ce. is A h e Y crashew I 83 Ephel Yao or : A h y of a 4 a v II 3. Espina r. ( or A . A a V 33 spha2b e. to A . A A EY: s Ephes et a A . A s A v 3.
Ephs is s A 3 A A. a as s y 3. shes : y if s
ephal to - st sh A2 to - Bhasa as s s s
36
3. sts s
Eph G to Er - 9 Patent Application Publication Feb. 23, 2006 Sheet 21 of 22 US 2006/0039904 A1
ph A. - - soc. I ex . . . . 3 3 . . . . .0 as a . - Ephiah32 - - vietEEEEEEEE Clar c A H c IcEEE v escork se Dixxv w EEEH RAA is c. 95.s shah ------sh - - - - a a a w w ------a as a sa w w w a s ph Aa - - essers S v A lsAx ph A53 - - - a so a V a J2EE AA. m phash - - As E. c is c a 3 A A sai A. E. B. ph A. - - are Al khsh Y 9. phhi - - EEESi S. is . Ephsphrah 23 - - ::::::::::::a Y a AA a K. R. sas his 3 - - K. a A K. K. 9. in 8 - - K. as Y: A 9. phs - - A R. L. I. I. I. A O O O O O O O
enal ------R 6 E.ph D E.g.,pf v c I e I s s pharh ------A sis ph R8 to go as c R. s phasa K. V. V - - - C hash K Vol. War - - - C ph A R A Qas H. J. G E G s ph 2 p. A QL r s a c e A H S Ephs to st A. 3. Ephra A air a , a a a K c a s c as a X c. ca. c a loa phtha A or GI sy s Eph. Aloid C. L. to s she 4 x s A ------G cc case A a Ephr 6 g or no c s sy 6
Signal Sequence SEANO. 6 Ephrie receptor ligand binding deman
i. . EEOIANO. EE3 IANO. T8 Tumorreorosis factor receptor (TNFR) domain splish sts Eph Ah SS E. AN3 = Fibonectin type III (3) domain A. w Ed ANO.8 " Erasherine domain Ephia gg EE0 IANO. 82 Ephia EEG IANO. 83 - Tyrosine kinase catalytic domain 88.is . . . is a sh EEGIANO.EEOIANO.8S 84 SAM domain (3terile pharmotif) Bhikh EEGIANO. 86 Ephs EEGIANO.8i
alh E XESEEA ANO. No. 8988 EEGSIANO. 90
FIG. 18D
US 2006/0039904 A1 Feb. 23, 2006
EPH RECEPTOR FC VARIANTS WITH Fc variant fusions that immunospecifically bind to at least ENHANCEDANTIBODY DEPENDENT one Eph receptor. Further, the invention provides pharma CELL-MEDIATED CYTOTOXCITY ACTIVITY ceutical formulations and kits for use in preventing, man aging, treating or ameliorating Eph receptor-mediated (and/ 1. CROSS REFERENCE TO RELATED or Ephrin-mediated) and/or associated diseases and APPLICATIONS disorders or one or more Symptoms thereof, including but not limited to cancer, inflammatory and autoimmune dis 0001) This application claims the benefit under 35 U.S.C. CSCS. S 119(e) of the following U.S. Provisional Application Nos.: 60/601,634, filed, Aug. 16, 2004 and 60/608,852, filed, Sep. 3. BACKGROUND OF THE INVENTION 13, 2004. The priority applications are hereby incorporated by reference herein in their entirety for all purposes. 3.1 Cancer 0003) A neoplasm, or tumor, is a neoplastic mass result 2. FIELD OF THE INVENTION ing from abnormal uncontrolled cell growth, which can be benign or malignant. Benign tumors generally remain local 0002) The present invention provides novel antibodies ized. Malignant tumors are collectively termed cancers. The comprising at least one antigen binding region and an Fc term “malignant” generally means that the tumor can invade region that further comprises at least one novel amino acid and destroy neighboring body structures and Spread to residue of the invention. The present invention also relates distant sites to cause death (for review, see Robbins and to novel antibodies comprising a variable region, or frag Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., ment thereof, that immunospecifically binds to at least one Philadelphia, pp. 68-122). Cancer can arise in many sites of Eph receptor and an Fc region that further comprises at least the body and behave differently depending upon its origin. one high effector function amino acid residue. The present Cancerous cells destroy the part of the body in which they invention further relates to novel variants of antibodies that originate and then spread to other part(s) of the body where immunospecifically bind to at least one Eph receptor which they start new growth and cause more destruction. contain one or more Substitutions in their Fc regions. Col lectively, these novel antibodies are referred to herein as “Fe 0004) More than 1.2 million Americans develop cancer variants of the invention” or “Fe variants.” In one embodi each year. Cancer is the Second leading case of death in the ment, the Fc variants of the invention have enhanced effector United States and if current trends continue, cancer is function. In another embodiment the Fc variants of the expected to be the leading cause of the death by the year invention have altered binding affinity to one or more Fc 2010. Lung and prostate cancer are the top cancer killers for ligands. In another embodiment, the Fc variants of the men in the United States. Lung and breast cancer are the top invention have enhanced binding to FcyRIIIA and increased cancer killers for women in the United States. One in two ability to mediate antibody dependent cell-mediated cyto men in the United States will be diagnosed with cancer at toxicity (ADCC). In another embodiment, the Fc variants some time during his lifetime. One in three women in the have reduced binding to FcyRIIIA and decreased ability to United States will be diagnosed with cancer at Some time mediate ADCC (referred to herein as “ADCC activity”). In during her lifetime. still another embodiment, the Fc variants have enhanced 0005) A reliable cure for cancer has yet to be found. binding to the C1q and increased ability to mediate comple Current treatment options, Such as Surgery, chemotherapy ment dependent cytotoxicity (CDC). In yet another embodi and radiation treatment, are oftentimes either ineffective or ment, the Fc variants have reduced binding to C1q and present serious side effects. decreased ability to mediate CDC. In particular, the present invention relates to Fc variants that can bind to one or more 3.2 Metastasis Eph receptors. In addition, the present invention provides 0006) The most life-threatening forms of cancer often methods and protocols for the application or use of Fc arise when a population of tumor cells gains the ability to variants, particularly for therapeutic purposes. Specifically, colonize distant and foreign sites in the body. These meta the methods and protocols involve the administration of a static cells survive by overriding restrictions that normally prophylactically or therapeutically effective amount of one constrain cell colonization into dissimilar tissues. For or more Fc variants alone or in combination with the example, typical mammary epithelial cells will generally not administration of one or more other therapies useful for the grow or survive if transplanted to the lung, yet lung treatment and/or prevention of Eph receptor-mediated (and/ metastases are a major cause of breast cancer morbidity and or Ephrin-mediated) and/or associated diseases and disor mortality. Recent evidence Suggests that dissemination of ders or one or more symptoms thereof, including but not metastatic cells through the body can occur long before limited to cancer, inflammatory and autoimmune diseases. clinical presentation of the primary tumor. These The Fc variants utilized for therapeutic purposes may or may micrometastatic cells may remain dormant for many months not be conjugated or fused to a moiety (e.g., a therapeutic or years following the detection and removal of the primary agent or drug). The methods of the invention are particularly tumor. Thus, a better understanding of the mechanisms that useful for the prevention, management, treatment or ame allow for the growth and Survival of metastatic cells in a lioration numerous forms of cancer. The invention also foreign microenvironment is critical for the improvement of provides methods for screening for an antibody that immu therapeutics designed to fight metastatic cancer and diag nospecifically binds to at least one Eph receptor as well as nostics for the early detection and localization of metastases. methods to manipulate the Fc region and thereby modulate the ability of said Fc region to mediate ADCC and/or CDC 3.3 Cancer Cell Signaling activity and/or the binding affinity for one or more Fc 0007 Cancer is a disease of aberrant signal transduction. ligands. The invention also provides methods for generating Aberrant cell signaling overrides anchorage-dependent con US 2006/0039904 A1 Feb. 23, 2006
Straints on cell growth and Survival (Rhim, et al., Critical ageal cancers (Ogawa, et al., 2000, Oncogene 19:6043; Reviews in Oncogenesis 8:305, 1997; Patarca, Critical Zelinski, et al., 2001, Cancer Res 61:2301; Walker-Daniels, Reviews in Oncogenesis 7:343, 1996; Malik, et al., Bio et al., 1999, Prostate 41:275; Easty, et al., 1995, IntJ Cancer chimica et Biophysica Acta 1287:73, 1996; Cance, et al., 60: 129; Nemoto, et al., 1997, Pathobiology 65:195). EphA4 Breast Cancer Res Treat 35:105, 1995). Tyrosine kinase is expressed in brain, heart, lung, muscle, kidney, placenta, activity is induced by ECM anchorage and indeed, the pancreas (Fox, et al., 1995, Oncogene 10:897) and melano expression or function of tyrosine kinases is usually cytes (Easty, et al., 1997, Int. J. Cancer 71:1061). EphA4 increased in malignant cells (Rhim, et al., Critical Reviews binds Ephrins A1, A2, A3, A4, A5, B2, and B3, (Pasquale, in Oncogenesis 8:305, 1997; Cance, et al., Breast Cancer 1997, Curr. Opin. in Cell Biology 9:608) also ligands B61, Res Treat 35:105, 1995; Hunter, Cell 88:333, 1997). Based AL1/RAGS, LERK4, Htk-L, and Elk-L3, (Martone, et al., on evidence that tyrosine kinase activity is necessary for 1997, Brain Research 771:238). Ligand binding leads to malignant cell growth, tyrosine kinases have been targeted EphA4 autophosphorylation on tyrosine residues (Ellis, et with new therapeutics (Levitzki, et al., Science 267: 1782, al., 1996, Oncogene 12:1727). EphA4 tyrosine phosphory 1995; Kondapaka, et al., Molecular & Cellular Endocrinol lation creates a binding region for proteins with Src Homol ogy 117:53, 1996; Fry, et al., Current Opinion in BioTech ogy 2/3 (SH2/SH3) domains, such as the cytoplasmic nology 6: 662, 1995). Unfortunately, obstacles associated tyrosine kinase p59fyn (Ellis, et al., Supra; Cheng, et al., with Specific targeting to tumor cells often limit the appli Cytokine and Growth Factor Reviews 13:75, 2002). Acti cation of these drugs. In particular, tyrosine kinase activity vation of EphA4 in Xenopus embryos leads to loss of is often vital for the function and survival of benign tissues cadherin-dependent cell adhesion (Winning, et al., Differ (Levitzki, et al., Science 267: 1782, 1995). To minimize entiation 70:46, 2002, Cheng, et al., Supra), Suggesting a role collateral toxicity, it is critical to identify and then target for EphA4 in tumor angiogenesis; however, the role of tyrosine kinases that are Selectively overexpressed in tumor EphA4 in cancer progression is unclear. EphA4 appears to cells. be upregulated in breast cancer, esophageal cancer, and pancreatic cancer (Kuang, et al., Nucleic Acids Res. 3.4 Eph Family of Receptor Tyrosine Kinases 26:1116, 1998; Meric, et al, Clinical Cancer Res. 8:361, 0008. The Eph family of receptors are the largest family 2002; Nemoto, et al., Pathobiology 65:195, 1997; Logsdon, of receptor tyrosine kinases (RTKS). The Eph receptors, and et al., Cancer Res. 63:2649, 2003), yet it is downregulated their membrane bound ephrin ligands are important media in melanoma tissue (Easty, et al., Supra). tors of cell-cell communication regulating cell attachment, shape, and mobility. Eph RTK Signaling events control 0010 EphB2 and EphB4 receptors are also overex multiple aspects of embryonic development, particularly in pressed in certain tumor tissues. EphB4 overexpression is the nervous system (reviewed in Kullander et al., 2002, Nat. mainly found in infiltrating ductal breast carcinomas with Rev. Mol. Cell Biol. 3:473 and Mamling et al., 2002, Trends high grade malignancy-2 (Berclaz et al., 1996, Biochem Biochem Sci 27:514-520. Receptors in the EPH subfamily Biophys Res Commun 226:869) while Eph B2 is overex typically have a single kinase domain and an extracellular pressed in a majority of gastric tumors (Kiynokawa et al., region containing a Cys-rich domain and 2 fibronectin type 1994, Cancer Res 54:3645). Both receptors are overex III repeats (see FIG. 18). The ephrin receptors are divided pressed in many tumor cell lines as well (Berclaz et al., into 2 groups based on the Similarity of their extracellular Supra; Kiynokawa et al., Supra; Bennett et al., 1995, PNAS domain Sequences and their affinities for binding ephrin-A USA92:1866). Both EphB2 and EphB4 are also upregulated and ephrin-B ligands. Many members of the Eph receptors in colon carcinoma tissue (Liu et al., 2002, Cancer 94:934; have been identified as important markers and/or regulators Stephenson et al., 2001, BMC Mol Biol 2:15). In addition, of the development and progression of cancer (see for Eph B2 and EphB4 are also important for vascular develop example Thaker et al., 2004, Clin. Cancer Res. 10:5145; Fox ment in the embryo and possibly in tumors (Wang et al., B Pet al., 2004, Biochem. Biophys. Res. Commun. 318:882; 1998, Cell 93:741; Gerety, S.S. et al. 1999 Mol Cell 4:403). Nakada et al., 2004, Cancer Res. 64.3179; Coffman et al., 2003, Cancer Res. 63:7907; also reviewed in Dodelet et al., 3.5 Cancer Therapy 2000, Oncogene 19:5614). Of the Eph receptors known to be 0011. One barrier to the development of anti-metastasis involved in cancer the role and expression patterns of agents has been the assay Systems that are used to design and EphA2 and EphA4 are among the best characterized. evaluate these drugs. Most conventional cancer therapies 0009 EphA2 is expressed in adult epithelia, where it is target rapidly growing cells. However, cancer cells do not found at low levels and is enriched within sites of cell-cell necessarily grow more rapidly but instead Survive and grow adhesion (Zantek, et al., 1999, Cell Growth & Diff 10:629; under conditions that are non-permissive to normal cells Lindberg, et al., 1990, Mol & Cell Biol 10: 6316). This (Lawrence and Steeg, 1996, World J. Urol. 14:124-130). Subcellular localization is important because EphA2 binds These fundamental differences between the behaviors of EphrinsA1 to A5 that are anchored to the cell membrane normal and malignant cells provide opportunities for thera (Eph. Nomenclature Committee, 1997, Cell 90:403; Gale, et peutic targeting. The paradigm that micrometastatic tumors al., 1997, Cell & Tissue Res 290: 227). The primary con have already disseminated throughout the body emphasizes Sequence of ligand binding is EphA2 autophosphorylation the need to evaluate potential chemotherapeutic drugs in the (Lindberg, et al., 1990, Supra). However, unlike other recep context of a foreign and three-dimensional microenviron tor tyrosine kinases, EphA2 retains enzymatic activity in the ment. Many Standard cancer drug assays measure tumor cell absence of ligand binding or phosphotyrosine content growth or Survival under typical cell culture conditions (i.e., (Zantek, et al., 1999, Supra). EphA2 and ephrin-A1 are monolayer growth). However, cell behavior in two-dimen upregulated in the transformed cells of a wide variety of Sional assays often does not reliably predict tumor cell tumors including breast, prostate, colon, Skin, and eSoph behavior in vivo. US 2006/0039904 A1 Feb. 23, 2006
0012 Currently, cancer therapy may involve Surgery, and are responsible for binding a number of natural proteins chemotherapy, hormonal therapy and/or radiation treatment to elicit important biochemical events. to eradicate neoplastic cells in a patient (See, for example, Stockdale, 1998, “Principles of Cancer Patient Manage 0017. The Fc region of an antibody interacts with a ment', in Scientific American: Medicine, Vol. 3, Rubenstein number of ligands including Fc receptors and other ligands, and Federman, eds., Chapter 12, Section IV). All of these imparting an array of important functional capabilities approaches pose significant drawbacks for the patient. Sur referred to as effector functions. An important family of Fc gery, for example, may be contraindicated due to the health receptors for the IgG class are the Fc gamma receptors of the patient or may be unacceptable to the patient. Addi (FcyRs). These receptors mediate communication between tionally, Surgery may not completely remove the neoplastic antibodies and the cellular arm of the immune System tissue. Radiation therapy is only effective when the neoplas (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181 tic tissue exhibits a higher Sensitivity to radiation than 220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). normal tissue, and radiation therapy can also often elicit In humans this protein family includes FcyRI (CID64), Serious Side effects. Hormonal therapy is rarely given as a including isoforms FcyRIA, FcyRIB, and FcyRIC; FcyRII Single agent and although can be effective, is often used to (CD32), including isoforms FcyRIIA, FcyRIIB, and Fcy RIIC; and Fcy RIII (CID16), including isoforms prevent or delay recurrence of cancer after other treatments FcyRIIIA and FcyRIIB (Jefferis et al., 2002, Immunol Lett have removed the majority of the cancer cells. 82:57-65). These receptors typically have an extracellular 0013 With respect to chemotherapy, there are a variety of domain that mediates binding to Fc, a membrane Spanning chemotherapeutic agents available for treatment of cancer. A region, and an intracellular domain that may mediate Some Significant majority of cancer chemotherapeutics act by signaling event within the cell. These different FcyR Sub inhibiting DNA synthesis (See, for example, Gilman et al., types are expressed on different cell types (reviewed in Goodman and Gilman's: The Pharmacological Basis of Ravetch et al., 1991, Annu Rev Immunol 9:457-492). For Therapeutics, Eighth Ed. (Pergamom Press, New York, example, in humans, FcyRIIIB is found only on neutrophils, 1990)). AS Such, chemotherapy agents are inherently non whereas FcyRIIIA is found on macrophages, monocytes, Specific. In addition almost all chemotherapeutic agents are natural killer (NK) cells, and a subpopulation of T-cells. toxic, and chemotherapy causes significant, and often dan gerous, Side effects, including Severe nausea, bone marrow 0018 Formation of the Fc/FcyR complex recruits effector depression, immunosuppression, etc. (See, for example, cells to Sites of bound antigen, typically resulting in Signal Stockdale, 1998, “Principles Of Cancer Patient Manage ing events within the cells and important Subsequent ment” in Scientific American Medicine, vol. 3, Rubenstein immune responses Such as release of inflammation media and Federman, eds., ch. 12, Sect. 10). Furthermore, even tors, B cell activation, endocytosis, phagocytosis, and cyto with administration of combinations of chemotherapeutic toxic attack. The ability to mediate cytotoxic and phagocytic agents, many tumor cells are resistant or develop resistance effector functions is a potential mechanism by which anti to the chemotherapeutic agents. bodies destroy targeted cells. The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcyRS rec 0.014 Recently, cancer therapy could also involve bio ognize bound antibody on a target cell and Subsequently logical therapy or immunotherapy. Biological therapies/ cause lysis of the target cell is referred to as antibody immunotherapies are limited in number and although more dependent cell-mediated cytotoxicity (ADCC) (Raghavanet Specific then chemotherapeutic agents many Still target both al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., health and cancerous cells. In addition, Such therapies may 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, produce Side effects Such as rashes or Swellings, flu-like Annu Rev Immunol 19:275-290). Notably, the primary cells Symptoms, including fever, chills and fatigue, digestive tract for mediating ADCC, NK cells, express only FcyRIIIA, problems or allergic reactions. whereas monocytes express FcyRI, FcyRII and FcyRIII 0.015 Thus, there is a significant need for alternative (Ravetch et al., 1991, Supra). cancer treatments, particularly for treatments that more 0019. Another important Fc ligand is the complement Specifically target cancer cells. The identification of mem protein C1q. Fc binding to C1q mediates a process called bers of the Eph receptor family as markers for tumor cells complement dependent cytotoxicity (CDC) (reviewed in makes them powerful targets for therapeutics. Thus, a cancer Ward et al., 1995, Ther Immunol 2:77-94). C1q is capable of treatment that would specifically target and destroy tumor binding Six antibodies, although binding to two IgGs is cells aberrantly expressing one or more members of the Eph Sufficient to activate the complement cascade. C1q forms a receptor family would be a powerful tool for the treatment complex with the C1r and C1s serine proteases to form the and prevention of cancers. C1 complex of the complement pathway. 3.6 Antibodies for the Treatment of Cancer 0020 Several key features of antibodies including but not limited to, Specificity for target, ability to mediate immune 0016 Antibodies are immunological proteins that bind a effector mechanisms, and long half-life in Serum, make Specific antigen. In most mammals, including humans and antibodies and related immunoglobulin molecules powerful mice, antibodies are constructed from paired heavy and light therapeutics. Numerous monoclonal antibodies are currently polypeptide chains. Each chain is made up of two distinct in development or are being used therapeutically for the regions, referred to as the variable (Fv) and constant (Fc) treatment of a variety of conditions including cancer. regions. The light and heavy chain Fv regions contain the Examples of these include Vitaxin(R) (MedImmune), a antigen binding determinants of the molecule and are humanized Integrin CEVfB3 antibody (e.g., PCT publication responsible for binding the target antigen. The Fc regions WO 2003/075957), Herceptin® (Genentech), a humanized define the class (or isotype) of antibody (IgG for example) anti-Her2/neu antibody approved to treat breast cancer (e.g., US 2006/0039904 A1 Feb. 23, 2006
U.S. Pat. No. 5,677,171), CNTO 95 (Centocor), a human inhibiting receptorS is an important consideration. For Integrin Civ antibody (PCT publication WO 02/12501), example, enhancing Fc binding to the positive regulators Rituxane (IDEC/Genentech/Roche), a chimeric anti-CD20 (e.g., FcyRIIIA) while leaving unchanged or even reducing antibody approved to treat Non-Hodgkin's lymphoma (e.g., Fc binding to the negative regulator FcyRIIB could result in U.S. Pat. No. 5,736,137) and Erbitux(R) (ImClone), a chi optimized effector function such as enhanced ADCC medi meric anti-EGFR antibody (e.g., U.S. Pat. No. 4,943,533). ated destruction of tumor cells. Another critical consider 0021. There are a number of possible mechanisms by ation is that Fc variants should be engineered Such that the which antibodies destroy tumor cells, including anti-prolif binding to FcyRs and/or C1q is modulated in the desired eration via blockage of needed growth pathways, intracel manner but So that they maintain their Stability, Solubility, lular Signaling leading to apoptosis, enhanced down regu Structural integrity as well as their ability to interact with lation and/or turnover of receptors, ADCC, CDC, and other important Fc ligands Such as FcRn and proteins A and promotion of an adaptive immune response (Cragg et al., G. 1999, Curr Opin Immunol 11:541-547; Glennie et al., 2000, 0024 Numerous mutagenesis studies have been carried Immunol Today 21:403-410). However, despite widespread out on the Fc domain (See for example, Duncan et al., 1988, use, antibodies are not yet optimized for clinical use and Nature 332:563-564; Lund et al., 1995, Faseb J 9:115-119; many have Suboptimal anticancer potency. Thus, there is a Lund et al., 1996, J Immunol 157:4963-4969; Armour et al., Significant need to enhance the capacity of antibodies to 1999, Eur J Immunol 29:2613-2624; Shields et al., 2001, J destroy targeted cancer cells. Methods for enhancing the Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol anti-tumor-potency of antibodies via enhancement of their Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans ability to mediate cytotoxic effector functions such as ADCC 30:487-490; U.S. Pat. Nos. 5,624,821, 5,885,573 and PCT and CDC are particularly promising. The importance of publication Nos. WO 00/42072, WO 99/58572 and WO FcyR-mediated effector functions for the anti-cancer activity 04/029207). While the vast majority of Substitutions reduce of antibodies has been demonstrated in mice (Clynes et al., or ablate Fc binding with FcyRs some have resulted in 1998, Proc Natl AcadSci U.S.A. 95:652-656; Clynes et al., higher FcyR affinity. However, most of the methods dis 2000, Nat Med 6:443-446), and the affinity of the interaction closed resulted in only modest improvements in FcRyIIIA between Fc and certain FcyRs correlates with targeted binding and ADCC activity. The present invention provides cytotoxicity in cell-based assays (Shields et al., 2001, J Biol for the first time a modified Fc of antibody that immuno Chem 276:6591-6604; Presta et al., 2002, Biochem Soc Specifically binds to one or more Eph receptor that has Trans 30:487-490; Shields et al., 2002, J Biol Chem increased binding to FcRyIIIA binding, significant enhance 277.26733-26740). Together these data suggest that manipu ment in ADCC and does not show an increase in FcRyIIB lating the binding ability of the Fc region of an IgG1 binding. antibody to certain FcyRs may enhance effector functions resulting in more effective destruction of cancer cells in 0025 Citation or discussion of a reference herein shall patients. Furthermore, because FcyRS can mediate antigen not be construed as an admission that Such is prior art to the uptake and processing by antigen presenting cells, enhanced present invention. Fc/FcyR affinity may also improve the capacity of antibody 4. SUMMARY OF THE INVENTION therapeutics to elicit an adaptive immune response. 0026. The present invention provides novel antibodies 0022 While enhancing effector function can increase the comprising immunologically active fragments of immuno capacity of antibodies to destroy target cells, for Some globulin molecules and an Fc region that further comprises antibody therapies reduced or eliminated effector function at least one novel amino acid residue of the invention (also may be more desirable. This is particularly true for those referred to herein as “high effector function amino acid antibodies designed to deliver a drug (e.g., toxins and residue(s))”. Said novel antibodies are referred to herein as isotopes) to the target cell where the Fc/FcyR mediated “Fc variants of the invention' or “Fe variants' or alterna effector functions bring healthy immune cells into the proX tively, a “modified antibody.” Fc binding interactions are imity of the deadly payload, resulting in depletion of normal essential for a variety of effector functions and downstream lymphoid tissue along with the target cells (Hutchins et al., Signaling events including, but not limited to, antibody 1995, PNAS USA92: 11980-11984; White et al., 2001, Annu dependent cell-mediated cytotoxicity (ADCC) and comple Rev Med 52:125-145). In these cases the use of Fc variants ment dependent cytotoxicity (CDC). Accordingly, the inven that poorly recruit complement or effector cells would be of tion provides Fc variants that exhibit altered binding affinity tremendous benefit (see for example, Wu et al., 2000, Cell for at least one or more Fc ligands (e.g., FcyRs, C1q) relative Immunol 200:16-26; Shields et al., 2001, J. Biol Chem to an antibody having the same amino acid Sequence as the 276:6591-6604; U.S. Pat. No. 6,194.551; U.S. Pat. No. molecule of the invention but not comprising the novel 5,885,573 and PCT publication WO 04/029207). amino acids residues of the invention (referred to herein as 0023 All FcyRs bind the same region on the Fc of the a “comparable molecule') Such as, for example, an antibody IgG subclass, but with different affinities (e.g., FcyRI is a comprising an unmodified Fc region containing naturally high affinity while FcyRII and FcyRIII are low affinity occurring amino acid residues at the corresponding position binders. Other differences between the FcyRs are mechanis in the Fc domain. In addition, the present invention provides tic. For example, FcyRI, Fcy RIA/C, and FcyRIIIA are novel Fc variants comprising a variable region, or fragment positive regulators of immune complex triggered activation, thereof, that immunospecifically binds to one or more Eph characterized by having an immunoreceptor tyrosine-based receptor and at least one high effector function amino acid activation motif (ITAM) while FcyRIIB has an immunore residue. ceptor tyrosine-based inhibition motif (ITIM) and is there 0027. The present invention further provides Fc variants fore inhibitory. Thus, the balance between activating and of antibodies that immunospecifically bind to one or more US 2006/0039904 A1 Feb. 23, 2006
Eph receptor, Said Fc variants comprising an Fc region in further embodiment, the Fc variants of the invention are which at least one amino acid residue has been Substituted. variants of an antibody that immunospecifically binds to one It is specifically contemplated that Said Fc variants may be or more Eph receptor. generated by methods well known to one skilled in the art. 0031. In a specific embodiment, Fc variants of the inven Briefly, such methods include but are not limited to, com tion comprise an Fc region comprising at least one high bining a variable region with the desired specificity (e.g., a effector function amino acid residue Selected from the group variable region isolated from a phage display or expression consisting of: 234E, 235R, 235A, 235W, 235P, 235V, 235Y, library or derived from a human or non-human antibody) 236E, 239D, 265L, 269S, 269G, 298I, 298T, 298F, 327N, with an Fc region containing at least one high effector 327G, 327W, 328S, 328V,329H,329Q, 330K, 330V, 330G, function amino acid residue. Alternatively, one skilled in the 330Y, 330T, 330L, 330I, 330R, 330C, 332E, 332H, 332S, art may generate an Fc variant by Substituting at least one 332W, 332F, 332D, and 332Y, wherein the numbering amino acid residue in the Fc region of an antibody. system is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Infor 0028. The present invention also provides Fc variants that mation Service, Springfield, Va.). have altered binding affinity for one or more Fc ligands (e.g., FcyRs, C1q) relative to a comparable molecule (e.g., an 0032. In another specific embodiment, Fc variants of the antibody having an original unmodified Fc region). In one invention comprise an Fc region comprising at least one embodiment, the Fc variants have higher binding affinity to high effector function amino acid residue Selected from the activating FcyRS (e.g., FcyRIIIA) and/or unchanged or lower group consisting of: 239D, 330K, 330V, 330G, 330Y, 330T, binding affinity to inhibitory FcyRs (e.g., FcyRIIB) relative 330L, 330I, 330R, 330C, 332E, 332H, 332S,332W, 332F, to a comparable molecule (e.g., an antibody having an 332D, and 332Y wherein the numbering system is that of the original unmodified Fc region). The present invention fur EU index as set forth in Kabat. ther provides Fc variants with enhanced ADCC function 0033. In still another specific embodiment, Fc variants of relative to a comparable molecule (e.g., an antibody having the invention comprise an Fc region comprising at least one an original unmodified Fc region). In another embodiment, high effector function amino acid residue Selected from the the Fc variants of the invention have enhanced ability to group consisting of 239D, 330L and 332E. In one embodi mediate ADCC (“referred to herein as ADCC activity”) in ment, Fc variants of the invention comprise an Fc region addition to the above changes in FcyR affinities relative to a comprising at least the high effector function amino acid comparable molecule (e.g., an antibody having an original residue 332E. In a specific embodiment, Fc variants of the unmodified Fc region). In a further embodiment, the Fc invention comprise an Fc region comprising the high effec variants of the invention are variants of an antibody that tor function amino acid residues 239D, 330L and 332E. immunospecifically binds to one or more Eph receptor (e.g., 0034. In a one embodiment, the Fc variants comprise at EphA2 and EphA4). In another embodiment, the Fc variants least one amino acid Substitution at a position Selected from of the invention do not have significantly altered antigen the group consisting of: 206, 207, 208, 209, 210, 211, 212, binding Specificity. 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 0029. The present invention also provides Fc variants 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, have lower binding affinity to activating FcyRs (e.g., 237, 239, 242, 246, 250, 251, 257, 259, 260,261,265, 269, FcyRIIIA) and/or increased binding affinity to inhibitory 273, 274, 275,277,281,282,284, 287, 291, 298,300,302, FcyRs (e.g., FcyRIIB) relative to a comparable molecule 304,306, 308, 310,314,316,318, 319, 321,323,327,328, (e.g., an antibody having an original unmodified Fc region). 329, 330, 332 and 336, wherein the numbering of the The present invention further provides Fc variants with residues in the Fc region is that of the EU index as set forth decreased ADCC activity relative to a comparable molecule in Kabat. (e.g., an antibody having an original unmodified Fc region 0035) In a specific embodiment, the Fc variants comprise )original antibodies. In one embodiment, the Fc variants of at least one Substitution Selected from the group consisting the invention exhibit decreased ADCC activity in addition to of: L234E, L235R, L235A, L235W, L235P, L235V, L235Y, the above changes in FcyR affinities relative to a comparable G236E, S239D, D265L, E269S, E269G, S298I, S298T, molecule (e.g., an antibody having an original unmodified S298F, A327N, A327G, A327W, L328S, L328V, P329H, Fc region). In another embodiment, the Fc variants of the P329O, A330K, A330V, A330O, A330Y, A330T, A330L, invention are variants of an antibody that immunospecifi A330I, A330R, A330C, 1332E, 1332H, 1332S, 1332W, cally binds to one or more Eph receptor. In a further 1332F, 1332D, and 1332Y, wherein the numbering system is embodiment, the Fc variants of the invention do not have that of the EU index as set forth in Kabat. In one embodi Significantly altered antigen binding Specificity. ment, the Fc variants comprise at least one Substitution selected from the group consisting of S239D, A330L and 0030 The present invention additionally provides Fc 1332E. In another embodiment, the Fc variants comprise at variants that have altered binding affinity to the complement least each of the following substitutions, S239D, A330L and protein C1q relative to a comparable molecule (e.g., an 1332E. In a further embodiment, the Fc variants have at least antibody having an original unmodified Fc region). In one the amino acid Substitution 1332E. embodiment, the Fc variants have enhanced binding affinity to C1q and enhanced ability to mediate CDC (referred to 0036. It is an object of the present invention to provide Fc herein as “CDC activity”). In another embodiment, the Fc variants that bind with greater affinity to one or more Fc variants have reduced binding affinity to C1q and reduced ligand (e.g., FcyRs, C1q). In one embodiment, said variants CDC activity relative to a comparable molecule (e.g., an have an affinity for one or more Fc ligand (e.g., FcyRs, C1q) antibody having an original unmodified Fc region). In a that is at least 2 fold greater than that of a comparable US 2006/0039904 A1 Feb. 23, 2006 molecule (e.g., an antibody prior to Fc modification). In a dissociation constant (K) that is decreased between about further embodiment, the Fc variants of the invention have 2 fold and about 10 fold, or between about 5 fold and about affinity for an Fc ligand (e.g., FcyR, C1q that is between 2 50 fold, or between about 25 fold and about 250 fold, or fold and 500 fold greater than that of a comparable molecule between about 100 fold and about 500 fold, relative to a (e.g., an antibody prior to Fc modification). In one specific comparable molecule. In a further specific embodiment, an embodiment, an Fc variant of the invention has a greater Fc variant of the invention has an equilibrium dissociation affinity for FcyRIIIA. In another specific embodiment, an Fc constant (K) that is decreased between 2 fold and 10 fold, variant of the invention has a greater affinity for FcyRIIB. In or between 5 fold and 50 fold, or between 25 fold and 250 yet another specific embodiment, an Fc variant of the fold, or between 100 fold and 500 fold, relative to a invention has a greater affinity for C1q. comparable molecule. In another Specific embodiment, an Fc variant of the invention has a ratio of FcyRIIIA/FcyRIIB 0037. It is a further object of the present invention to equilibrium dissociation constants (K) that is decreased provide a Fc variants that bind with reduced affinity to one and enhanced ADCC activity relative to a comparable or more Fc ligand (e.g., FcyRs, C1q). In one embodiment, molecule. the Fc variants of the invention have affinity for one or more Fc ligand (e.g., FcyRs, C1q) that is between about 2 fold and 0041. In one embodiment, an Fc variant of the invention about 500 fold lower than that of a comparable molecule has an increased affinity for FcyRIIIA and an affinity for (e.g., an antibody prior to Fc modification). In another FcyRIIB that is unchanged or preferably reduced, an affinity embodiment, the Fc variants of the invention have affinity for C1q that is reduced and enhanced ADCC activity relative for one or more Fc ligand (e.g., FcyRs, C1q) that is between to a comparable molecule (e.g., an antibody prior to Fc 2 fold and 500 fold lower than that of a comparable molecule modification). (e.g., an antibody prior to Fc modification). In a specific embodiment, the Fc variants of the invention have an affinity 0042. In another embodiment, an Fc variant of the inven for FcyRIIB that is either unchanged, or more preferably tion has a decreased affinity for FcyRIIIA and an affinity for reduced. In another specific embodiment, the Fc variants of FcyRIIB that is increased and reduced ADCC activity rela the invention have an affinity for FcyRIIIA that is reduced. tive to a comparable molecule (e.g., an antibody prior to Fc In yet another embodiment, the Fc variants of the invention modification). In still another embodiment, an Fc variant of have an affinity for C1q that is reduced. the invention has a ratio of FcyRIIIA/FcyRIIB equilibrium dissociation constants (K) that is increased and reduced 0038. It is a further object of the present invention to ADCC activity relative to a comparable molecule. provide Fc variants that have enhanced ADCC and/or CDC activity. In one embodiment, Fc variants of the invention 0043. The binding properties of a receptor for its ligand, have ADCC and/or CDC activity that is at least about 2 fold may be determined by a variety of methods well-known in greater then that of a comparable molecule (e.g., an antibody the art, including but not limited to, equilibrium methods prior to Fc modification). In another embodiment, the Fc (e.g., enzyme-linked immunoabsorbent assay (ELISA) or variants of the invention have ADCC and/or CDC activity radioimmunoassay (RIA)), or kinetics (e.g., BIACORE(R) that is between about 2 fold and about 100 fold greater than analysis), and other methods Such as indirect binding assays, that of a comparable molecule. In a further embodiment, Fc competitive inhibition assays, fluorescence resonance variants of the invention have ADCC and/or CDC activity energy transfer (FRET), gel electrophoresis and chromatog that is at least 2 fold greater then that of a comparable raphy (e.g., gel filtration). These and other well-known molecule (e.g., an antibody prior to Fc modification). In yet methods may utilize a label on one or more of the compo a further embodiment, the Fc variants of the invention have nents being examined and/or employ a variety of detection ADCC and/or CDC activity that is between 2 fold and 100 methods including but not limited to chromogenic, fluores fold greater than that of a comparable molecule. cent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., 0039. It is a further object of the present invention to ed., Fundamental Immunology, 4" Ed., Lippincott-Raven, provide Fc variants that have reduced ADCC and/or CDC Philadelphia (1999), which focuses on antibody-immunogen activity. In one embodiment, Fc variants of the invention interactions. have ADCC and/or CDC activity that is at least 2 fold lower than that of a comparable molecule (e.g., an antibody prior 0044) The Fc variants of the present invention may be to Fc modification). In another embodiment, the Fc variants combined with other Fc modifications (e.g., other amino of the invention have ADCC and/or CDC activity that is acid Substitutions, altered glycosylation, etc.), including but between 2 fold and 100 fold lower than that of a comparable not limited to modifications that alter Fc ligand binding molecule. In a further embodiment, Fc variants of the and/or effector function. The invention encompasses com invention have ADCC and/or CDC activity that is at least bining an Fc variant of the invention with other Fc modifi about 2 fold lower than that of a comparable molecule (e.g., cations to provide additive, Synergistic, or novel properties an antibody prior to Fc modification). In another embodi in antibodies or Fc fusions. Preferably, the other Fc modi fications enhance the phenotype of the Fc variants of the ment, the Fc variants of the invention have ADCC and/or present invention (e.g., Fc variant comprising at least one CDC activity that is between about 2 fold and about 100 fold high effector function amino acid) with which they are lower than that of a comparable molecule. combined. For example, if an Fc variant (i.e., incorporating 0040. In one specific embodiment, an Fc variant of the a hinge modification of the invention) is combined with a invention has an increased affinity for FcyRIIIA and an mutant known to bind FcyRIIIA with a higher affinity than affinity for FcyRIIB that is unchanged or preferably reduced a comparable molecule comprising a wild type Fc region, and enhanced ADCC activity. In another specific embodi the combination results in a greater fold enhancement in ment, an Fc variant of the invention has an equilibrium FcyRIIIA affinity. US 2006/0039904 A1 Feb. 23, 2006
004.5 The invention encompasses molecules that com EphA4. In still another embodiment, the Fc variant of the prise homodimerS or heterodimers of Fc regions wherein at invention immunoreacts with more then one Eph receptor. In least one Fc region incorporates at least one high effector one embodiment, the Fc variant of the invention binds both function amino acid of the invention. Heterodimers com EphA4 and Epha2. In yet another embodiment, the Fc prising Fc regions refer to molecules where the two Fc variant of the invention binds and is an agonist of both chains have different Sequences. In Some embodiments, in EphA4 and Epha2. The Fc variant may have the same the heterodimeric molecules comprising an Fc region incor immunoreactivity for more than one Eph receptor or alter porating at least one high effector function amino acid and/or natively, the Fc variant may immunoreact more Strongly other Fc modification, each chain has one or more different with one Eph receptor versus another. It is specifically modifications from the other chain. In other embodiments, in contemplated that Fc variants of the invention may be the heterodimeric molecules comprising an Fc region incor bispecific antibodies. It is also contemplated that an Fc porating a hinge modification, one chain contains the wild variant of the invention may bind to a common epitope type Fc region and the other chains comprises one or more shared by more then one Eph receptor. modifications. Methods of engineering heterodimeric Fc containing molecules are known in the art and encompassed 0049. The present invention also encompasses Fc vari within the invention. ants with modified binding affinity to one or more Fc ligand (e.g., FcyRs, C1q) and altered ADCC and/or CDC activity 0046. In one embodiment, the Fc variants of the inven that immunospecifically bind to at least one Eph receptor tion with modified binding affinity to one or more Fc ligand conjugated or fused to a moiety (e.g., therapeutic agent or (e.g., FcyRs, C1q) and altered ADCC and/or CDC activity drug). immunospecifically bind to one or more Eph receptor. In another embodiment, Said Fc variants are antagonists of one 0050. The present invention also encompasses the use of or more Eph receptor. An antagonist of one or more Eph Fc variants with modified binding affinity to one or more Fc receptor is any molecule that blocks, inhibits, reduces or ligand (e.g., FcyRs, C1q) and altered ADCC and/or CDC neutralizes the function, activity and/or expression of one or activity that immunospecifically bind to at least one Eph more Eph receptor. Antagonists may act by interfering with receptor for the prevention, management, treatment or ame the binding of a receptor to a ligand and Vice versa, by lioration of Eph receptor-mediated (and/or ephrin-mediated) incapacitating or killing cells which have been activated by and/or associated diseases and disorders or one or more a ligand, and/or by interfering with receptor or ligand Symptoms thereof, including but not limited to cancer, activation (e.g., tyrosine kinase activation) or signal trans inflammatory and autoimmune diseases either alone or in duction after ligand binding to a cellular receptor. The combination with other therapies. The invention also antagonist may completely block receptor-ligand interac encompasses the use of Fc variants with modified binding tions or may Substantially reduce Such interactions. Thus, an affinity to one or more Fc ligand (e.g., FcyRS, C1q) and antagonist of an Eph receptor can block Eph receptor altered ADCC and/or CDC activity that immunospecifically Signaling. bind to at least one Eph receptor conjugated or fused to a moiety (e.g., therapeutic agent or drug) for preventing, 0047. In yet another embodiment, the Fc variants of the managing, treating or ameliorating Eph receptor-mediated invention are agonists of one or more Eph receptor. An and/or associated diseases and disorders or one or more agonist of an Eph receptor is any molecule that can increase Symptoms thereof, including but not limited to cancer, the activity, activation or function of an Eph receptor. inflammatory and autoimmune diseases either alone or in Agonists may, for example, act by activating a target mol combination with other therapies. ecule and/or mediating Signal transduction. In a preferred embodiment, Said Fc variant is a variant of an antibody that 0051. The invention further encompasses treatment pro immunospecifically binds one or more Eph receptor, Such as tocols that enhance the prophylactic or therapeutic effect of those described herein. Additional Eph receptor Specific Fc variants with altered binding affinity to to one or more Fc antibodies have been described in PCT Publication Nos. WO ligand (e.g., FcyRs, C1q) and altered ADCC and/or CDC 04/014292, WO 03/094859 and U.S. patent application Ser. activity that immunospecifically bind to at least one Eph No. 10/863,729 all of which are incorporated by reference receptor. herein in their entireties. 0052 The invention provides methods for the identifica 0.048. In one embodiment, an Fc variant of the invention tion and/or generation of antibodies that immunospecifically with modified binding affinity to one or more Fc ligand (e.g., bind to at least one Eph receptor. In addition, the invention FcyRs, C1q) and altered ADCC and/or CDC activity pref provides methods for the Screening and identification of erentially binds one or more Eph receptor (e.g., Eph A1, A2, antibodies that bind to at least one Eph receptor and are A3a, A3b, A4, A5a, A5b, A6, A7, A8, B1, B2a, B2b, B3, B4 either antagonists or agonists of at least one Eph receptor and B6) versus other receptor tyrosine kinases. In one including but not limited to assays that monitor Eph receptor embodiment, Eph receptors of the invention include but are activity (e.g., cell adhesion, angiogenesis, tumor cell growth not limited to those described in section 6.1 entitled “Fc and tumor progression) and/or plasma concentration. Fur Variants that Immunospecifically Bind to an Eph Receptor', ther, the invention provides for a method to manipulate both infra, and those shown in FIG. 18. In another embodiment, the ADCC and or CDC activity as well as the binding said Fc variant of the invention preferentially binds Eph A2 affinities for FcyR and/or C1q of antibodies identified using over other Eph receptors. In another embodiment, the Fc Such Screening methods. The antibodies identified and variant of the invention do not bind Eph A2. In another manipulated utilizing Such methods can be used for the embodiment, the Fc variant of the invention preferentially prevention, treatment, management or amelioration of Eph binds EphA4 over other Eph receptors. In yet another receptor-mediated diseases and disorders or one or more embodiment, an Fc variant of the invention does not bind Symptoms thereof, including but not limited to cancer, US 2006/0039904 A1 Feb. 23, 2006 inflammatory and autoimmune diseases either alone or in 0062 FIG. 9. Cell-based ADCC assay of Vitaxin(R) and combination with other therapies. the I332E (Vitaxin-1M) Fc variant using 50:1 ratio of 0053. The present invention provides kits comprising one effector to target cells at a variety of antibody concentrations or more Fc variants with modified binding affinity to to one from 0.4 to 1000 ng/ml. The I332E Fc variant shows higher or more Fc ligand (e.g., FcyRs, C1q) and altered ADCC ADCC activity over a wide range of antibody concentra and/or CDC function that immunospecifically bind to at tions. least one Eph receptor conjugated or fused to a detectable 0063 FIG. 10. Cell-based ADCC assay of Vitaxin(R) and agent, therapeutic agent or drug, in one or more containers, the I332E (Vitaxin-1M) Fc variant using different ratios of for use in the prevention, treatment, management, amelio effector to target cells and different amounts of antibody ration, detection, monitoring or diagnosis of Eph receptor ranging from 2.5 ng to 200 ng per well. The I332E Fc variant mediated and/or associated diseases and disorders including shows higher ADCC activity over a wide range of antibody but not limited to cancer, inflammatory and autoimmune concentrations at all E:T ratioS. diseases. 0064 FIG. 11. Cell-based ADCC assay of Vitaxin(R) and 5. BRIEF DESCRIPTION OF THE FIGURES the Vitaxin S239D/A330L/I332E (Vitaxin-3M) Fc variant 0.054 FIG. 1. The nucleotide and deduced amino acid against Several target cell lines expressing different levels of sequence of the variable region of the antibody Vitaxin(R), Integrin CVB3, A498 (moderate), DU145 (low), M21 (high) (A) heavy chain variable region (SEQ ID NO: 1 and SEQID and ACHN (moderate), using two different E:T ratios and NO:3, respectively) (B) light chain variable region (SEQID antibody amounts ranging from 4 ng to 400 ng per well. In NO: 2 and SEQ ID NO: 4, respectively). The CDRs are all cases the S239D/A330L/I332E (Vitaxin-3M) Vitaxin Fc underlined. variant shows higher ADCC activity. 0055 FIG. 2. The nucleotide and deduced amino acid 0065 FIG. 12. ELISA analysis of the wild type anti Sequence of the variable region of the anti-EphA2 antibody EphA2 antibody 3F2 and the 3F2 I332E (3F2-1M) and 3F2 12G3H11 (abbreviated “12G3) (A) heavy chain variable S239D/A330L/I332E (3F2-3M) Fc variants binding to region (SEQ ID NO: 62 and SEQ ID NO: 64, respectively) FcyRIIIA tetramer (panel A), FcyRIIIA monomer (panel B) (B) light chain variable region (SEQ ID NO: 63 and SEQID and C1q (panel C). Both the 3F2-1M and 3F2-3M Fc NO: 65, respectively). The CDRs are underlined. variants bind better to FcyRIIIA monomers and tetramers, 0056 FIG. 3. The nucleotide and deduced amino acid although the 3F2-3M Fc variant binds the monomer signifi Sequence of the variable region of the anti-EphA2 antibody cantly better then either the wild type antibody or 3F2-1M 3F2 (A) heavy chain variable region (SEQ ID NO: 66 and Fc variant. In contrast both the 3F2-1M and 3F2-3M Fc SEQID NO: 68, respectively) (B) light chain variable region variants did not bind C1q to the same degree as the wild type (SEQ ID NO: 67 and SEQ ID NO: 69, respectively). The antibody with the 3M Fc variant showing the largest CDRS are underlined. decrease in binding. 0057 FIG. 4. Map of the expression plasmid used for the 0.066 FIG. 13. FACS analysis of anti-EphA2 antibody production of full length IgGs. SmaI/BsiWI restriction sites 3F2-WT, 3F2-1M and 3F2-3M binding to cells via Fc used to clone the light chain variable region, Xbal/Apal domain interactions. THP-1 and NK cells were stained with restriction sites used to clone variable region of heavy chain antibodies to FcyRI, FcyRII and FcyRIII (also commonly and Apal/Not restriction Sites were used to replace the referred to CD64, CD32 and CD16, respectively). THP-1 constant region of the heavy chain. cells have high levels of CD32 on their cell Surface, mod erate levels of CD64 and very low levels of CD16 (panel A). 0.058 FIG. 5. Screening of Vitaxin Fc variant clones by NK cells however show the opposite profile, high levels of characterizing their relative binding to FcyRIIIA compared CD16 and low levels of CD32 and CD64 (panel B). All three to parental sclv-Fc as determined by ELISA. Numerous versions of 3F2 (wt, 1M and 3M) bound to a similar degree clones were seen to have improved binding. to THP-1 cells (panel C). However, the variants were seen 0059 FIG. 6. Relative ADCC activity of several Vitaxin to bind to a greater extent to NK cells, with the 3F2-3M Fc Fc variant clones against M21 cells as determined by a variant showing the largest increase in binding (panel D). cell-based assay. Several Fc variants, including I332E, 0067 FIG. 14. Cell-based ADCC assay of 12C3H11 showed improved ADCC activity relative to the parental (anti-EphA2 antibody) and its I332E Fc variant using 50:1 ScFV-Fc. ratio of effector to A549 target cells (panel A) and a similar 0060 FIG. 7. All 20 amino acids were substituted at study using two different E:T ratios from (panel B). In both position 332 of Vitaxin. The relative binding affinities of Studies the amount of antibody ranged from 4 ng to 400 ng each position 332 Fc variant to FcyRIIIA was determined by per well. The I332E Fc variant shows higher ADCC activity ELISA (panel A). The relative ADCC activity of each over a wide range of antibody concentrations at all E:T position 332 Fc variant was determined by a cell-based ratioS. assay (panel B). The I322E Fc variant was seen to provide 0068 FIG. 15. Cell-based ADCC assay of anti-EphA2 the greatest improvement in both binding and in ADCC antibody 3F2 and the 3F2-1M and 3F2-3M Fc variants to activity. target cells expressing high (T231, A549) levels of EphA2. 0061 FIG. 8. Binding of Vitaxin(R) and the I332E In each assay the antibody concentration ranged from 0.02 (Vitaxin-1M) Fe variant to FcyRIIIA(A) and FcyRIIB (B) as ug/ml to 2 tug/ml. E:T ratios varied from 12.5:1 to 100:1 determined by ELISA. The binding of Vitaxin-1M Fc vari depending on the assay. The 3F2-3M Fc variant was seen to ant to Fc FcyRIIIA is improved while the binding to FcyRIIB have the highest ADCC activity against all cell types. appears unchanged. Although the 3F2-1M Fc variant showed higher ADCC US 2006/0039904 A1 Feb. 23, 2006 activity against more cell types than the 3F2 wild type, it was Sequence as the molecule of the invention but not compris generally not as active as the 3F2-3M Fc variant. ing the novel amino acids residues of the invention (referred 0069 FIG. 16. Cell-based ADCC assay of anti-EphA2 to herein as a “comparable molecule') Such as an antibody antibody 3F2-WT, 3F2-1M and 3F2-3M Fc variants to target comprising an unmodified Fc region containing naturally cells expressing high (Hey8) and moderate (SKOV3) levels occurring amino acid residues at the corresponding position of EphA2. The antibody concentration and E:T ratios are the in the Fc domain. In particular, the present invention pro same as for FIG. 15. The 3F2-3M Fc variant was seen to vides Fc variants comprising a variable region, or fragment have the highest ADCC activity against all cell types. thereof, that immunospecifically binds to at least one Eph Although the 3F2-1M Fc variant showed higher ADCC receptor and a Fc region that further comprises at least one activity against most cell types than the 3F-WT, it was high effector function amino acid residue. generally not as active as the 3F2-3M Fc variant. 0074 The present invention further provides Fc variants of antibodies that immunospecifically bind to at least one 0070 FIG. 17. Cell-based ADCC assay of anti-EphA2 Eph receptor, Said Fc variants comprising an Fc region in antibody 3F2, 3F2-1M and 3F2-3M Fc variants to target which at least one amino acid residue has been Substituted. cells expressing low (A498, SKMEL28) levels of EphA2. The present invention also relates to Fc variants with altered The SKMEL28 cells express Integrin C.VB5 as were also binding affinity to their FcyRs compared to that of a com used as target cells for the Vitaxin and Vitaxin-3M antibod parable molecule (e.g., an antibody having an original ies. The antibody concentration and E.T ratios are the same unmodified Fc region). In one embodiment, the Fc variants as for FIG. 15. None of the 3F2 antibodies were seen to have have higher binding affinity to activating FcyRs (e.g., activity against SKMEL28 cells. Although both Vitaxin and FcyRIIIA). In a specific embodiment, the Fc variants of the the Vitaxin-3M antibodies had activity, the Vitaxin-3M Fc invention have equilibrium dissociation constants (K) that variant was significantly more active. are decreased relative to a comparable molecule. In another 0071 FIG. 18A-D. Alignment of the currently known embodiment the Fc variants have higher binding affinity to human Eph family of receptors. The following protein activating FcyRS and unchanged or lower binding affinity to motifs are underlined as described in the key: Signal inhibitory FcyRs (e.g., FcyRIIB). In a further embodiment, Sequence; ephrin receptor ligand binding domain; fibronec are Fc variants which have a ratio of FcyRIIIA/FcyRIIB tin type III domain; transmembrane domain; tyrosine kinase equilibrium dissociation constants (K) that are decreased catalytic domain; SAM domain. relative to a comparable molecule. In yet a further embodi ment, the Fc variants of the invention also exhibit increased 0072 FIG. 19. Alignment of the currently known human ADCC activity when compared to a comparable molecule Ephrin family of Eph receptor ligands. The following pro (e.g., an antibody having an original unmodified Fc region) tein motifs are underlined as described in the key: Signal in addition to the above changes in FcyR affinities. In Sequence; Ephrin domain; transmembrane domain (B-fam another embodiment, the Fc variants of the invention are ily only). variants of an antibody that immunospecifically binds to at least one Eph receptor (e.g., EphA1, A2, A3a, A3b, A4, A5a, 6. DETAILED DESCRIPTION OF THE A5b, A6, A7, A8, B 1, B2a, B2b, B3, B4 and B6). In a INVENTION Specific embodiment, the Fc variants of the invention immu 0073. The present invention provides certain amino acids nospecifically bind least one Eph receptor and are antago residues in the Fc region of an IgG antibody that correlate nists of at least one Eph receptor. In another specific with high effector function. Further, the invention provides embodiment, the Fc variants of the invention immunospe high effector function residues in the Fc region of an cifically bind least one Eph receptor and are agonists of at antibody which exhibit high binding affinity for the Fc least one Eph receptor. receptor, FcyRIIIA. In further embodiments, the invention 0075. The antibodies of the present invention may be encompasses the introduction of at least one of the high produced “de novo' by combining a variable domain, or effector amino acid residues of the invention that does not fragment thereof, that immunospecifically binds at least one result in a concomitant increase in binding the FcyRIIB Eph receptor with an Fc domain comprising one or more of receptor. In another embodiment, the invention encompasses the high effector function residues disclosed herein, or may the introduction of at least one of the high effector amino be produced by modifying an Fc domain-containing anti acid residues of the invention that results in a concomitant body that binds an at least one Eph receptor by introducing decrease in binding the FcyRIIB receptor and/or C1q. In still one or more high effector function and/or other modulatory another embodiment, the introduction of at least one of the amino acid residues into the Fc domain. high effector amino acid residues of the invention that results in a concomitant increase in binding to both the FcyRIIIA 0076. The present invention also relates to novel Fc and FcyRIIB receptors. In a preferred embodiment, the ratio variants with a higher binding affinity to inhibitory FcyRs of FcyRIIIA/FcyRIIB equilibrium dissociation constants and a lower binding affinity to activating FcyRS (e.g., (K), is decreased. Furthermore, the presence of at least one FcyRIIIA) reative to a comparable molecule (e.g., an anti of the high effector amino acid residue of the invention body having an original unmodified Fc region). In one results in antibodies with an enhanced antibody dependent embodiment, said Fc variants will also exhibit a reduced cell-mediated cytotoxicity (ADCC) activity. Accordingly, ability to mediate ADCC activity compared to a comparable the invention provides Fc variants that exhibit altered effec molecule (e.g., an antibody having an original unmodified tor function (e.g., ADCC, CDC, etc.) and/or altered binding Fc region). In another embodiment, the Fc variants of the affinity for at least one Fc ligand (e.g., FcyRIIIA, FcyRIIB, invention are variants of an antibody that immunospecifi C1q, etc.) relative to an antibody (or other Fc-domain cally binds to at least one Eph receptor. In a specific containing polypeptide) having the same amino acid embodiment, the Fc variants of the invention with a higher US 2006/0039904 A1 Feb. 23, 2006
binding affinity to inhibitory FcyRs and a lower binding EphA4 is not bound to ligand, either as a result of decreased affinity to activating FcyRS immunospecifically bind least cell-cell contacts, altered Subcellular localization, or one Eph receptor and are Antagonists of at least one Eph increases in amount of EphA4 relative to EphA4-ligand. In receptor. In another Specific embodiment, the Fc variants of yet a further embodiment, the hyperproliferative cells over the invention with a higher binding affinity to inhibitory express EphA2. In another embodiment, Some EphA2 is not FcyRs and a lower binding affinity to activating FcyRs bound to ligand, either as a result of decreased cell-cell immunospecifically bind least one Eph receptor and are Eph contacts, altered Subcellular localization, or increases in receptor agonists. amount of EphA2 relative to EphA2-ligand. 0077. In addition, the present invention further provides 0081. As used herein, the terms “antibody' and “anti novel Fc variants with altered binding to C1q relative to a bodies' refer to monoclonal antibodies, multispecific anti comparable molecule (e.g., an antibody having an original bodies, human antibodies, humanized antibodies, camelised unmodified Fc region). Specifically, the Fc variants of the antibodies, chimeric antibodies, single-chain FVS (ScFv), invention may exhibit a higher binding affinity for C1q and disulfide-linked FVs (sdFv), Fab fragments, F(ab') frag increased CDC activity. Alternatively, the Fc variants of the ments, and anti-idiotypic (anti-Id) antibodies (including, invention may exhibit a lower binding affinity for C1q and e.g., anti-Id antibodies to antibodies of the invention), and reduced CDC activity. In other situations, the Fc variants of epitope-binding fragments of any of the above. In particular, the invention with altered binding to C1q exhibit CDC antibodies include immunoglobulin molecules and immu activity that is unchanged relative to a comparable molecule. nologically active fragments of immunoglobulin molecules, It is specifically contemplated that Fc variants with alter i.e., molecules that contain an antigen binding site, these ations in C1q binding and CDC activity may also exhibit fragments may or may not be fused to another immunoglo alterations in binding to one or more FcyRs and/or ADCC bulin domain including but not limited to, an Fc region or activity. In one embodiment, the Fc variants of the invention fragment thereof. As outlined herein, the terms “antibody” are variants of an antibody that immunospecifically binds to and “antibodies' specifically include the Fc variants at least one Eph receptor. In a Specific embodiment, the Fc described herein, full length antibodies and variant Fc variants of the invention altered binding to C1q immuno Fusions comprising Fc regions, or fragments thereof, com Specifically bind at least one Eph receptor and are antago prising at least one novel amino acid residue described nists of at least one Eph receptor. herein fused to an immunologically active fragment of an immunoglobulin or to other proteins as described herein. 0078. In a further specific embodiment, the Fc variants of Such variant Fc fusions include but are not limited to, the invention altered binding to C1q immunospecifically scFv-Fc fusions, variable region (e.g., VL and VH)-Fc bind at least one Eph receptor and are agonists of at least one fusions, scFv-scFv-Fc fusions. Immunoglobulin molecules Eph receptor. can be of any type (e.g., IgG, IgE, IgM, Ig|D, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or 007.9 The Fc variants of the invention may be useful to Subclass. prevent, treat, or manage metastasis of tumors or the inhi bition of other functions mediated or influenced by an Eph 0082 As used herein, the term “Eph receptor(s)" encom receptor, including but not limited to cell proliferation, cell passes a family of polypeptides comprising proteins that are attachment, cell migration, granulation tissue development, defined by a certain degree of homology to the known Eph tumor growth, tumor cell invasion and/or inflammation. receptor tyrosine kinases (RTKs). Eph receptors include, but Although not intending to be bound by any mechanism of are not limited to Eph A1 (also known as ephrin type-A action, Fc variants of the invention that immunospecifically receptor 1, erythropoietin-producing hepatoma amplified bind to at least one Eph receptor may function through a Sequence and exemplified by GenBank Acc. No. variety of mechanisms including but not limited to, targeting NP 005223.2), EphA2 (also known as epithelial cell recep a cell aberrantly expressing an Eph receptor for destruction, tor protein tyrosine kinase and exemplified by GenBank or acting as an agonist or antagonist of one or more Eph Acc. No. NP 004422.2), EphA3 (also known as human receptor activity. Also encompassed by the invention are Fc embryo kinase 1, eph-like tyrosine kinase 1, TYRO4 protein variants that inhibit or stimulate the functional activity of at tyrosine kinase and exemplified by GenBank Acc. Nos. least one Eph receptor resulting in the inhibition of Eph NP 005224.2 and NP 872585.1, isoforms 3a and 3b receptor-mediated pathologies. Agonistic antibodies of respectively), EphA4 (also known as ephrin type-A receptor 4, TYRO1 protein tyrosine kinase, tyrosine-protein kinase EphA2 and EphA4 have been described in PCT Publication receptor SEK, receptor protein-tyrosine kinase HEK8 and Nos. WO 04/014292, WO 03/094859 and U.S. patent appli exemplified by GenBank Acc. No. NP 004429.1), EphA5 cation Ser. No. 10/863,729, all of which are incorporated by (also known as Eph homology kinase-1, ephrin type-A reference herein in their entireties. receptor 5, receptor protein-tyrosine kinase HEK7, tyrosine 0080. In other embodiments, the Fc variants of the inven protein kinase receptor EHK-1 and exemplified by GenBank tion that immunospecifically bind an Eph receptor are used Acc. Nos. NP 004430.2 and NP 872272 isoforms. 5a and to treat, prevent and/or manage a disease or disorder asso 5b respectively), EphA6 (exemplified by GenBank Acc. No. ciated with cell hyperproliferation, Such as but not limited to XP 114973.4), EphA7 (also known as Eph homology cancer, asthma, chronic obstructive pulmonary disease, kinase-3, ephrin type-A receptor 7, receptor protein-tyrosine inflammatory diseases of the bowel, intestine, Stomach, and kinase HEK11, tyrosine-protein kinase receptor EHK-3 and other vital organs, restenosis (Smooth muscle and/or endot exemplified by GenBank Acc. No. NP 004431.1), EphA8 helial), Crohn's disease, psoriasis, and other non-metastatic (also known as tyrosylprotein kinase, protein-tyrosine diseases. In one embodiment, the hyperproliferative cells are kinase, hydroxyaryl-protein kinase, ephrin type-A receptor 8 epithelial. In another embodiment, the hyperproliferative precursor, eph- and elk-related tyrosine kinase, tyrosine cells overexpress EphA4. In a further embodiment, Some protein kinase receptor eek and exemplified by GenBank US 2006/0039904 A1 Feb. 23, 2006
Acc. No. NP 065387.1), EphB1 (also known as eph affinity of an antibody for its FcyRs and/or the complement tyrosine kinase 2 and exemplified by GenBank Acc. No. protein C1q by modulating one or more of the factors that NP 004432.1), Eph B2 (also known as eph tyrosine kinase regulate protein-protein interactions (e.g., receptor-ligand 3, elk-related tyrosine kinase, developmentally-regulated and antibody-antigen interactions). Such factors include but eph-related tyrosine kinase and exemplified by GenBank are not limited to, factors affecting protein folding or three Acc. Nos. NP 059145.1 and NP 004.433.2 isoforms 2a dimensional configuration Such as hydrogen bonds, hydro and 2b respectively), Eph B3 (also known as human embryo phobic interactions, ionic interactions, Von der Waals forces kinase 2, EPH-like tyrosine kinase-2 and exemplified by and/or disulfide bonds as well as factors affecting allosteric GenBank Acc. No. NP 004434.2), EphB4 (also known as hepatoma transmembrane kinase and exemplified by Gen interactions, Solubility and covalent modifications. Bank Acc. No. NP 004.435.3) and B6 (exemplified by 0086. Without wishing to be bound by any particular GenBank Acc. No. NM 004445.1). An alignment of several theory, the amino acid Substitutions of the invention modu Eph receptor polypeptides contemplated by the present late the ADCC and/or CDC activity of an antibody by invention is shown in FIG. 18. altering one more of the factors that influence downstream 0.083 AS used herein, the term “immunospecifically effector function including but not limited to, the affinity of binds to an Eph receptor' and analogous terms refer to the antibody for its FcyRs and/or to C1q, ability to mediate peptides, polypeptides, proteins, fusion proteins and anti cytotoxic effector and/or complement cascade functions, bodies or fragments thereof that Specifically bind to at least protein stability, antibody half life and recruitment of effec one Eph receptor or a fragment thereof. A peptide, polypep tor cells and/or molecules. tide, protein, or antibody that immunospecifically binds to at 0087. It will be understood that Fc region (also referred least one Eph receptor or a fragment thereof may bind to to herein as “Fc' and “Fc polypeptide') as used herein other peptides, polypeptides, or proteins with lower affinity includes the polypeptides comprising the constant region of as determined by, e.g., immunoassays, BIAcore, or other an antibody excluding the first constant region immunoglo assays known in the art. Antibodies or fragments that bulin domain. Thus Fc refers to the last two constant region immunospecifically bind to at least one Eph receptor or a immunoglobulin domains of IgA, Ig), and IgG, and the last fragment thereof may be croSS-reactive with related anti three constant region immunoglobulin domains of IgE and gens. Preferably, antibodies or fragments that immunospe IgM, and the flexible hinge N-terminal to these domains. For cifically bind to at least one Eph receptor or a fragment IgA and IgM Fc may include the J chain. For IgG, Fc thereof preferentially bind to at least one Eph receptor over comprises immunoglobulin domains Cgamma2 and other antigens. However, the present invention Specifically Cgamma3 (Cy2 and Cy3) and the hinge between Cgamma1 encompasses antibodies with multiple specificities (e.g., an (CY1) and Cgamma2 (Cy2). Although the boundaries of the antibody with Specificity for two or more discrete antigens Fc region may vary, the human IgG heavy chain Fc region (reviewed in Cao et al., 2003, Adv Drug Deliv Rev 55:171; is usually defined to comprise residues C226 or P230 to its Hudson et al., 2003, Nat Med 1:129)) in the definition of an carboxyl-terminus, wherein the numbering is according to antibody that “immunospecifically binds to an Eph recep the EU index as in Kabat et al. (1991, NIH Publication tor.” For example, bispecific antibodies contain two different 91-3242, National Technical Information Service, Spring binding Specificities fused together. In the Simplest case a field, Va.). The “EU index as set forth in Kabat” refers to the bispecific antibody would bind to two adjacent epitopes on residue numbering of the human IgG1 EU antibody as a single target antigen, Such an antibody would not croSS described in Kabat et al. Supra. Fc may refer to this region react with other antigens (as described Supra). Alternatively, in isolation, or this region in the context of an antibody, bispecific antibodies can bind to two different antigens. Such antibody fragment, or Fc fusion protein. Note: Polymor an antibody immunospecifically binds to two different mol phisms have been observed at a number of Fc positions, ecules, but not to other unrelated molecules. Another class of including but not limited to Kabat 270, 272,312,315, 356, multispecific antibodies may recognize a shared Subunit of and 358, and thus slight differences between the presented multi-Subunit complexes in the context of one or more Sequence and Sequences in the prior art may exist. Specific complexes. In addition, an antibody that immuno Specifically binds an Eph receptor may croSS-react with 0088. It will be understood that the complementarity related Eph receptors or RTKs. determining regions (CDRS) residue numbers referred to 0084 Antibodies or fragments that immunospecifically herein are those of Kabat et al. (1991, NIH Publication bind to an Eph receptor or a fragment thereof can be 91-3242, National Technical Information Service, Spring identified, for example, by immunoassays, BIAcore, or other field, Va.). Specifically, residues 24-34 (CDR1), 50-56 techniques known to those of skill in the art. An antibody or (CDR2) and 89-97 (CDR3) in the light chain variable fragment thereof binds Specifically to an Eph receptor or a domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 fragment thereof when it binds to an Eph receptor or a (CDR3) in the heavy chain variable domain. Note that CDRs fragment thereof with higher affinity than to any croSS vary considerably from antibody to antibody (and by defi reactive antigen as determined using experimental tech nition will not exhibit homology with the Kabat consensus niques, Such as radioimmunoassays (RIA) and enzyme Sequences). Maximal alignment of framework residues fre linked immunosorbent assays (ELISAS). See, e.g., Paul, ed., quently requires the insertion of “spacer” residues in the 1989, Fundamental Immunology Second Edition, Raven numbering system, to be used for the Fv region. It will be Press, New York at pages 332-336 for a discussion regarding understood that the CDRS referred to herein are those of antibody Specificity. Kabat et al. Supra. In addition, the identity of certain individual residues at any given Kabat Site number may vary 0085. Without wishing to be bound by any particular from antibody chain to antibody chain due to interspecies or theory, the amino acid Substitutions of the invention alter the allelic divergence. US 2006/0039904 A1 Feb. 23, 2006
0089. In one embodiment, Fc variants of the invention 0096. In another embodiment, the Fc variants comprise at will have at least one amino acid Substitution of the Fc least at least 2, or at least 3, or at least 4, or at least 5, or at region wherein Said antibody variant has a modified binding least 6, or at least 10, or at least 20, or at least 30, or at least affinity for its FcyRs and/or for C1q relative to a comparable 40, or at least 50, or at least 60, or at least 70, or at least 80, molecule (e.g., the original antibody without said Substitu or at least 90, or at least 100, or at least 200 amino acid tion). Substitutions of the Fc region. 0090. In a specific embodiment, Fc variants comprise an Fc region comprising at least one high effector function TABLE 1. amino acid reside Selected from the group consisting of: 234E, 235R, 235A, 235W, 235P, 235V, 235Y, 236E, 239D, Preferred Amino Acid Residues with High Effector Function (HEF 265L, 269S, 269G, 298I, 298T, 298F, 327N, 327G, 327W, Position Amino Acid HEF Residue(s) 328S, 328V, 329H,329Q, 330K, 330V,330G, 330Y, 330Y, 234 L E 330T, 330L, 330I, 330R, 330C, 332E, 332H, 332S,332W, 235 L R, A, W, P. V. Y 332F, 332D, and 332Y, wherein the numbering system is that 236 G E of the EU index as set forth in Kabat. Contemplated high 239 S D effector function amino acid residues of the invention are 265 D L 269 E S, G also set forth in Table 1. 298 S I, T, F 327 A. N, G, W 0.091 In another embodiment, the Fc variants comprise 328 L S, V an Fc region comprising at least 2, or at least 3, or at least 329 P H, Q 4, or at least 5, or at least 6, or at least 10, or at least 20, or 330 A. K, V, G, Y, T, L, I, at least 30, or at least 40, or at least 50, or at least 60, or at R, C least 70, or at least 80, or at least 90, or at least 100, or at 332 I E, H, S, W, F, Y, D least 200 high effector function amino acid residues. "heavy chain position number and amino acid residue amino acid residue present in naturally occurring antibody 0092. In another specific embodiment, Fc variants of the residues that can be engineered into corresponding position to generate an invention comprise an Fc region comprising at least one Fc region with high effector function. high effector function amino acid residue Selected from the group consisting of: 239D, 330K, 330V, 330G, 330Y, 330T, 0097. In one embodiment, the Fc variants comprise at 330L, 330I, 330R, 330C, 332E, 332H, 332S,332W, 332F, least one Substitution Selected from the group consisting of 332D, and 332Y, wherein the numbering system is that of S239D, A330L and I332E. In another embodiment, the Fc the EU index as set forth in Kabat. variants comprise at least each of the following Substitu 0093. In still another specific embodiment, Fc variants of tions, S239D, A330L and I332E. In a further embodiment, the invention comprise an Fc region comprising at least one the Fc variants of the invention have at least the high effector high effector function amino acid residue Selected from the amino acid 332E. group consisting of 239D, 330L and 332E. In one embodi ment, Fc variants of the invention comprise an Fc region 0098. It is specifically contemplated that conservative comprising at least the high effector function amino acid amino acid Substitutions may be made for Sa amino acid residue 332E. In a specific embodiment, Fc variants of the substitutions in the Fc of the antibody of interest, described invention comprise an Fc region comprising the high effec supra (see Table 1). It is well known in the art that “con tor function amino acid residues 239D, 330L and 332E. Servative amino acid Substitution” refers to amino acid Substitutions that Substitute functionally-equivalent amino 0094. In a specific embodiment, Fc variants will have one acids. Conservative amino acid changes result in Silent or more amino acid Substitutions at positions Selected from changes in the amino acid Sequence of the resulting peptide. the group consisting of 206, 207, 208, 209, 210, 211, For example, one or more amino acids of a similar polarity 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, act as functional equivalents and result in a Silent alteration 224, 225, 226, 227, 228, 229, 230, 231, 232,233,234, 235, within the amino acid Sequence of the peptide. Substitutions 236, 237,239, 242, 246, 250, 251, 257, 259,260, 261, 265, that are charge neutral and which replace a residue with a 269, 273, 274, 275, 277, 281, 282, 284, 287, 291, 298,300, Smaller residue may also be considered “conservative Sub 302,304,306, 308, 310,314,316, 318, 319,321, 323,327, Stitutions' even if the residues are in different groups (e.g., 328, 329, 330, 332 and 336, of the Fc region wherein the replacement of phenylalanine with the Smaller isoleucine). numbering of the residues in the Fc region is that of the EU Families of amino acid residues having Similar side chains index as set forth in Kabat. have been defined in the art. Several families of conservative 0.095. In another specific embodiment, the Fc variants amino acid Substitutions are shown in Table 2. comprise at least one Substitution Selected from the group consisting of: L234E, L235R, L235A, L235W, L235P, TABLE 2 L235V, L235Y, G236E, S239D, D265L, E269S, E269C; S298I, S298T, S298F, A327N, A327G, A327W, L328S, Families of Conservative Amino Acid Substitutions L328V, P329H, P329Q, A330K, A330V, A330G, A330Y, Family Amino Acids A330T, A330L, A330I, A330R, A330C, I332E, I332H, non-polar Trp, Phe, Met, Leu, Ile, Val, I332S, I332W, I332F, I332D, and I332Y, wherein the num Ala, Pro bering system is that of the EU index as set forth in Kabat. uncharged polar Gly, Ser, Thr, Asn., Gln, Tyr, Specific amino acid Substitutions of the invention are also Cys set forth in Table 1. US 2006/0039904 A1 Feb. 23, 2006
binding assays, competitive inhibition assays, fluorescence TABLE 2-continued resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other meth Families of Conservative Amino Acid Substitutions ods may utilize a label on one or more of the components Family Amino Acids being examined and/or employ a variety of detection meth ods including but not limited to chromogenic, fluorescent, acidic/negatively charged Asp, Glu basic/positively charged Arg, Lys, His luminescent, or isotopic labels. A detailed description of Beta-branched Thr, Val, Ile binding affinities and kinetics can be found in Paul, W. E., residues that influence chain orientation Gly, Pro ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, aromatic Trp, Tyr, Phe, His Philadelphia (1999), which focuses on antibody-immunogen interactions. 0099] The term “conservative amino acid substitution” 0103) In one embodiment, the Fc variants of the inven also refers to the use of amino acid analogs or variants. tion bind FcyRIIIA with increased affinity relative to a Guidance concerning how to make phenotypically Silent comparable molecule. In another embodiment, the Fc Vari amino acid substitutions is provided in Bowie et al., “Deci ants of the invention bind FcyRIIIA with increased affinity phering the Message in Protein Sequences: Tolerance to and bind FcyRIIB with a binding affinity that is unchanged Amino Acid Substitutions.” (1990, Science 247: 1306-1310). when relative to a comparable molecule. In Still another embodiment, the Fc variants of the invention bind FcyRIIIA 0100. In another embodiment, the Fc variants have at with increased affinity and bind FcyRIIB with a decreased least the high effector amino acid 332D. affinity relative to a comparable molecule. In yet another 0101 One skilled in the art will understand that that the embodiment, the Fc variants of the invention have a ratio of Fc variants of the invention may have altered FcyR and/or FcyRIIIA/FcyRIIB equilibrium dissociation constants (K) C1q binding properties (examples of binding properties that is decreased relative to a comparable molecule. include but are not limited to, binding specificity, equilib 0104. In a preferred embodiment, the Fc variants of the rium dissociation constant (K), dissociation and associa invention bind FcyRIIIA with increased affinity and bind tion rates (Koff and Kon respectively), binding affinity FcyRIIB with a decreased affinity when relative to a com and/or avidity) and that certain alterations are more or less parable molecule and immunospecifically bind an Eph desirable. It is well known in the art that the equilibrium receptor. dissociation constant (KD) is defined as kirk. It is gen erally understood that a binding molecule (e.g., and anti 0105. In one embodiment, said Fc variants bind with body) with a low K is preferable to a binding molecule increased affinity to FcyRIIIA. In another embodiment said (e.g., and antibody) with a high K. However, in Some Fc variants have affinity for FcyRIIIA that is at least 2 fold, instances the value of the k or k may be more relevant or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least than the value of the K. One skilled in the art can determine 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 which kinetic parameter is most important for a given fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, antibody application. For example a modification that or at least 80 fold, or at least 90 fold, or at least 100 fold, or enhances Fc binding to one or more positive regulators (e.g., at least 200 fold greater than that of a comparable molecule. FcyRIIIA) while leaving unchanged or even reducing Fc In a further embodiment said Fc variants have affinity for binding to the negative regulator FcyRIIB would be more FcyRIIIA that is at least about 2 fold, or at least about 3 fold, preferable for enhancing ADCC activity. Alternatively, a or at least about 5 fold, or at least about 7 fold, or a least modification that reduced binding to one or more positive about 10 fold, or at least about 20 fold, or at least about 30 regulator and/or enhanced binding to FcyRIIB would be fold, or at least about 40 fold, or at least about 50 fold, or at preferable for reducing ADCC activity. Accordingly, the least about 60 fold, or at least about 70 fold, or at least about ratio of binding affinities (e.g., equilibrium dissociation 80 fold, or at least about 90 fold, or at least about 100 fold, constants (K)) can indicate if the ADCC activity of an Fc or at least about 200 fold greater than that of a comparable variant is enhanced or decreased. For example a decrease in molecule. the ratio of FcyRIIIA/FcyRIIB equilibrium dissociation con stants (K), will correlate with improved ADCC activity, 0106. In another embodiment, an Fc variant of the inven while an increase in the ratio will correlate with a decrease tion has an equilibrium dissociation constant (K) that is in ADCC activity. Additionally, modifications that enhanced decreased between about 2 fold and about 10 fold, or binding to C1q would be preferable for enhancing CDC between about 5 fold and about 50 fold, or between about 25 activity while modification that reduced binding to C1q. fold and about 250 fold, or between about 100 fold and about would be preferable for reducing or eliminating CDC activ 500 fold, or between about 250 fold and about 1000 fold ity. relative to a comparable molecule. In a further embodiment, an Fc variant of the invention has an equilibrium dissocia 0102) The binding affinities and properties of an Fc tion constant (K) that is decreased between 2 fold and 10 domain for its ligand, may be determined by a variety of in fold, or between 5 fold and 50 fold, or between 25 fold and vitro assay methods (biochemical or immunological based 250 fold, or between 100 fold and 500 fold, or between 250 assays) known in the art for determining Fc-FcyR interac fold and 1000 fold relative to a comparable molecule. In a tions, i.e., specific binding of an Fc region to an FcyR Specific embodiment, Said Fc variants have an equilibrium including but not limited to, equilibrium methods (e.g., dissociation constants (K) for FcyRIIIA that is reduced by enzyme-linked immunoabsorbent assay (ELISA, See at least 2 fold, or at least 3 fold, or at least 5 fold, or at least Example 3 or radioimmunoassay (RIA)), or kinetics (e.g., 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 BIACORE(R) analysis), and other methods such as indirect fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, US 2006/0039904 A1 Feb. 23, 2006
or at least 70 fold, or at least 80 fold, or at least 90 fold, or about 3 fold, or at least about 5 fold, or at least about 7 fold, at least 100 fold, or at least 200 fold, or at least 400 fold, or or a least about 10 fold, or at least about 20 fold, or at least at least 600 fold, relative to a comparable molecule. In a about 50 fold or at least about 100 fold, greater than that of further specific embodiment, Said Fc variants have an equi a comparable molecule. librium dissociation constants (K) for FcyRIIIA that is reduced by at least about 2 fold, or at least about 3 fold, or 0110. In still another embodiment, the Fc variants have at least about 5 fold, or at least about 7 fold, or a least about an equilibrium dissociation constants (K) for FcyRIIIA that 10 fold, or at least about 20 fold, or at least about 30 fold, are increased by at least 1 fold, or by at least 3 fold, or by or at least about 40 fold, or at least about 50 fold, or at least at least 5 fold or by at least 10 fold or by at least 20 fold, or about 60 fold, or at least about 70 fold, or at least about 80 by at least 50 fold when compared to that of the original fold, or at least about 90 fold, or at least about 100 fold, or antibody without the substituted Fc. In a further embodi at least about 200 fold, or at least about 400 fold, or at least ment, the Fc variants have an equilibrium dissociation about 600 fold, relative to a comparable molecule. constants (K) for FcyRIIIA that are increased by at least about 1 fold, or by at least about 3 fold, or by at least about 0107. In one embodiment, said Fc variant binds to 5 fold or by at least about 10 fold or by at least about 20 fold, FcyRIIB with an affinity that is unchanged or reduced. In or by at least about 50 fold when compared to that of the another embodiment said Fc variants have affinity for original antibody without the substituted Fc. In another FcyRIIB that is unchanged or reduced by at least 1 fold, or embodiment Said Fc variants have equilibrium dissociation by at least 3 fold, or by at least 5 fold or by at least 10 fold constants (K) for FcyRIIB that are decreased at least 2 fold, or by at least 20 fold, or by at least 50 fold relative to a or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least comparable molecule. In a further embodiment said Fc 10 fold, or at least 20 fold, or at least 50 fold or at least 100 variants have affinity for FcyRIIB that is unchanged or fold, relative to a comparable molecule. In a further embodi reduced by at least about 1 fold, or by at least about 3 fold, ment Said Fc variants have equilibrium dissociation con or by at least about 5 fold or by at least about 10 fold or by stants (K) for FcyRIIB that are decreased at least about 2 at least about 20 fold, or by at least about 50 fold relative to fold, or at least about 3 fold, or at least about 5 fold, or at a comparable molecule. least about 7 fold, or a least about 10 fold, or at least about 0108. In another embodiment, said Fc variants have an 20 fold, or at least about 50 fold or at least about 100 fold, equilibrium dissociation constants (K) for FcyRIIB that is relative to a comparable molecule. unchanged or increased by at least at least 2 fold, or at least 0111 "Antibody-dependent cell-mediated cytotoxicity' 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or "ADCC” refers to a form of cytotoxicity in which or at least 20 fold, or at least 30 fold, or at least 40 fold, or Secreted Ig bound onto Fc receptors (FcRS) present on at least 50 fold, or at least 60 fold, or at least 70 fold, or at certain cytotoxic cells (e.g., Natural Killer (NK) cells, least 80 fold, or at least 90 fold, or at least 100 fold, or at neutrophils, and macrophages) enables these cytotoxic least 200 fold relative to a comparable molecule. In a further effector cells to bind Specifically to an antigen-bearing target embodiment, Said Fc variants have an equilibrium dissocia cell and Subsequently kill the target cell with cytotoxins. tion constants (K) for FcyRIIB that is unchanged or Specific high-affinity IgG antibodies directed to the surface increased by at least at least about 2 fold, or at least about of target cells "arm' the cytotoxic cells and are absolutely 3 fold, or at least about 5 fold, or at least about 7 fold, or a required for Such killing. Lysis of the target cell is extracel least about 10 fold, or at least about 20 fold, or at least about lular, requires direct cell-to-cell contact, and does not 30 fold, or at least about 40 fold, or at least about 50 fold, involve complement. or at least about 60 fold, or at least about 70 fold, or at least about 80 fold, or at least about 90 fold, or at least about 100 0112 The ability of any particular antibody to mediate fold, or at least about 200 fold relative to a comparable lysis of the target cell by ADCC can be assayed. To assess molecule. ADCC activity an antibody of interest is added to target cells in combination with immune effector cells, which may be 0109. In another embodiment, the Fc variants of the activated by the antigen antibody complexes resulting in invention bind FcyRIIIA with decreased affinity and bind cytolysis of the target cell. Cytolysis is generally detected by FcyRIIB with increased affinity when compared to the the release of label (e.g. radioactive Substrates, fluorescent original antibodies without the Substituted Fc. In one dyes or natural intracellular proteins) from the lysed cells. embodiment, said Fc variants have affinity for FcyRIIIA that Useful effector cells for such assays include peripheral blood is reduced by at least 1 fold, or by at least 3 fold, or by at mononuclear cells (PBMC) and Natural Killer (NK) cells. least 5 fold or by at least 10 or by at least 20 fold, or by at Specific examples of in vitro ADCC assays are described in least 50 fold when compared to that of the original antibody Wisecarver et al., 1985, 79:277; Bruggemann et al., 1987, J without the Substituted Fc. In another embodiment, said Fc Exp Med 166:1351; Wilkinson et al., 2001, J Immunol variants have affinity for FcyRIIIA that is reduced by at least Methods 258:183; Patel et al., 1995 J Immunol Methods about 1 fold, or by at least about 3 fold, or by at least about 184:29 (each of which is incorporated by reference) and 5 fold or by at least about 10 or by at least about 20 fold, or herein (see example 3). Alternatively, or additionally, ADCC by at least about 50 fold when compared to that of the activity of the antibody of interest may be assessed in Vivo, original antibody without the substituted Fc. In a further e.g., in a animal model Such as that disclosed in Clynes et al., embodiment, said Fc variants have affinity for FcyRIIB that 1998, PNAS USA 95:652 (incorporated by reference). is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 50 fold 0113. In one embodiment, the Fc variants of the invention or at least 100 fold, greater than that of a comparable are also characterized by in Vitro functional assays for molecule. In yet a further embodiment, Said Fc variants have determining one or more FcyR mediator effector cell func affinity for FcyRIIB that is at least about 2 fold, or at least tions (See Example 3). In another embodiment, the mol US 2006/0039904 A1 Feb. 23, 2006 ecules of the invention have similar binding properties and complexed with a cognate antigen. To assess complement effector cell functions in in Vivo models (Such as those activation, a CDC assay, e.g. as described in Gazzano described and disclosed herein) as those in in vitro based Santoro et al., 1996, J. Immunol. Methods, 202:163, may be assayS. However, the present invention does not exclude performed. molecules of the invention that do not exhibit the desired 0119) The present invention further provides Fc variants phenotype in in vitro based assays but do exhibit the desired with enhanced CDC function. In one embodiment, the Fc phenotype in Vivo. variants of the invention have increased CDC activity. In 0114. The present invention further provides Fc variants another embodiment, said Fc variants have CDC activity with enhanced ADCC function. In one embodiment, the Fc that is at least 2 fold, or at least 3 fold, or at least 5 fold or variants of the invention have increased ADCC activity. In at least 10 fold or at least 50 fold or at least 100 fold greater one embodiment said Fc variants have ADCC activity that is than that of a comparable molecule. In a further embodi at least 2 fold, or at least 3 fold, or at least 5 fold or at least ment, said Fc variants have CDC activity that is at least 10 fold or at least 50 fold or at least 100 fold greater than that about 2 fold, or at least about 3 fold, or at least about 5 fold of a comparable molecule. In another embodiment Said Fc or at least about 10 fold or at least about 50 fold or at least variants have ADCC activity that is at least about 2 fold, or about 100 fold greater than that of a comparable molecule. at least about 3 fold, or at least about 5 fold or at least about In another embodiment, an Fc variant of the invention binds 10 fold or at least about 50 fold or at least about 100 fold C1q with an affinity that is at least 2 fold, or at least 3 fold, greater than that of a comparable molecule. In a specific or at least 5 fold, or at least 7 fold, or a least 10 fold, or at embodiment, Fc variants of the invention bind FcyRIIIA least 20 fold, or at least 50 fold or at least 100 fold, greater with increased affinity, bind FcyRIIB with decreased affinity than that of a comparable molecule. In a further embodi and have enhanced ADCC activity relative to a comparable ment, an Fc variant of the invention binds C1q with an molecule. affinity that is at least about 2 fold, or at least about 3 fold, 0115) In a further embodiment, the Fc variants of the or at least about 5 fold, or at least about 7 fold, or a least invention have enhanced ADCC activity and immunospe about 10 fold, or at least about 20 fold, or at least about 50 cifically bind to at least one Eph receptor. In another fold or at least about 100 fold, greater than that of a embodiment are Fc variants of the invention have enhanced comparable molecule. In a specific embodiment, Fc variants ADCC activity and have a ratio of FcyRIIIA/FcyRIIB equi of the invention bind C1q with increased affinity; have librium dissociation constants (K) that is decreased relative enhanced CDC activity and immunospecifically bind to at to a comparable molecule and immunospecifically bind to at least one Eph receptor. least one Eph receptor. In still another embodiment, the Fc 0120) The present invention also provides Fc variants variants of the invention have enhanced ADCC activity, bind with reduced CDC function. In one embodiment, the Fc activating FcyRs (e.g., FcyRIIIA) with higher affinity and variants of the invention have reduced CDC activity. In bind inhibitory FcyRs (e.g., FcyRIIB) with unchanged or another embodiment, said Fc variants have CDC activity lower affinity and immunospecifically bind to at least one that is at least 2 fold, or at least 3 fold, or at least 5 fold or Eph receptor. at least 10 fold or at least 50 fold or at least 100 fold less than that of a comparable molecule. In a further embodiment, 0116. The present invention also provides Fc variants said Fc variants have CDC activity that is at least about 2 with reduced ADCC function. In one embodiment, the Fc fold, or at least about 3 fold, or at least about 5 fold or at least variants of the invention have reduced ADCC activity. In about 10 fold or at least about 50 fold or at least about 100 another embodiment, said Fc variants have ADCC activity fold less than that of a comparable molecule. In another that is at least 2 fold, or at least 3 fold, or at least 5 fold or embodiment, an Fc variant of the invention binds C1q with at least 10 fold or at least 50 fold or at least 100 fold less than an affinity that is reduced by at least 1 fold, or by at least 3 that of a comparable molecule. In a further embodiment, fold, or by at least 5 fold or by at least 10 or by at least 20 said Fc variants have ADCC activity that is at least about 2 fold, or by at least 50 fold relative to a comparable molecule. fold, or at least about 3 fold, or at least about 5 fold or at least In another embodiment, an Fc variant of the invention binds about 10 fold or at least about 50 fold or at least about 100 C1q with an affinity that is reduced by at least about 1 fold, fold less than that of a comparable molecule. In a specific or by at least about 3 fold, or by at least about 5 fold or by embodiment, Fc variants of the invention bind FcyRIIIA at least about 10 or by at least about 20 fold, or by at least with decreased affinity, bind FcyRIIB with increased affinity about 50 fold relative to a comparable molecule. In a specific and have reduced ADCC activity. embodiment, Fc variants of the invention bind to at least one 0117. In one embodiment, the Fc variants of the invention Eph receptor, binds C1q with decreased affinity have have reduced ADCC activity and immunospecifically bind reduced CDC activity and immunospecifically bind to at to at least one Eph receptor. In another embodiment, the least one Eph receptor antibody variants of the invention have reduced ADCC activity, bind activating FcyRs (e.g., FcyRIIIA) with lower 0121. It is also specifically contemplated that the Fc affinity, bind inhibitory FcyRs (e.g., FcyRIIB) with higher variants of the invention may contain inter alia one or more affinity and immunospecifically bind to at least one Eph additional amino acid residue Substitutions, mutations and/ receptor. or modifications which result in an antibody with preferred characteristics including but not limited to: increased Serum 0118 “Complement dependent cytotoxicity” and “CDC" half life, increase binding affinity, reduced immunogenicity, refer to the lysing of a target cell in the presence of increased production, altered Fc ligand binding, enhanced or complement. The complement activation pathway is initi reduced ADCC or CDC activity, altered glycosylation and/ ated by the binding of the first component of the complement or disulfide bonds and modified binding specificity (for System (C1q) to a molecule, an antibody for example, examples see infra). The invention encompasses combining US 2006/0039904 A1 Feb. 23, 2006
an Fc variant of the invention with other Fc modifications to reference in its entirety. Additional methods are described in provide additive, Synergistic, or novel properties in antibod Section 6.2 entitled "Antibodies of the Invention' below. ies or Fc fusions. In one embodiment, the other Fc modifi 0124. The receptor tyrosine kinases (RTKs) are trans cations enhance the phenotype of the Fc variant with which membrane molecules which transduce Signals from the they are combined. For example, if an Fc variant of the extracellular environment into the cytoplasm. The Eph fam invention is combined with a mutant known to bind ily of RTKs is the largest subfamily of RTKs. This group is FcyRIIIA with a higher affinity than a comparable molecule distinguished by a cysteine-rich region and two fibronectin comprising a wild type Fc region; the combination with a type III repeats in the extracellular domain. The Eph recep mutant of the invention results in a greater fold enhancement tors are activated by a Second family of cell Surface in FcyRIIIA affinity. anchored proteins, the ephrins. Members of both the Eph 0122) In one embodiment, the Fc variants of the present tyrosine kinases and the ephrin ligands mediate Signaling invention may be combined with other known Fc variants after receptor-ligand interaction (Bruckner et al., 1997, Such as those disclosed in Ghetie et al., 1997, Nat Biotech. Science 275:1640; Holland et al., 1996, Nature 383:722). 15:637-40; Duncan et al., 1988, Nature 332:563-564; Lund This bi-directional Signaling are known to affect processes et al., 1991, J. Immunol 147:2657-2662; Lund et al., 1992, involving cellular interaction, like cell adhesion, cell migra Mol Immunol 29:53-59; Alegre et al., 1994, Transplantation tion and tissue border formation (Boyd et al., 2001 Sci 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci STKE RE.20; Schmucher et al., 2001, Cell 105: 7014, Kul USA 92:11980-11984; Jefferis et al., 1995, Immunol Lett. lander et al., 2002 Nat. Rev. Mol. Cell Biol. 3:475). More 44:111-117; Lund et al., 1995, FasebJ 9:115-119; Jefferis et recently, the Eph receptors have been linked to the devel al, 1996, Immunol Lett 54:101-104; Lund et al., 1996, J opment and progression of cancers. AS cell Surface mol Immunol 157:4963-4969; Armour et al., 1999, EurJ Immu ecules the Eph receptors are readily accessible target mol nol 29:2613-2624, Idusogie et al., 2000, J Immunol ecules for antibody directed therapies. In one embodiment, 164:41.78-4184; Reddy et al., 2000, J Immunol 164:1925 the Fc variants of the invention are variants of an antibody 1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et that immunospecifically binds to at least one Eph receptor. al, 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Eph receptors to which the Fc variant of the invention Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol Lett immunospecifically binds to include but are not limited to 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487 EphA1, EphA2, EphA3a, EphA3b, EphA4, EphA5a, 490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; EphA5b, EphA6, EphA7, EphA8, EphB1, EphB2a, 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; EphB2b, Eph B3, EphB4 and EphB6. 5,648,260; 6,194.551; 6,737,056; 6,821.505; 6,277,375; 0.125 The skilled artisan will appreciate that an Eph U.S. patent application Ser. Nos. 10/370,749 and PCT receptor of the invention is a molecule that exhibits a Publications WO 94/2935; WO 99/58572; WO 00/42072; Substantial degree of homology to known Eph receptors WO 02/060919, WO 04/029207, each of which is incorpo (see, e.g., Supra), Such that it has been or can be classified as rated herein by reference in its entirety. an Eph receptor family molecule based upon, its amino acid Sequence. Pairwise comparisons of the known human Eph 0123. In some embodiments, the Fc variants of the receptors were performed using the MegaAlign program present invention comprises one or more engineered glyco (DNASTAR) with the Clustal W algorithm (Thompson et forms, i.e., a carbohydrate composition that is covalently al., 1994 Nucleic Acids Res 22:4673-80). The results (FIG. attached to a molecule comprising an Fc region. Engineered 18) show that there are multiple regions each protein that glycoforms may be useful for a variety of purposes, includ share a high degree of Similarity among the Eph receptor ing but not limited to enhancing or reducing effector func tion. Engineered glycoforms may be generated by any family members. It is specifically contemplated that one method known to one skilled in the art, for example by using skilled in the art could generate antibodies to regions of an engineered or variant expression Strains, by co-expression Eph receptor that would allow for cross reactivity of said with one or more enzymes, for example DI N-acetylglu antibody between family members or a more restricted cosaminyltransferase III (GnTI11), by expressing a mol Specificity Such that Said antibody immunospecifically ecule comprising an Fc region in various organisms or cell bound only one family member with high affinity. To iden lines from various organisms, or by modifying carbohy tify potential immunogenic peptides for use in generating drate(s) after the molecule comprising Fc region has been antibodies that could be either protein specific or would bind expressed. Methods for generating engineered glycoforms with one or more Eph receptors, the antigenic index of each are known in the art, and include but are not limited to those protein can be examined using the Protean program (DNAS described in Umana et al., 1999, Nat. Biotechnol 17:176-180; TAR) with the Jameson-Wolf algorithm. The regions with Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields the highest antigenic indices among all members of the Eph et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., receptor family can be identified and those regions which are 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; highly conserved among one or more family members and U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT would be excellent candidates for raising an antibody which WO 00/61739A1; PCT WO 01/292246A1; PCT WO recognizes more then one family member. While the use of 02/311140A1; PCT WO 02/30954A1; PotillegentTM tech leSS conserved regions would likely generate an antibody nology (Biowa, Inc. Princeton, N.J.); GlycoMAbTM glyco Specific for one Eph receptor family member. Sylation engineering technology (GLYCART biotechnology 0.126 In one embodiment, the Fc variants of the inven AG, Zurich, Switzerland); each of which is incorporated tion preferentially bind to an Eph receptor present on a herein by reference in its entirety. See, e.g., WO 00061739; tumor cell and do not bind to an Eph receptor present on a EAO1229125; US 2003.01.15614; Okazaki et al., 2004, JMB, non-tumor cell. In another embodiment, the Fc variants of 336: 1239-49 each of which is incorporated herein by the invention do not stain normal tissues including but not US 2006/0039904 A1 Feb. 23, 2006 limited to, brain, lung, pancreas, liver, prostate, heart, ovary, less than 10's". In still another embodiment, an Fc skin, kidney, intestine and Stomach. Antibody binding and variant of the invention that immunospecifically binds to Specific Staining patterns can be readily determined by least on Eph receptor has a k rate (Fc variant (Ab)+antigen immunological labeling methods well known in the art (Ag)'s Ab-Ag) of less than about 10's", less than about including but not limited to, immunohistochemistry and 5x10's", less than about 10's", less than about 5x10 Fluorescence Activated Cell Scanning/Sorting (FACS). Spe 2s, less than about 10s, less than about 5x10's", less cific methods and protocols are found in Polak and Van than about 10's", less than about 5x10's", less than Noorden (1997) Introduction to Immunocytochemistry, sec about 10s, less than about 5x10s, less than about ond edition, Springer Verlag, N.Y. and in Haugland (2004) 10's", less than about 5x10's", less than about 10's", Handbook of Fluorescent Probes and Research Chemicals, less than about 5x10's", less than about 10's", less than ninth edition, a combined handbook and catalogue Pub about 5x10s, less than about 10's", less than about lished by Molecular Probes, Inc., Eugene, Oreg. among 5x10's", or less than about 10's. In a further others. embodiment, an Fc variant that immunospecifically binds to least on Eph receptor has a kit, of less than 5x10's", less O127) than 10s, less than 5x10s, less than 10's", less 0128. In another embodiment, the Fc variants of the than 5x10's", less than 10's", less than 5x10's", less invention are variants of antibodies that immunospecifically than 10's", less than 5x10s, less than 10's", less bind EphA2 and/or EphA4, their derivatives, analogs and than 5x10's", or less than 10's". In another embodi epitope-binding fragments thereof, Such as but not limited ment, an Fc variant that immunospecifically binds to least on to, those disclosed herein and in PCT Publication Nos. WO Eph receptor has a kit, of less than about 5x10's", less 04/014292, WO 03/094859 and U.S. patent application Ser. than about 10s, less than about 5x10s, less than No. 10/863,729, each of which is incorporated herein by about 10's", less than about 5x10's", less than about reference in its entirety and any of the antibodies listed in 10's", less than about 5x10's", less than about 10s', Table 4. In a specific embodiment, the Fc variants of the less than about 5x10's", less than about 10's", less than invention are antibodies that immunospecifically bind about 5x10's", or less than about 10's". EphA2 and/or EphA4 which comprise all or a portion of the 0131. In another embodiment, an Fc variant of the inven variable region (e.g., one or more CDR) from 12G3H11, tion that immunospecifically binds to least on Eph receptor and/or 3F2 and/or any of the antibodies listed in Table 4. has an affinity constant or K, (k./k,n) of at least 10M, at least 5x10'M', at least 10M, at least 5x10M, at least 0129. The present invention further encompasses the use 10'M', at least 5x10'M', at least 10M, at least of Fc variants of the invention that have a high binding 5x10M, at least 10M, at least 5x10'M', at least affinity for at least on Eph receptor. In a Specific embodi 107M, at least 5x10'M', at least 10M, at least ment, an Fc variant of the invention that immunospecifically 5x10M, at least 10M, at least 5x1O'M', at least binds to at least one Eph receptor has an association rate 10'M', at least 5x10'M', at least 10'M', at least constant or k, rate (Fc variant (Ab)+antigen (Ag)'s Ab 5x10'M', at least 10'M', at least 5x1O'M, at least Ag) of at least 10M's', at least 5x10Ms', at least 1O'M', at least 5x1O'M', at least 10''M', at least 10M's, at least 5x1OM's', at least 107M's, at least 5x10'M', at least 10'M', or at least 5x10'M'. In a 5x107M's', or at least 10MT's. In a further specific further embodiment, an Fc variant of the invention that embodiment, an Fc variant of the invention that immuno immunospecifically binds to least on Eph receptor has an Specifically binds to at least one Eph receptor has an affinity constant or K, (k/ki) of at least about 10M, at association rate constant or k rate (Fc variant (Ab)+antigen least about 5x10M, at least about 10M, at least about (Ag)'s Ab-Ag) of at least about 10M's', at least about 5x10M, at least about 10"M", at least about 5x10'M', 5x10M's, at least about 10°M's, at least about at least about 10M, at least about 5x10M, at least 5x10°M's', at least about 107M's', at least about about 10M, at least about 5x10M, at least about 5x107M's', or at least about 10M's. In another 107M, at least about 5x10'M', at least about 10M, at embodiment, an Fc variant that immunospecifically binds to least about 5x10M, at least about 10M, at least about at least one Eph receptor has a k, of at least 2x10M's', 5x1O'M', at least about 10'M', at least about 5x10'M', at least 5x10M's', at least 10M's', at least 5x10M at least about 10'M', at least about 5x10'M', at least 1s', at least 107M's', at least 5x107M's', or at least about 10'M', at least about 5x10'’M, at least about 10M's'. In a further embodiment, an Fc variant that 1O'M', at least about 5x1O'M', at least about 10'M', immunospecifically binds to at least one Eph receptor has a at least about 5x10'M', at least about 10'M', or at least k of at least about 2x10M's', at least about 5x10M about 5x1O'M'. 1s', at least about 10M's', at least about5x10'M's', at least about 107M's', at least about 5x107M's', or at 0.132. In yet another embodiment, an Fc variant that least about 10M's'. immunospecifically binds to least on Eph receptor has a dissociation constant or Ka (k. k.) of less than 10 M, less 0130. In another embodiment, an Fc variant of the inven than 5x10°M, less than 10M, less than 5x10M, less tion that immunospecifically binds to least on Eph receptor than 10"M, less than 5x10"M, less than 10M, less than has a k, rate (Fc variant (Ab)+antigen (Ag)'s Ab-Ag) of 5x10M, less than 10M, less than 5x10M, less than less than 10's", less than 5x10's", less than 10°s, less 10M, less than 5x107M, less than 10M, less than than 5x10°s', less than 10s, less than 5x10s, less 5x10M, less than 10M, less than 5x10M, less than than 10's, less than 5x10's", less than 10s, less 10'M, less than 5x10'M, less than 10'M, less than than 5x10s, less than 10's", less than 5x10's", less 5x10'M, less than 10°M, less than 5x10'M, less than than 10's", less than 5x10's", less than 10s, less 10M, less than 5x10M, less than 10"M, less than than 5x10's", less than 10's", or less than 5x10's", or 5x10"M, less than 10M, or less than 5x10'M. In a US 2006/0039904 A1 Feb. 23, 2006 further embodiment, an Fc variant that immunospecifically incorporated herein by reference in its entirety), and binds to least on Eph receptor has a dissociation constant or 12G3H11 (infra) and analogs, derivatives, or fragments Ka (kt/k.) of less than about 10 M, less than about thereof. It is specifically contemplated that the Fc variants of 5x10°M, less than about 10M, less than about 5x10M, the invention may comprise all or a portion of the variable less than about 10"M, less than about 5x10"M, less than region (e.g., one or more CDR) from 12G3H11 (see Table 3) about 10M, less than about 5x10M, less than about and/or any of the antibodies listed in Table 4. 10M, less than about 5x10M, less than about 107M, less than about 5x107M, less than about 10M, less than 0135) In one embodiment, the Fc variant is an Fc variant about 5x10M, less than about 10M, less than about of 12G3H11, a humanized agonistic monoclonal antibody 5x10M, less than about 10'M, less than about 5x10 that binds EphA2. The DNA and deduced amino acid 1OM, less than about 10M, less than about 5x10'M, less Sequence of the variable region of the heavy and light chains than about 10 M, less than about 5x10'M, less than of 12C3H11 are shown in FIGS. 2A and 2B respectively. about 10M, less than about 5x10'M, less than about The amino acid Sequences for the heavy chain variable 10'M, less than about 5x10"M, less than about 10M, region and light chain variable region are provided herein as or less than about 5x10'M. SEQ ID NO: 64 and SEQ ID NO: 65, respectively (FIGS. 2A and 2B). The nucleotide sequence for the heavy chain 0133) Other RTK molecules contemplated as targets for variable and light chain variable region are provided herein Fc variants include Anaplastic Lymphoma Kinase (ALK), an as SEQID NO: 62 and SEQID NO: 63, respectively (FIGS. orphan RTK. ALK was originally identified as a fusion 2A and 2B). In another embodiment, Fc variant of the protein with nucleophosmin (npm/ALK) due to a t2:5) present invention binds to the same epitope as 12G3H11 or translocation (Morris et al., 1994) this fusion results in competes with 12G3H11 for binding to EphA2. In an constitutive activation of the intracellular ALK kinase and alternative embodiment, the Fc variant of the invention that was shown to induce anaplastic lymphoma. The full-length immuno-specifically binds to an Eph receptor is not an Fc ALK receptor has been shown to be highly expressed in the variant of 12G3H11. developing nervous System and down-regulated postnatally (Iwahara et al., 1997). It was recently discovered that ALK 0.136. In one embodiment, the Fc variant is an Fc variant is a cell receptor for pleiotrophin (PTN) in humans and that of 3F2, a humanized agonistic monoclonal antibody that this tyrosine kinase receptor is over expressed in human binds EphA2. The DNA and deduced amino acid sequence glioblastoma and is rate-limiting for the growth of a of the variable region of the heavy and light chains of 3F2 Xenograft model of glioblastoma. (U.S. patent publication are shown in FIGS. 3A and 3B respectively. The amino acid No. 2002/034768). In one embodiment, the Fc variants of Sequences for the heavy chain variable region and light the invention immunospecifically bind to ALK (e.g., Gen chain variable region are provided herein as SEQ ID NO: 68 bank Acc. No.:Q9UM73). In a specific embodiment, vari and SEQ ID NO: 69, respectively (FIGS. 3A and 3B). The ants of the invention immunospecifically bind to the extra nucleotide Sequence for the heavy chain variable and light cellular domain of ALK. In another embodiment, the Fc chain variable region are provided herein as SEQ ID NO: 66 variants of the invention immunospecifically bind to PTN and SEQ ID NO: 67, respectively (FIGS. 3A and 3B). In (e.g., Genbank Acc. No. NP 002816). In a specific embodi another embodiment, Fc variant of the present invention ment, the Fc variants of the invention block the binding of binds to the same epitope as 3F2 or competes with 3F2 for PTN to ALK. binding to EphA2. In an alternative embodiment, the Fc variant of the invention that immuno-specifically binds to an 6.1 Fe Variants that Immunospecifically Bind to an Eph receptor is not an Fc variant of 3F2. Eph Receptor 0.137 In a specific embodiment, an Fc of the invention is 0134. As discussed above, the invention encompasses Fc generated by combining a antigen binding domain (e.g., variants comprising a variable region that immunospecifi variable region) or fragment thereof of an antibody or cally binds to at least one Eph receptor and a Fc region that fragment thereof that immunospecifically binds an Eph further comprises at least one high effector function amino receptor (examples Supra) with an Fc region comprising at acid residue (e.g., 239D,330L,332E wherein the numbering least one high effector function amino acid residue. Methods of the residues is that of the EU index as set forth in Kabat). for generating Such a recombinant antibody are well know to The invention further encompasses Fc variants that immu one skilled in the art and are further described infra. nospecifically bind to at least one Eph receptor, have altered ADCC and/or CDC activity and modified binding affinities 0.138. In one embodiment, the Fc variant of the invention for one or more Fc ligand (e.g., FcyRS, C1q) relative to a preferentially binds EphA2 over other Eph receptors. In comparable molecule. The invention specifically encom another embodiment, the Fc variant of the invention pref passes Fc variants derived from anti-Eph receptor antibodies erentially binds EphA4 over other Eph receptors. In still or fragments thereof including, but not limited to, EphO99B another embodiment, the Fc variant of the invention immu 102.147 (ATCC access No. PTA-4572), EphO99B-208.261 noreacts with one or more Eph receptor complex (e.g., an (ATCC access No. PTA-4573), Eph099B-210.248 (ATCC Eph receptor-Ephrin ligand complex). In still another access No. PTA-4574), EphO99B-233.152 (ATCC access embodiment, an Fc variant of the invention immunospecifi No. PTA-5194), (PCT Publication No. WO 03/094859 cally binds more then one Eph receptor. Combinations of which is incorporated herein by reference in its entirety); Eph receptorS bound by an Fc variant that immunospecifi EA2 (ATCC access No. PTA-4380), EA3, EA4, EA5 (ATCC cally binds more then one Eph receptor are represented by access No. PTA-4381), (PCT Publication No. WO the following formulas, EphA(x)+EphB(y); EphA(x)+ 04/014292 which is incorporated herein by reference in its EphA(x); EphB(y)+EphB(y); wherein (x) is 1, 2, 3, 3a, 3b, entirety); LX-13 and scFv EA44 (ATCC access No. PTA 4, 5,5a, 5b, 6, 7 or 8 and (y) is 1, 2, 2a, 2b, 3, 4, 5 or 6. In 6044), (U.S. patent application Ser. No. 10/863,729 which is a Specific embodiment, an Fc variant that Specifically immu US 2006/0039904 A1 Feb. 23, 2006 noreacts with more then one Eph receptor binds to, e.g., immunospecifically binds to an Eph receptor, Fc variants EphA2+EphA4, or EphA2+EphA3, or EphA2+EphB4, or comprising any combination of some or all of the VHCDRs EphA4+EphA3, or EphA4+EphB4. It is specifically con and VLCDRS listed in Table 3 infra. templated that an Fc variant that immunospecifically binds more then one Eph receptor is a bispecific antibody. It is TABLE 3 further contemplated that an Fc variant that immunospecifi cally binds more then one Eph receptor is an antibody that CDR Secuences Of 12G3H11 and 3F2 binds a common epitope between two or more Eph recep CDR Sequence SEQ ID NO : tors. It is further contemplated that an Fc variant that immunospecifically binds more then one Eph receptor is an 12G3H11 WH1 DYSMN 5 antibody that cross-reacts with one or more Eph receptorS. In addition, the Fc variant of the invention may have the 12G3H11 WH2 FIRNKANDYTTEYADSWKG 6 Same immunoreactivity for more then one Eph receptor 12G3H11 WH3 YPRHHAMDS 7 (e.g., Eph A2 and EphA4) or alternatively, the Fc variant may immunoreact more Strongly with one Eph receptor then with 12G3H11 WL1 RASQSISNNLH 8 another. 12G3H11 WL2 YAFOSIS 9 0.139. The present invention encompasses Fc variants that immunospecifically bind to EphA2, Said antibodies com 12G3H11 WL3 QQANSWPLT 10 prising a variable heavy (“VH') domain having an amino 3F2 WH1 DYSMN 70 acid sequence of the VH domain of 12C3H11, EphO99B 102.147, EphO99B-208.261, EphO99B-210.248, EphO99B 3F2 WH2 FIRNKANAYTTEYSASWKG 71. 233.152, EA2, EA3, EA4, or EA5. The present invention 3F2 WH3 YPRYHAMDS 72 also encompasses Fc variants that immunospecifically bind to EphA2, Said antibodies comprising a variable light 3F2 WL1 RASQSISNNLH 73 (“VL) domain having an amino acid sequence of the VL domain of 12G3H11, EphO99B-102.147, EphO99B 3F2 WL2 YGFOSIS 74 208.261, EphO99B-210.248, Eph099B-233.152, EA2, EA3, 3F2 WL3 QQANSWPLT 75 EA4, or EA5. The invention further encompasses Fc vari ants that immunospecifically bind to EphA2, said antibodies comprising a VH domain disclosed herein combined with a 0142) VL domain disclosed herein, or other VL domain. The present invention further encompasses Fc variants Fc vari TABLE 4 ants that immunospecifically bind to EphA2, Said Fc vari anti-Eph receptor antibodies ants comprising a VL domain disclosed herein combined with a VH domain disclosed herein, or other VH domain. Antibody/ ATCC Patent Hybridoma EphR No. Date of deposit App. No. 0140. The present invention encompasses Fc variants that EphO99B-102.147 EphA2 PTA-4572 Aug. 7, 2002 WO O3/094.859 immunospecifically bind to EphA4, Said antibodies com EphO99B208.261 EphA2 PTA-4573 Aug. 7, 2002 WO O3/094.859 prising a variable heavy (“VH') domain having an amino EphO99B-210.248 EphA2 PTA-4574 Aug. 7, 2002 WO O3/094.859 acid sequence of the VH domain of LX-13 or scFv EA44. EphO99B-233.152 EphA2 PTA-5194 May 12, 2003 WO O3/094.859 The present invention also encompasses Fc variants that EA2 EphA2 PTA-4380 May 22, 2002 WO O4/O14292 immunospecifically bind to EphA4, Said antibodies com EAS EphA2 PTA-4381 May 22, 2002 WO O4/O14292 prising a variable light (“VL) domain having an amino acid EA44 EphA4 PTA-6044 Jun. 4, 2004 10/863,729 sequence of the VL domain of LX-13 or scFv EA44. The invention further encompasses Fc variants that immunospe 0143. The present invention also encompasses Fc vari cifically bind to EphA4, said antibodies comprising a VH ants that compete with 12G3H11, EphO99B-102.147, domain disclosed herein combined with a VL domain dis Eph099B-208.261, EphO99B-210.248, EphO99B-233.152, closed herein, or other VL domain. The present invention EA2, EA3, EA4, EA5, LX-13 or scFv EA44 or an antigen further encompasses Fc variants Fc variants that immuno binding fragment thereof for binding to an Eph receptor. Specifically bind to EphA4, Said Fc variants comprising a VL Competition assays, which can be used to identify Such domain disclosed herein combined with a VH domain dis antibodies, are well known to one skilled in the art. In a closed herein, or other VH domain. particular embodiment, 1 lug/ml of an antibody of the 0.141. The present invention encompasses Fc variants that invention prevents 75%, 80%, 85% or 90% of ORIGEN immunospecifically bind to an Eph receptor, Said antibodies TAG labeled 12G3H11, EphO99B-102.147, EphO99B comprising a VH CDR having an amino acid Sequence of 208.261, EphO99B-210.248, Eph099B-233.152, EA2, EA3, any one of the VHCDRs listed in Table 3 infra. The present EA4, EA5, LX-13 or scFv EA44 from binding to biotin invention also encompasses Fc variants that immunospecifi labeled Eph receptor as measured by well-known ORIGEN cally bind to an Eph receptor, Said antibodies comprising a analysis. VLCDR having an amino acid Sequence of any one of the 0144. The present invention also provides Fc variants that VLCDRs listed in Table 3 infra. The present invention also comprise a framework region known to those of Skill in the encompasses Fc variants that immunospecifically bind to an art. In one embodiment, the fragment region of an antibody Eph receptor, Said Fc variants comprising one or more VH of the invention or fragment thereof is human or humanized. CDRS and one or more VL CDRS listed in Table 3. The 0145 The present invention encompasses Fc variants present invention further encompasses Fc variants that comprising the amino acid Sequence of 12G3H11, 3F2, US 2006/0039904 A1 Feb. 23, 2006
Eph099B-102.147, EphO99B-208.261, EphO99B-210.248, CDRS, VL domains, or VL CDRS described herein that Eph099B-233.152, EA2, EA3, EA4, EA5, LX-13 or scEv immunospecifically bind to an Eph receptor. Standard tech EA44 with mutations (e.g., one or more amino acid Substi niques known to those of skill in the art can be used to tutions) in the framework or variable regions in addition to introduce mutations (e.g., additions, deletions, and/or Sub any other Substitutions or changes (e.g., Fc Substitution(s) as Stitutions) in the nucleotide Sequence encoding an antibody described Supra). In one embodiment, mutations in these of the invention, including, for example, Site-directed antibodies maintain or enhance the avidity and/or affinity of mutagenesis and PCR-mediated mutagenesis are routinely the antibodies for the Eph receptor to which they immuno used to generate amino acid Substitutions. In one embodi Specifically bind. Standard techniques known to those ment, the VH and/or VLCDRS derivatives include less than skilled in the art (e.g., immunoassays) can be used to assay 25 amino acid Substitutions, less than 20 amino acid Sub the affinity of an antibody for a particular antigen. Stitutions, less than 15 amino acid Substitutions, leSS than 10 amino acid Substitutions, leSS than 5 amino acid Substitu 0146 The present invention encompasses the use of a tions, less than 4 amino acid Substitutions, less than 3 amino nucleic acid molecule(s), generally isolated, encoding an Fc acid Substitutions, or less than 2 amino acid Substitutions in variant that immunospecifically binds to an Eph receptor. In the relative to the original VH and/or VLCDRs. In another a specific embodiment, an isolated nucleic acid molecule embodiment, the VH and/or VL CDRS derivatives have encodes an Fc variant that immunospecifically binds to an conservative amino acid Substitutions (e.g. Supra) are made Eph receptor, Said Fc variant having the amino acid at one or more predicted non-essential amino acid residues sequence of 12C3H11, EphO99B-102.147, EphO99B (i.e., amino acid residues which are not critical for the 208.261, EphO99B-210.248, Eph099B-233.152 EA2, EA3, antibody to immunospecifically bind to an Eph receptor). EA4, EA5, LX-13 or scrv EA44 containing one or more Fc Alternatively, mutations can be introduced randomly along Substitution (e.g. Supra). In another embodiment, an isolated all or part of the VH and/or VLCDR coding sequence, such nucleic acid molecule encodes an Fc variant that immuno as by Saturation mutagenesis, and the resultant mutants can Specifically binds to and Eph receptor, Said Fc variant be Screened for biological activity to identify mutants that comprising a VH domain having the amino acid Sequence of retain activity. Following mutagenesis, the encoded anti the VH domain of 12G3H11, EphO99B-102.147, EphO99B body can be expressed and the activity of the antibody can 208.261, EphO99B-210.248, Eph099B-233.152, EA2, EA3, be determined. EA4, EA5, LX-13 scFv EA44. In another embodiment, an isolated nucleic acid molecule encodes an Fc variant that 0150. The present invention encompasses Fc variants of immunospecifically binds to an Eph receptor, said antibody 12G3H11, Eph099B-102.147, Eph099B-208,261, comprising a VL domain having the amino acid Sequence of Eph099B-210.248, EphO99B-233.152, EA2, EA3, EA4, the VL domain of 12G3H11, EphO99B-102.147, EphO99B EA5, LX-13 or ScFv EA44 with one or more additional 208.261, EphO99B-210.248, Eph099B-233.152, EA2, EA3, amino acid residue substitutions in the variable light (VL) EA4, EA5, LX-13 schv EA44. domain and/or variable heavy (VH) domain. The present invention also encompasses Fc variants of 12G3H11, 0147 The invention encompasses the use of an isolated Eph099B-102.147, EphO99B-208.261, EphO99B-210.248, nucleic acid molecule encoding an Fc variant that immuno Eph099B-233.152, EA2, EA3, EA4, EA5, LX-13 or scEv Specifically binds to an Eph receptor, Said Fc variant com EA44 with one or more additional amino acid residue prising a VH CDR having the amino acid Sequence of any Substitutions in one or more VLCDRS and/or one or more of the VH CDRS listed in Table 3 and/or derived from the VH CDRs. The antibody generated by introducing substi heavy chain of any of the antibodies listed in Table 4. In tutions in the VH domain, VHCDRS, VL domain and/or VL particular, the invention encompasses the use of an isolated CDRs of an Fc variant of 12G3H11, EphO99B-102.147, nucleic acid molecule encoding an Fc variant that immuno Eph099B-208.261, EphO99B-210.248, EphO99B-233.152, Specifically binds to an Eph receptor, Said antibody com EA2, EA3, EA4, EA5, LX-13 or ScFv EA4 can be tested in prising one, two, or more VHCDRS having the amino acid vitro and in vivo, for example, for its ability to bind to an sequence of any of the VHCDRs listed in Tables 3 and/or Eph receptor and/or FcDRS (by, e.g., immunoassays includ derived from the heavy chain of any of the antibodies listed ing, but not limited to ELISAS and BIAcore), or for its in Table 4. ability to mediate ADCC, prevent, treat, manage or amelio 0.148. The present invention encompasses the use of an rate cancer or one or more Symptoms thereof. isolated nucleic acid molecule encoding an Fc variant that 0151. The present invention also encompasses the use of immunospecifically binds to an Eph receptor, Said Fc variant Fc variants that immunospecifically bind to at least one Eph comprising a VL CDR having an amino acid Sequence of receptor or a fragment thereof, Said Fc variants comprising any of the VLCDRs listed in Table 3, and/or derived from an amino acid Sequence of a variable heavy chain and/or the light chain of any of the antibodies listed in Table 4. In variable light chain that is at least 45%, at least 50%, at least particular, the invention encompasses the use of an isolated 55%, at least 60%, at least 65%, at least 70%, at least 75%, nucleic acid molecule encoding an Fc variant that immuno at least 80%, at least 85%, at least 90%, at least 95%, or at Specifically binds to an Eph receptor, Said antibody com least 99% identical to the amino acid sequence of the prising one, two or more VLCDRS having the amino acid variable heavy chain and/or light chain of 12G3H11 (i.e., sequence of any of the VLCDRs listed in Table 3 and/or SEQ ID NO: 64 and/or SEQ ID NO: 65), 3F2 (i.e., SEQ ID derived from the light chain of any of the antibodies listed NO: 68 and/or SEQ ID NO: 69), Eph099B-102.147, in Table 4. Eph099B-208.261, EphO99B-210.248, EphO99B-233.152, EA2, EA3, EA4, EA5, LX-13 or scFv EA44. The present 0149 8JThe present invention encompasses the use of Fc invention also encompasses the use of Fc variants that variants that immunospecifically bind to an Eph receptor, Fc immunospecifically bind to at least one Eph receptor or a variants comprising derivatives of the VH domains, VH fragment thereof, said Fc variants comprising an amino acid US 2006/0039904 A1 Feb. 23, 2006
Sequence of a variable heavy chain and/or variable light Publishing Associates, Inc. and John Wiley and Sons, Inc., chain that is at least about 45%, at least about 50%, at least NY at pages 6.3.1 to 6.3.6 and 2.10.3). about 55%, at least about 60%, at least about 65%, at least 0153. Set forth below, is a more detailed description of about 70%, at least about 75%, at least about 80%, at least the antibodies encompassed within the various aspects of the about 85%, at least about 90%, at least about 95%, or at least invention. about 99% identical to the amino acid sequence of the variable heavy chain and/or light chain of 12G3H11 (i.e., 6.2 Antibodies of the Invention SEQ ID NO: 64 and/or SEQ ID NO: 65), 3F2 (i.e., SEQ ID NO: 68 and/or SEQ ID NO: 69), Eph099B-102.147, 0154 Fc variants of the invention may include, but are Eph099B-208.261, EphO99B-210.248, EphO99B-233.152, not limited to, Synthetic antibodies, monoclonal antibodies, EA2, EA3, EA4, EA5, LX-13 or scFv EA44. The present oligoclonal antibodies recombinantly produced antibodies, invention further encompasses the use of Fc variants that intrabodies, multispecific antibodies, bispecific antibodies, immunospecifically bind to at least one Eph receptor or a human antibodies, humanized antibodies, chimeric antibod fragment thereof, Said antibodies or antibody fragments ies, Synthetic antibodies, single-chain FvFcs (ScFvFc), comprising an amino acid Sequence of one or more CDRS Single-chain FVS (ScFV), and anti-idiotypic (anti-Id) antibod that is at least 45%, at least 50%, at least 55%, at least 60%, ies. In particular, antibodies used in the methods of the at least 65%, at least 70%, at least 75%, at least 80%, at least present invention include immunoglobulin molecules and 85%, at least 90%, at least 95%, or at least 99% identical to immunologically active portions of immunoglobulin mol the amino acid sequence of one or more CDRs of 12G3H11, ecules. The antibodies of the invention can be of any type Eph099B-102.147, EphO99B-208.261, EphO99B-210.248, (e.g., IgG, IgE, IgM, Ig|D, IgA and IgY), class (e.g., IgG1, Eph099B-233.152, EA2, EA3, EA4, EA5, LX-13 or scEv IgG, IgG, IgG, IgA and IgA) or Subclass of immuno EA44. The present invention further encompasses the use of globulin molecule. Fc variants that immunospecifically bind to at least one Eph O155 Fc variants of the invention may be from any receptor or a fragment thereof, said antibodies or antibody animal origin including birds and mammals (e.g., human, fragments comprising an amino acid Sequence of one or murine, donkey, sheep, rabbit, goat, guinea pig, camel, more CDRS that is at least about 45%, at least about 50%, horse, or chicken). Preferably, the antibodies are human or at least about 55%, at least about 60%, at least about 65%, humanized monoclonal antibodies. AS used herein, “human” at least about 70%, at least about 75%, at least about 80%, antibodies include antibodies having the amino acid at least about 85%, at least about 90%, at least about 95%, Sequence of a human immunoglobulin and include antibod or at least about 99% identical to the amino acid sequence ies isolated from human immunoglobulin libraries or from of one or more CDRs of 12G3H11, EphO99B-102.147, mice that express antibodies from human genes. Eph099B-208.261, EphO99B-210.248, EphO99B-233.152, EA2, EA3, EA4, EA5, LX-13 or scFv EA44. The determi 0156 Antibodies like all polypeptides have an Isoelectric nation of percent identity of two amino acid Sequences can Point (pl), which is generally defined as the pH at which a be determined by any method known to one skilled in the art, polypeptide carries no net charge. It is known in the art that including BLAST protein searches. protein solubility is typically lowest when the pH of the Solution is equal to the isoelectric point (pl) of the protein. 0152 The present invention also encompasses the use of It is possible to optimize solubility by altering the number Fc variants that immunospecifically bind to at least one Eph and location of ionizable residues in the antibody to adjust receptor or fragments thereof, where Said Fc variants are the pl. For example the p of a polypeptide can be manipu encoded by a nucleotide Sequence that hybridizes to the lated by making the appropriate amino acid Substitutions nucleotide sequence of 12G3H11 (i.e., SEQ ID NO: 62 (e.g., by Substituting a charged amino acid Such as a lysine, and/or SEQ ID NO: 63), 3F2 (i.e., SEQ ID NO: 66 and/or for an uncharged residue. Such as alanine). Without wishing SEQ ID NO: 67), EphO99B-102.147, Eph099B-208.261, to be bound by any particular theory, amino acid Substitu Eph099B-210.248, EphO99B-233.152, EA2, EA3, EA4, tions of an antibody that result in changes of the p of Said EA5, LX-13 or scFv EA44 under stringent conditions. In antibody may improve solubility and/or the stability of the another preferred embodiment, the invention encompasses antibody. One skilled in the art would understand which Fc variants that immunospecifically bind to an Eph receptor amino acid Substitutions would be most appropriate for a or a fragment thereof, Said Fc variants comprising one or particular antibody to achieve a desired pl. The p of a more CDRs encoded by a nucleotide sequence that hybrid protein may be determined by a variety of methods includ izes under Stringent conditions to the nucleotide Sequence of ing but not limited to, isoelectric focusing and various one or more CDRs of 12C3H11, EphO99B-102.147, computer algorithms (see for example Bjelldvist et al., 1993, Eph099B-208.261, EphO99B-210.248, EphO99B-233.152, Electrophoresis 14:1023). In one embodiment, the p of the EA2, EA3, EA4, EA5, LX-13 or scFv EA44. Stringent Fc variants of the invention is between is higher then about hybridization conditions include, but are not limited to, 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. hybridization to filter-bound DNA in 6xsodium chloride/ In another embodiment, the p of the Fc variants of the sodium citrate (SSC) at about 45° C. followed by one or invention is between is higher then 6.5, 7.0, 7.5, 8.0, 8.5, or more washes in 0.2xSSC/0.1% SDS at about 50-65 C., 9.0. In one embodiment, Substitutions resulting in alterations highly Stringent conditions Such as hybridization to filter in the p of the Fc variant of the invention will not signifi bound DNA in 6xSSC at about 45° C. followed by one or cantly diminish its binding affinity for an Eph receptor. It is more washes in 0.1XSSC/0.2% SDS at about 60° C., or any specifically contemplated that the Substitution(s) of the Fc other Stringent hybridization conditions known to those region that result in altered binding to FcyR (described skilled in the art (see, for example, Ausubel, F. M. et al., eds. Supra) may also result in a change in the pl. In a preferred 1989 Current Protocols in Molecular Biology, vol. 1, Green embodiment, Substitution(s) of the Fc region are specifically US 2006/0039904 A1 Feb. 23, 2006 22 chosen to effect both the desired alteration in FcyR binding Similar procedures are disclosed in WO 93/08829, and in and any desired change in p. AS used herein the p value is Traunecker et al., 1991, EMBO J., 10:3655-3659. A more defined as the p of the predominant charge form. The p of directed approach is the generation of a Di-diabody a a protein may be determined by a variety of methods tetravalent bispecific antibody. Methods for producing a including but not limited to, isoelectric focusing and various Di-diabody are known in the art (see e.g., Lu et al., 2003, J computer algorithms (see, e.g., Bjelldvist et al., 1993, Elec Immunol Methods 279:219–32; Marvin et al., 2005, Acta trophoresis 14:1023). Pharmacolical Sinica 26:649). 0157. The Tm of the Fab domain of an antibody, can be 0160 According to a different approach, antibody vari a good indicator of the thermal Stability of an antibody and able domains with the desired binding specificities (anti may further provide an indication of the shelf-life. A lower body-antigen combining sites) are fused to immunoglobulin Tm indicates more aggregation/leSS Stability, whereas a constant domain Sequences. The fusion preferably is with an higher Tm indicates leSS aggregation/more Stability. Thus, immunoglobulin heavy chain constant domain, comprising antibodies having higher Tm are preferable. In one embodi at least part of the hinge, CH2, and CH3 regions. In one ment, the Fab domain of an Fc variant has a Tm value higher embodiment, the first heavy-chain constant region (CH1) than at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., containing the Site necessary for light chain binding is 80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., present in at least one of the fusions. DNAS encoding the 115 C. or 120° C. In another embodiment, the Fab domain immunoglobulin heavy chain fusions and, if desired, the of an Fc variant has a Tm value higher than at least about 50 immunoglobulin light chain, are inserted into Separate C., about 55 C., about 60° C., about 65° C., about 70° C., expression vectors, and are co-transfected into a Suitable about 75 C., about 80° C., about 85°C., about 90° C., about host organism. This provides for great flexibility in adjusting 95° C., about 100° C., about 105 C., about 110° C., about the mutual proportions of the three polypeptide fragments in 115 C. or about 120° C. Thermal melting temperatures embodiments when unequal ratioS of the three polypeptide (Tm) of a protein domain (e.g., a Fab domain) can be chains used in the construction provide the optimum yields. measured using any Standard method known in the art, for It is, however, possible to insert the coding Sequences for example, by differential Scanning calorimetry (see, e.g., two or all three polypeptide chains in one expression vector Vermeer et al., 2000, Biophys.J. 78:394-404; Vermeer et al., when, the expression of at least two polypeptide chains in 2000, Biophys. J. 79: 2150-2154). equal ratioS results in high yields or when the ratios are of no particular Significance. 0158 Fc variants of the invention may be monospecific, bispecific, trispecific or have greater multispecificity. Mul 0.161 In one embodiment of this approach, the bispecific tispecific antibodies may immunospecifically bind to differ antibodies are composed of a hybrid immunoglobulin heavy ent epitopes of desired target molecule or may immunospe chain with a first binding specificity in one arm (e.g., an Eph cifically bind to both the target molecule as well as a receptor), and a hybrid immunoglobulin heavy chain-light heterologous epitope, Such as a heterologous polypeptide or chain pair (providing a second binding specificity) in the Solid Support material. See, e.g., International Publication other arm. It was found that this asymmetric structure Nos. WO 94/04690; WO 93/17715; WO 92/08802; WO facilitates the Separation of the desired bispecific compound 91/00360; and WO92/05793; Tutt, et al., 1991, J. Immunol. from unwanted immunoglobulin chain combinations, as the 147:60-69; U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, presence of an immunoglobulin light chain in only one half 5,573,920, and 5,601,819; and Kostelny et al., 1992, J. of the bispecific molecule provides for a facile way of Immunol. 148:1547; each of which is incorporated herein by separation. This approach is disclosed in WO 94/04690 reference in their entireties). In one embodiment, one of the (incorporated herein by reference in its entirety). For further binding Specificities is for an Eph receptor, the other one is details of generating bispecific antibodies See, for example, for any other antigen (i.e., another Eph receptor, an Ephrin, Suresh et al., 1986, Methods in Enzymology, 121:210 (incor a signaling or effector molecule). porated herein by reference in its entirety). According to 0159 Multispecific antibodies have binding specificities another approach described in WO96/27011 (incorporated for at least two different antigens. While such molecules herein by reference in its entirety), a pair of antibody normally will only bind two antigens (i.e. bispecific anti molecules can be engineered to maximize the percentage of bodies, BSAbs), antibodies with additional specificities such heterodimers which are recovered from recombinant cell as trispecific antibodies are encompassed by the instant culture. The preferred interface comprises at least a part of invention. Examples of BSAbs include without limitation the CH3 domain of an antibody constant domain. In this those with one arm directed against a Integrin O?3 and the method, one or more Small amino acid Side chains from the other arm directed against any other antigen. Methods for interface of the first antibody molecule are replaced with making bispecific antibodies are known in the art. Tradi larger side chains (e.g. tyrosine or tryptophan). Compensa tional production of full-length bispecific antibodies is based tory "cavities” of identical or Similar size to the large Side on the coexpression of two immunoglobulin heavy chain chain(s) are created on the interface of the Second antibody light chain pairs, where the two chains have different Speci molecule by replacing large amino acid Side chains with ficities (Millstein et al., 1983, Nature, 305:537-539 which is Smaller ones (e.g. alanine or threonine). This provides a incorporated herein by reference in its entirety). Because of mechanism for increasing the yield of the heterodimer Over the random assortment of immunoglobulin heavy and light other unwanted end-products Such as homodimerS. chains, these hybridomas (quadromas) produce a potential 0162 Bispecific antibodies include cross-linked or “het mixture of different antibody molecules, of which only one eroconjugate' antibodies. For example, one of the antibodies has the correct bispecific structure. Purification of the correct in the heteroconjugate can be coupled to avidin, the other to molecule, which is usually done by affinity chromatography biotin. Such antibodies have, for example, been proposed to Steps, is rather cumberSome, and the product yields are low. target immune system cells to unwanted cells (U.S. Pat. No. US 2006/0039904 A1 Feb. 23, 2006 23
4,676.980), and for treatment of HIV infection (WO body fragments and/or reduces the concentration of Said 91/00360, WO 92/200373, and EP 03089) The above ref antibodies or antibody fragments to be administered. Anti erences are each incorporated herein by reference in their bodies having increased in Vivo half-lives can be generated entireties. Heteroconjugate antibodies may be made using by techniques known to those of skill in the art. For example, any convenient croSS-linking methods. Suitable croSS-link antibodies with increased in Vivo half-lives can be generated ing agents are well known in the art, and are disclosed in by modifying (e.g., Substituting, deleting or adding) amino U.S. Pat. No. 4,676,980, along with a number of cross acid residues identified as involved in the interaction linking techniques. Each of the above references is incor between the Fc domain and the FcRn receptor (See, e.g., porated herein by reference in its entirety. International Publication Nos. WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737056 and U.S. patent Publi 0163 Antibodies with more than two valencies incorpo cation No. 2003/0190311, each of which are incorporated rating at least one hinge modification of the invention are contemplated. For example, trispecific antibodies can be herein by reference in their entireties). prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991), 0167. In one embodiment, the Fc variants of the inven which is incorporated herein by reference. tion may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to 0164. The Fc variants of the invention encompass single alter its glycosylation, again to alter one or more functional domain antibodies, including camelized single domain anti properties of the antibody. bodies (see e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; 0.168. In still another embodiment, the glycosylation of Reichmann and Muyldermans, 1999, J. Immunol. Meth. the Fc variants of the invention is modified. For example, an 231:25; International Publication Nos. WO 94/04678 and aglycoslated antibody can be made (i.e., the antibody lacks WO 94/25591; U.S. Pat. No. 6,005,079; which are incorpo glycosylation). Glycosylation can be altered to, for example, rated herein by reference in their entireties). increase the affinity of the antibody for a target antigen. Such carbohydrate modifications can be accomplished by, for 0.165 Other antibodies specifically contemplated are example, altering one or more sites of glycosylation within "oligoclonal' antibodies. AS used herein, the term "oligo the antibody Sequence. For example, one or more amino acid clonal” antibodies” refers to a predetermined mixture of Substitutions can be made that result in elimination of one or distinct monoclonal antibodies. See, e.g., PCT publication more variable region framework glycosylation Sites to WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163 thereby eliminate glycosylation at that Site. Such aglycosy which are incorporated by reference herein. Preferably oli lation may increase the affinity of the antibody for antigen. goclonal antibodies consist of a predetermined mixture of antibodies against one or more epitopes are generated in a Such an approach is described in further detail in U.S. Pat. Single cell. More preferably oligoclonal antibodies comprise Nos. 5,714,350 and 6,350,861, each of which is incorporated a plurality of heavy chains capable of pairing with a com herein by reference in its entirety. mon light chain to generate antibodies with multiple Speci 0169. Additionally or alternatively, an Fc variant can be ficities (e.g., PCT publication WO 04/009618 which is made that has an altered type of glycosylation, Such as a incorporated by reference herein). Oligoclonal antibodies hypofucosylated antibody having reduced amounts of fuco are particularly useful when it is desired to target multiple Syl residues or an antibody having increased bisecting epitopes on a single target molecule (e.g., Integrin Clf). GlcNAc structures. Such altered glycosylation patterns have Those skilled in the art will know or can determine what been demonstrated to increase the ADCC ability of antibod type of antibody or mixture of antibodies is applicable for an ies. Such carbohydrate modifications can be accomplished intended purpose and desired need. by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glyco 0166 In one embodiment, antibodies of the present Sylation machinery have been described in the art and can be invention also encompass Fc variants that have half-lives used as host cells in which to express recombinant antibod (e.g., Serum half-lives) in a mammal, preferably a human, of ies of the invention to thereby produce an antibody with greater than 5 days, greater than 10 days, greater than 15 altered glycosylation. See, for example, Shields, R. L. et al. days, preferably greater than 20 days, greater than 25 days, (2002) J. Biol. Chem. 277:26733-26740; Umana et al. greater than 30 days, greater than 35 days, greater than 40 (1999) Nat. Biotech. 17:176-1, as well as, European Patent days, greater than 45 days, greater than 2 months, greater No: EP 1,176,195; PCT Publications WO 03/035835; WO than 3 months, greater than 4 months, or greater than 5 99/54342, each of which is incorporated herein by reference months. In another embodiment, antibodies of the present in its entirety. invention also encompass Fc variants that have half-lives (e.g., Serum half-lives) in a mammal, preferably a human, of 0170 In still another embodiment, the glycosylation of greater than about 5 days, greater than about 10 days, greater an Fc variant of the invention is modified. For example, an than about 15 days, preferably greater than about 20 days, aglycoslated antibody can be made (i.e., the antibody lacks greater than about 25 days, greater than about 30 days, glycosylation). Glycosylation can be altered to, for example, greater than about 35 days, greater than about 40 days, increase the affinity of the antibody for a target antigen. Such greater than about 45 days, greater than about 2 months, carbohydrate modifications can be accomplished by, for greater than about 3 months, greater than about 4 months, or example, altering one or more sites of glycosylation within greater than about 5 months. The increased half-lives of the the antibody Sequence. For example, one or more amino acid antibodies of the present invention in a mammal, preferably Substitutions can be made that result in elimination of one or a human, results in a higher Serum titer of Said antibodies or more variable region framework glycosylation Sites to antibody fragments in the mammal, and thus, reduces the thereby eliminate glycosylation at that Site. Such aglycosy frequency of the administration of Said antibodies or anti lation may increase the affinity of the antibody for antigen. US 2006/0039904 A1 Feb. 23, 2006 24
Such an approach is described in further detail in U.S. Pat. to, peptides, polypeptides, proteins, fusion proteins, nucleic Nos. 5,714,350 and 6,350,861, each of which is incorporated acid molecules, Small molecules, mimetic agents, Synthetic herein by reference in its entirety. drugs, inorganic molecules, and organic molecules. 0171 Additionally or alternatively, an Fc variant can be 0.175. In one embodiment, the present invention encom made that has an altered type of glycosylation, Such as a passes the use of antibodies or fragments thereof recombi hypofucosylated Fc variant having reduced amounts of nantly fused or chemically conjugated (including both cova fucosyl residues or an Fc variant having increased bisecting lent and non-covalent conjugations) to a heterologous GlcNAc structures. Such altered glycosylation patterns have protein or polypeptide (or fragment thereof, preferably to a been demonstrated to increase the ADCC ability of antibod polypeptide of at least 10, at least 20, at least 30, at least 40, ies. Such carbohydrate modifications can be accomplished at least 50, at least 60, at least 70, at least 80, at least 90 or by, for example, expressing the antibody in a host cell with at least 100 amino acids) to generate fusion proteins. In altered glycosylation machinery. Cells with altered glyco another embodiment, the present invention encompasses the Sylation machinery have been described in the art and can be use of antibodies or fragments thereof recombinantly fused used as host cells in which to express recombinant antibod or chemically conjugated (including both covalent and non ies of the invention to thereby produce an antibody with covalent conjugations) to a heterologous protein or polypep altered glycosylation. See, for example, Shields, R. L. et al. tide (or fragment thereof, preferably to a polypeptide of at (2002) J. Biol. Chem. 277:26733-26740; Umana et al. least about 10, at least about 20, at least about 30, at least (1999) Nat. Biotech. 17:176-1, as well as, European Patent about 40, at least about 50, at least about 60, at least about No: EP 1,176, 195; PCT Publications WO 03/035835; WO 70, at least about 80, at least about 90 or at least about 100 99/54342, each of which is incorporated herein by reference amino acids) to generate fusion proteins. The fusion does not in its entirety. necessarily need to be direct, but may occur through linker Sequences. For example, antibodies may be used to target 6.3 Antibody Conjugates and Derivatives heterologous polypeptides to particular cell types, either in Vitro or in Vivo, by fusing or conjugating the antibodies to 0172 Fc variants of the invention include derivatives that antibodies Specific for particular cell Surface receptors. Anti are modified (i.e., by the covalent attachment of any type of bodies fused or conjugated to heterologous polypeptides molecule to the antibody such that covalent attachment). For may also be used in in vitro immunoassays and purification example, but not by way of limitation, the antibody deriva methods using methods known in the art. See e.g., Interna tives include antibodies that have been modified, e.g., by tional publication No. WO 93/21232; European Patent No. glycosylation, acetylation, pegylation, phosphorylation, EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91 amidation, derivatization by known protecting/blocking 99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS groups, proteolytic cleavage, linkage to a cellular ligand or 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446 other protein, etc. Any of numerous chemical modifications 2452, which are incorporated by reference in their entireties. may be carried out by known techniques, including, but not limited to, Specific chemical cleavage, acetylation, formy 0176) The present invention further includes formula lation, metabolic Synthesis of tunicamycin, etc. Additionally, tions comprising heterologous proteins, peptides or polypep the derivative may contain one or more non-classical amino tides fused or conjugated to antibody fragments. For acids. example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, 0173 Antibodies or fragments thereof with increased in F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, Vivo half-lives can be generated by attaching to Said anti a VL CDR, or fragment thereof. Methods for fusing or bodies or antibody fragments polymer molecules Such as conjugating polypeptides to antibody portions are well high molecular weight polyethyleneglycol (PEG). PEG can known in the art. See, e.g., U.S. Pat. Nos. 5,336,603, be attached to Said antibodies or antibody fragments with or 5,622,929, 5,359,046, 5,349,053, 5,447.851, and 5,112,946; without a multifunctional linker either through Site-specific European Pat. Nos. EP 307.434 and EP 367,166; Interna conjugation of the PEG to the N- or C-terminus of said tional publication Nos. WO 96/04388 and WO 91/06570; antibodies or antibody fragments or via epsilon-amino Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: groups present on lysine residues. Linear or branched poly 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590 mer derivatization that results in minimal loSS of biological 5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA activity will be used. The degree of conjugation will be 89:11337-11341 (said references incorporated by reference closely monitored by SDS-PAGE and mass spectrometry to in their entireties). ensure proper conjugation of PEG molecules to the antibod 0177 Additional fusion proteins, e.g., of antibodies that ies. Unreacted PEG can be separated from antibody-PEG immunospecifically bind an Eph receptor (e.g., Supra), may conjugates by, e.g., Size exclusion or ion-exchange chroma be generated through the techniques of gene-shuffling, tography. motif-shuffling, exon-shuffling, and/or codon-Shuffling (col 0.174 Further, antibodies can be conjugated to albumin in lectively referred to as “DNA shuffling”). DNA shuffling order to make the antibody or antibody fragment more stable may be employed to alter the activities of antibodies of the in vivo or have a longer half life in vivo. The techniques are invention or fragments thereof (e.g., antibodies or fragments well known in the art, See e.g., International Publication thereof with higher affinities and lower dissociation rates). Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830, European Patent No. EP 413, 622, all of which are incor 721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr. porated herein by reference. The present invention encom Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends passes the use of antibodies or fragments thereof conjugated Biotechnol. 16(2): 76-82; Hansson, et al., 1999, J. Mol. Biol. or fused to one or more moieties, including but not limited 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques US 2006/0039904 A1 Feb. 23, 2006 25
24(2): 308-313 (each of these patents and publications are toxic agent includes any agent that is detrimental to cells. hereby incorporated by reference in its entirety). Antibodies Examples include paclitaxel, cytochalasin B, gramicidin D, or fragments thereof, or the encoded antibodies or fragments ethidium bromide, emetine, mitomycin, etopoSide, tenopo thereof, may be altered by being Subjected to random Side, Vincristine, vinblastine, colchicin, doxorubicin, dauno mutagenesis by error-prone PCR, random nucleotide inser rubicin, dihydroxy anthracin dione, mitoxantrone, mithra tion or other methods prior to recombination. One or more mycin, actinomycin D, 1-dehydrotestosterone, portions of a polynucleotide encoding an antibody or anti glucocorticoids, procaine, tetracaine, lidocaine, propranolol, body fragment, which portions immunospecifically bind to puromycin, epirubicin, and cyclophosphamide and analogs an Eph receptor may be recombined with one or more or homologs thereof. Therapeutic agents include, but are not components, motifs, Sections, parts, domains, fragments, limited to, antimetabolites (e.g., methotrexate, 6-mercap etc. of one or more heterologous molecules. topurine, 6-thioguanine, cytarabine, 5-fluorouracil decarba Zine), alkylating agents (e.g., mechlorethamine, thioepa 0.178 Moreover, the antibodies or fragments thereof can chlorambucil, melphalan, carmustine (BCNU) and lomus be fused to marker Sequences, Such as a peptide to facilitate tine (CCNU), cyclothosphamide, busulfan, dibromomanni purification. In preferred embodiments, the marker amino tol, Streptozotocin, mitomycin C, and cisdichlorodiamine acid Sequence is a hexa-histidine peptide, Such as the tag platinum (II) (DDP) cisplatin), anthracyclines (e.g., dauno provided in a pCE vector (QIAGEN, Inc., 92.59 Eton rubicin (formerly daunomycin) and doxorubicin), antibiotics Avenue, Chatsworth, Calif., 91311), among others, many of (e.g., dactinomycin (formerly actinomycin), bleomycin, which are commercially available. AS described in Gentz et mithramycin, and anthramycin (AMC)), and anti-mitotic al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for agents (e.g., Vincristine and vinblastine). A more extensive instance, hexa-histidine provides for convenient purification list of therapeutic moieties can be found in PCT publications of the fusion protein. Other peptide tags useful for purifi WO 03/075957; which is incorporated herein by reference in cation include, but are not limited to, the hemagglutinin its entirety. “HA' tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 0182 Further, an antibody or fragment thereof may be 37:767) and the “flag” tag. conjugated to a therapeutic agent or drug moiety that modi fies a given biological response. Therapeutic agents or drug 0179. In other embodiments, Fc variants of the present moieties are not to be construed as limited to classical invention or analogs or derivatives thereof are conjugated to chemical therapeutic agents. For example, the drug moiety a diagnostic or detectable agent. Such antibodies can be may be a protein or polypeptide possessing a desired bio useful for monitoring or prognosing the development or logical activity. Such proteins may include, for example, a progression of a cancer as part of a clinical testing proce toxin Such as abrin, ricin A, Onconase (or another cytotoxic dure, Such as determining the efficacy of a particular therapy. RNase), pseudomonas exotoxin, cholera toxin, or diphtheria Such diagnosis and detection can be accomplished by cou toxin; a protein Such as tumor necrosis factor, C.-interferon, pling the antibody to detectable Substances including, but B-interferon, nerve growth factor, platelet derived growth not limited to various enzymes, Such as but not limited to factor, tissue plasminogen activator, an apoptotic agent, e.g., horseradish peroxidase, alkaline phosphatase, beta-galac TNF-C, TNF-B, AIM I (see, International Publication No. tosidase, or acetylcholinesterase; prosthetic groups, Such as WO 97/33899), AIM II (see, International Publication No. but not limited to streptavidinlbiotin and avidin/biotin; fluo WO 97/34911), Fas Ligand (Takahashi et al., 1994, J. rescent materials, Such as but not limited to, umbelliferone, Immunol., 6:1567), and VEGI (see, International Publica fluorescein, fluorescein isothiocynate, rhodamine, dichloro tion No. WO 99/23105), a thrombotic agent or an anti triazinylamine fluorescein, dansyl chloride or phycoeryth angiogenic agent, e.g., angiostatin or endostatin; or, a bio rin; luminescent materials, Such as but not limited to, logical response modifier Such as, for example, a luminol, bioluminescent materials, Such as but not limited lymphokine (e.g., interleukin-1 (“IL-1'), interleukin-2 (“IL to, luciferase, luciferin, and aequorin; radioactive materials, 2’), interleukin-6 (“IL-6”), granulocyte macrophage colony such as but not limited to iodine (131I, 125I, 123I, 121I.), stimulating factor (“GM-CSF"), and granulocyte colony carbon (14C), sulfur (35S), tritium (3H), indium (115In, Stimulating factor (“G-CSF)), or a growth factor (e.g., 113In, 112In, 111 In.), and technetium (99Tc), thallium growth hormone (“GH')). (201T), gallium (68Ga, 67Ga), palladium (103Pd), molyb denum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 0183 Moreover, an antibody can be conjugated to thera 177Lu, 159Gd, 149Pm, 14OLa, 175Yb, 166Ho, 90Y, 47Sc, peutic moieties Such as a radioactive materials or macrocy 186Re, 188Re, 142 Pr, 105Rh, 97.Ru, 68Ge, 57Co, 65Zn, clic chelators useful for conjugating radiometal ions (see 85Sr., 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and above for examples of radioactive materials). In certain 117Tin; positron emitting metals using various positron embodiments, the macrocyclic chelator is 1,4,7,10-tetraaza emission tomographies, noradioactive paramagnetic metal cyclododecane-N,N',N',N"-tetraacetic acid (DOTA) which ions, and molecules that are radiolabelled or conjugated to can be attached to the antibody via a linker molecule. Such Specific radioisotopes. linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4:2483; 0180. The present invention further encompasses uses of Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zim Fc variants of the invention or fragments thereof conjugated merman et al., 1999, Nucl. Med. Biol. 26:943; each incor to a therapeutic agent. porated by reference in their entireties. 0181 An antibody or fragment thereof may be conju 0.184 Techniques for conjugating therapeutic moieties to gated to a therapeutic moiety Such as a cytotoxin, e.g., a antibodies are well known. Moieties can be conjugated to cytostatic or cytocidal agent, a therapeutic agent or a radio antibodies by any method known in the art, including, but active metal ion, e.g., alpha-emitters. A cytotoxin or cyto not limited to aldehyde/Schiff linkage, Sulphydryl linkage, US 2006/0039904 A1 Feb. 23, 2006 26 acid-labile linkage, cis-aconityl linkage, hydraZone linkage, 0.190 Polyclonal antibodies to an Eph receptor can be enzymatically degradable linkage (see generally Garnett, produced by various procedures well known in the art. For 2002, Adv Drug Deliv Rev 53:171). Techniques for conju example, an Eph receptor or immunogenic fragments gating therapeutic moieties to antibodies are well known, thereof can be administered to various host animals includ See, e.g., Amon et al., “Monoclonal Antibodies For Immu ing, but not limited to, rabbits, mice, rats, etc. to induce the notargeting Of Drugs In Cancer Therapy”, in Monoclonal production of Sera containing polyclonal antibodies Specific Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. for an Eph receptor. Various adjuvants may be used to 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Anti increase the immunological response, depending on the host bodies For Drug Delivery', in Controlled Drug Delivery Species, and include but are not limited to, Freund's (com (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, plete and incomplete), mineral gels Such as aluminum Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents hydroxide, Surface active Substances Such as lySolecithin, In Cancer Therapy: A Review', in Monoclonal Antibodies pluronic polyols, polyanions, peptides, oil emulsions, key 84: Biological And Clinical Applications, Pinchera et al. hole limpet hemocyanins, dinitrophenol, and potentially (eds.), pp. 475-506 (1985); "Analysis, Results, And Future useful human adjuvants such as BCG (bacille Calmette Prospective Of The Therapeutic Use Of Radiolabeled Anti Guerin) and corynebacterium parvum. Such adjuvants are body In Cancer Therapy”, in Monoclonal Antibodies For also well known in the art. Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 0191 Monoclonal antibodies can be prepared using a 303-16 (Academic Press 1985), and Thorpe et al., 1982, wide variety of techniques known in the art including the use Immunol. Rev. 62:119; each incorporated by reference in of hybridoma, recombinant, and phage display technologies, their entireties. or a combination thereof. For example, monoclonal anti 0185. Methods for fusing or conjugating antibodies to bodies can be produced using hybridoma techniques includ polypeptide moieties are known in the art. See, e.g., U.S. ing those known in the art and taught, for example, in Pat. No. 5,336,603; 5,622,929; 5,359,046; 5,349,053, 5,447, Harlow et al., Antibodies: A Laboratory Manual, (Cold 851, and 5,112,946; EP 307434; EP 367,166; PCT Publi Spring Harbor Laboratory Press, 2nd ed. 1988); Hammer cations WO 96/04388 and WO 91/06570; Ashkenazi et al., ling, et al., in: Monoclonal Antibodies and T-Cell Hybrido 1991, PNAS USA 88:10535; Zheng et al., 1995, J 1mmunol mas 563-681 (Elsevier, N.Y., 1981) (said references incor 154:5590; and Vilet al., 1992, PNAS USA 89:11337; each porated by reference in their entireties). The term incorporated by reference in their entireties. The fusion of an “monoclonal antibody” as used herein is not limited to antibody to a moiety does not necessarily need to be direct, antibodies produced through hybridoma technology. The but may occur through linker Sequences. Such linker mol term “monoclonal antibody” refers to an antibody that is ecules are commonly known in the art and described in derived from a single clone, including any eukaryotic, Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson et prokaryotic, or phage clone, and not the method by which it al., 1999, Bioconjug Chem 10:553; Zimmerman et al., 1999, is produced. Nucl Med Biol 26:943; Garnett, 2002, Adv. Drug Deliv Rev 0.192 Methods for producing and screening for specific 53:171, each of which is incorporated herein by reference in antibodies using hybridoma technology are routine and well its entirety. known in the art. Briefly, mice can be immunized with an Eph receptor or a domain thereof (e.g., the extracellular 0186 Alternatively, an antibody can be conjugated to a domain) and once an immune response is detected, e.g., Second antibody to form an antibody heteroconjugate as antibodies Specific for an Eph receptor are detected in the described by Segal in U.S. Pat. No. 4,676,980, which is mouse Serum, the mouse Spleen is harvested and Splenocytes incorporated herein by reference in its entirety. isolated. The splenocytes are then fused by well known 0187 Antibodies may also be attached to solid supports, techniques to any Suitable myeloma cells, for example cells which are particularly useful for immunoassays or purifica from cell line SP20 available from the ATCC. Additionally, tion of the target antigen. Such Solid Supports include, but a RIMMS (repetitive immunization, multiple sites) tech are not limited to, glass, cellulose, polyacrylamide, nylon, nique can be used to immunize an animal (Kilpatrick et al., polystyrene, polyvinyl chloride or polypropylene. 1997, Hybridoma 16:381-9, incorporated herein by refer ence in its entirety). Hybridomas are selected and cloned by 0188 The therapeutic moiety or drug conjugated to an limited dilution. The hybridoma clones are then assayed by antibody or fragment thereof that immunospecifically binds methods known in the art for cells that secrete antibodies to an Eph receptor Should be chosen to achieve the desired capable of binding a polypeptide of the invention. AScites prophylactic or therapeutic effect(s) for a particular disorder fluid, which generally contains high levels of antibodies, can in a Subject. A clinician or other medical perSonnel should be generated by immunizing mice with positive hybridoma consider the following when deciding on which therapeutic clones. moiety or drug to conjugate to an antibody or fragment 0193 Accordingly, monoclonal antibodies can be gener thereof that immunospecifically binds to an Eph receptor: ated by culturing a hybridoma cell Secreting an antibody, the nature of the disease, the Severity of the disease, and the wherein the hybridoma is generated by fusing Splenocytes condition of the Subject. isolated from a mouse immunized with an Eph receptor or immunogenic fragments thereof, with myeloma cells and 6.4 Methods of Generating Antibodies then Screening the hybridomas resulting from the fusion for 0189 The Fc variants of the invention can be produced hybridoma clones that secrete an antibody able to bind an by any method known in the art for the synthesis of Eph receptor. antibodies, in particular, by chemical Synthesis or by recom 0194 The Fc variants of the invention contain novel binant expression techniques. amino acid residues in their Fc regions. Fc variants can be US 2006/0039904 A1 Feb. 23, 2006 27 generated by numerous methods well known to one skilled et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science in the art. Non-limiting examples include, isolating antibody 240: 1041-1043 (said references incorporated by reference in coding regions (e.g., from hybridoma) and making one or their entireties). more desired Substitutions in the Fc, region of the isolated 0198 To generate whole antibodies, PCR primers includ antibody coding region. Alternatively, the variable regions ing VH or VL nucleotide Sequences, a restriction site, and a may be Subcloned into a vector encoding an Fc region flanking Sequence to protect the restriction site can be used comprising one or more high effector function amino acid to amplify the VH or VL sequences in scFv clones. Utilizing residues. Additional methods and details are provided below. cloning techniques known to those of Skill in the art, the PCR amplified VH domains can be cloned into vectors 0.195 Antibody fragments that recognize specific an Eph expressing a VH constant region, e.g., the human gamma receptor epitopes may be generated by any technique known constant, and the PCR amplified VL domains can be cloned to those of skill in the art. For example, Fab and F(ab')2 into Vectors expressing a VL constant region, e.g., human fragments of the invention may be produced by proteolytic kappa or lamba constant regions. In one embodiment, the cleavage of immunoglobulin molecules, using enzymes Such constant region is an Fc region containing at least one high as papain (to produce Fab fragments) or pepsin (to produce effector function amino acid. In another embodiment, the F(ab')2 fragments). F(ab')2 fragments contain the variable vectors for expressing the VH or VL domains comprise a region, the light chain constant region and the CH1 domain promoter, a Secretion signal, a cloning site for both the of the heavy chain. Further, the antibodies of the present variable and constant domains, as well as a Selection marker invention can also be generated using various phage display such as neomycin. The VH and VL domains may also be methods known in the art. cloned into one vector expressing the desired constant regions. The heavy chain conversion vectors and light chain 0196. In phage display methods, functional antibody conversion vectors are then co-transfected into cell lines to domains are displayed on the Surface of phage particles that generate Stable or transient cell lines that express full-length carry the polynucleotide Sequences encoding them. In par antibodies, e.g., IgG, using techniques known to those of ticular, DNA sequences encoding VH and VL domains are skill in the art. amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues). The DNA 0199 Achimeric antibody is a molecule in which differ encoding the VH and VL domains are recombined together ent portions of the antibody are derived from different with an ScFv linker by PCR and cloned into a phagemid immunoglobulin molecules. Methods for producing chi vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is meric antibodies are known in the art. See e.g., Morrison, electroporated in E. coli and the E. coli is infected with 1985, Science 229:1202; Oi et al., 1986, BioTechniques helper phage. Phage used in these methods are typically 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191 filamentous phage including fl and M13 and the VH and VL 202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4.8 16397, domains are usually recombinantly fused to either the phage and 6,311,415, which are incorporated herein by reference in gene III or gene VIII. Phage expressing an antigen binding their entirety. domain that binds to the an Eph receptor epitope of interest 0200 For some uses, including in vivo use of antibodies can be selected or identified with antigen, e.g., using labeled in humans and in vitro detection assays, it may be preferable antigen or antigen bound or captured to a Solid Surface or to use human or chimeric antibodies. Completely human bead. Examples of phage display methods that can be used antibodies are particularly desirable for therapeutic treat to make the antibodies of the present invention include those ment of human Subjects. Human antibodies can be made by disclosed in Brinkman et al., 1995, J. Immunol. Methods a variety of methods known in the art including phage 182:41-50; Ames et al., 1995, J. Immunol. Methods display methods described above using antibody libraries 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. derived from human immunoglobulin Sequences. See also 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT Publica al., 1994, Advances in Immunology 57:191-280; PCT Pub tion Nos. WO 98/46645, WO 98/50433, WO 98/24893, lication Nos. WO 90/02809, WO 91/10737, WO92/01047, WO98/16654, WO 96/34096, WO 96/33735, and WO WO 92/18619, WO 93/11236, WO 95/15982, WO 91/10741; each of which is incorporated herein by reference 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, in its entirety. 5,223,409, 5,403.484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 0201 Ahumanized antibody is an antibody or its variant 5,658,727, 5,733,743 and 5,969,108; each of which is incor or fragment thereof which is capable of binding to a prede porated herein by reference in its entirety. termined antigen and which comprises a framework region having Substantially the amino acid Sequence of a human 0.197 As described in the above references, after phage immunoglobulin and a CDR having Substantially the amino Selection, the antibody coding regions from the phage can be acid Sequence of a non-human immunoglobulin. A human isolated and used to generate whole antibodies, including ized antibody comprises Substantially all of at least one, and human antibodies, or any other desired antigen binding typically two, variable domains (Fab, Fab', F(ab')2, Fabc, fragment, and expressed in any desired host, including Fv) in which all or substantially all of the CDR regions mammalian cells, insect cells, plant cells, yeast, and bacte correspond to those of a non-human immunoglobulin (i.e., ria, e.g., as described below. Techniques to recombinantly donor antibody) and all or substantially all of the framework produce Fab, Fab' and F(ab')2 fragments can also be regions are those of a human immunoglobulin consensus employed using methods known in the art Such as those Sequence. In one embodiment, a humanized antibody also disclosed in International Publication No. WO 92/22324; comprises at least a portion of an immunoglobulin constant Mullinax et al., 1992, BioTechniques 12(6): 864-869; Sawai region (Fc), typically that of a human immunoglobulin. US 2006/0039904 A1 Feb. 23, 2006 28
Ordinarily, the antibody will contain both the light chain as and Sequence comparison to identify unusual framework well as at least the variable domain of a heavy chain. The residues at particular positions. (See, e.g., Queen et al., U.S. antibody also may include the CH1, hinge, CH2, CH3, and Pat. No. 5,585,089; and Riechmann et al., 1988, Nature CH4 regions of the heavy chain. The humanized antibody 332:323, which are incorporated herein by reference in their can be Selected from any class of immunoglobulins, includ entireties.) ing IgM, IgG, Ig), IgA and IgE, and any isotype, including 0202 Human antibodies can also be produced using IgG1, IgG2, IgG3 and IgG4. Usually the constant domain is transgenic mice which are incapable of expressing func a complement fixing constant domain where it is desired that tional endogenous immunoglobulins, but which can express the humanized antibody exhibit cytotoxic activity, and the human immunoglobulin genes. For example, the human class is typically IgG.Sub.1. Where Such cytotoxic activity is heavy and light chain immunoglobulin gene complexes may not desirable, the constant domain may be of the IgG.Sub.2 be introduced randomly or by homologous recombination class. The humanized antibody may comprise Sequences into mouse embryonic Stem cells. Alternatively, the human from more than one class or isotype, and Selecting particular variable region, constant region, and diversity region may be constant domains to optimize desired effector functions is introduced into mouse embryonic Stem cells in addition to within the ordinary skill in the art. The framework and CDR the human heavy and light chain genes. The mouse heavy regions of a humanized antibody need not correspond pre and light chain immunoglobulin genes may be rendered cisely to the parental Sequences, e.g., the donor CDR or the non-functional Separately or Simultaneously with the intro consensus framework may be mutagenized by Substitution, duction of human immunoglobulin loci by homologous insertion or deletion of at least one residue so that the CDR recombination. In particular, homozygous deletion of the JH or framework residue at that Site does not correspond to region prevents endogenous antibody production. The modi either the consensus or the import antibody. Such mutations, fied embryonic Stem cells are expanded and microinjected however, will not be extensive. In one embodiment, at least into blastocysts to produce chimeric mice. The chimeric 75% of the humanized antibody residues will correspond to mice are then bred to produce homozygous offspring that those of the parental framework region (FR) and CDR express human antibodies. The transgenic mice are immu sequences. In another embodiment at least 90% of the nized in the normal fashion with a Selected antigen, e.g., an humanized antibody residues will correspond to those of the Eph receptor or immunogenic fragments thereof. Mono parental framework region (FR) and CDR sequences. In a clonal antibodies directed against the antigen can be further embodiment, greater than 95% of the humanized obtained from the immunized, transgenic mice using con antibody residues will correspond to those of the parental ventional hybridoma technology. The human immunoglo framework region (FR) and CDR sequences. In yet another bulin transgenes harbored by the transgenic mice rearrange embodiment, at least about 75% of the humanized antibody during B cell differentiation, and Subsequently undergo class residues will correspond to those of the parental framework Switching and Somatic mutation. Thus, using Such a tech region (FR) and CDR sequences. In a further embodiment at nique, it is possible to produce therapeutically usefull IgG, least about 90% of the humanized antibody residues will IgA, IgM and IgE antibodies. For an overview of this correspond to those of the parental framework region (FR) technology for producing human antibodies, See Lonberg and CDR Sequences. In yet a further embodiment, greater and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a than about 95% of the humanized antibody residues will detailed discussion of this technology for producing human correspond to those of the parental framework region (FR) antibodies and human monoclonal antibodies and protocols and CDR Sequences. Humanized antibody can be produced for producing Such antibodies, See, e.g., International Pub using variety of techniques known in the art, including but lication Nos. WO 98/24893, WO 96/34096, and WO not limited to, CDR-grafting (European Pat. No. EP239, 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633, 400; International Publication No. WO 91/09967; and U.S. 425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939, Pat. Nos. 5.225,539, 5,530,101, and 5,585,089), veneering 598, which are incorporated by reference herein in their or resurfacing (European Patent Nos. EP 592,106 and EP entirety. In addition, companies Such as Abgenix, Inc. (Free 519,596; Padlan, 1991, Molecular Immunology 28(4/5): mont, Calif.), Genpharm (San Jose, Calif.) and Medarex 489-498; Studnicka et al., 1994, Protein Engineering 7(6): (Princeton, N.J.) can be engaged to provide human antibod 805-814; and Roguska et al., 1994, PNAS 91:969-973), ies directed against a Selected antigen using technology chain shuffling (U.S. Pat. No. 5,565,332), and techniques similar to that described above. disclosed in, e.g., U.S. Pat. No. 6,407.213, U.S. Pat. No. 5,766,886, WO 93.17105, Tan et al., J. Immunol. 169:1119 0203 Further, the antibodies of the invention can, in turn, 25 (2002), Caldas et al., Protein Eng. 13(5): 353-60 (2000), be utilized to generate anti-idiotype antibodies that "mimic' Morea et al., Methods 20(3): 267-79 (2000), Baca et al., J. an Eph receptor using techniques well known to those Biol. Chem. 272(16): 10678-84 (1997), Roguska et al., skilled in the art. (See, e.g., Greenspan & Bona, 1989, Protein Eng. 9(10): 895-904 (1996), Couto et al., Cancer FASEB J. 7(5): 437-444; and Nissinoff, 1991, J. Immunol. Res. 55 (23 Supp): 5973s -5977s (1995), Couto et al., Cancer 147(8): 2429-2438). For example, antibodies of the inven Res. 55(8): 1717-22 (1995), Sandhu J S, Gene 150(2): tion which bind to and competitively inhibit the binding of 409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3): an Eph receptor (as determined by assays well known in the 959-73 (1994). Often, framework residues in the framework art and disclosed infra) to its ligands can be used to generate regions will be Substituted with the corresponding residue anti-idiotypes that "mimic' an Eph receptor binding from the CDR donor antibody to alter, preferably improve, domains and, as a consequence, bind to and neutralize an antigen binding. These framework Substitutions are identi Eph receptor and/or its ligands. Such neutralizing anti fied by methods well known in the art, e.g., by modeling of idiotypes or Fab fragments of Such anti-idiotypes can be the interactions of the CDR and framework residues to used in therapeutic regimens to neutralize an Eph receptor. identify framework residues important for antigen binding The invention provides methods employing the use of US 2006/0039904 A1 Feb. 23, 2006 29 polynucleotides comprising a nucleotide Sequence encoding amino acid Substitutions or deletions of one or more variable an antibody of the invention or a fragment thereof. region cysteine residues participating in an intrachain dis ulfide bond to generate antibody molecules lacking one or 0204. In a preferred embodiment, the nucleotide more intrachain disulfide bonds. Other alterations to the Sequence encoding an antibody that immunospecifically polynucleotide are encompassed by the present invention binds an Eph receptor is obtained and used to generate the and within the skill of the art. Fc variants of the invention. The nucleotide Sequence can be obtained from sequencing hybridoma clone DNA. If a clone 6.5 Polypeptides and Fusion Proteins that Bind to containing a nucleic acid encoding a particular antibody or an Eph Receptor an epitope-binding fragment thereof is not available, but the Sequence of the antibody molecule or epitope-binding frag 0208. The present invention encompasses polypeptides ment thereof is known, a nucleic acid encoding the immu and fusion proteins that immunospecifically bind to an Eph noglobulin may be chemically Synthesized or obtained from receptor. a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly 0209. In a one embodiment, a polypeptide or a fusion A+RNA, isolated from any tissue or cells expressing the protein that immuno-specifically binds to an Eph receptor antibody, Such as hybridoma cells Selected to express an inhibits or reduces the interaction between an Eph receptor antibody) by PCR amplification using synthetic primers that and its ligands by about 25%, 30%, 35%, 45%, 50%, 55%, hybridize to the 3' and 5' ends of the sequence or by cloning 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% in an using an oligonucleotide probe Specific for the particular in vivo or in vitro assay described herein or well-known to gene Sequence to identify, e.g., a cDNA clone from a cDNA one of skill in the art. In this context “about” means plus or library that encodes the antibody. Amplified nucleic acids minus 0.1% to 2.5%. In alternative embodiment, a polypep generated by PCR may then be cloned into replicable tide or a fusion protein that immunospecifically binds to an cloning vectors using any method well known in the art. Eph receptor does not significantly inhibit the interaction between an Eph receptor and its ligands in an in Vivo or in 0205 Once the nucleotide sequence of the antibody is vitro assay described herein or well-known to one of skill in determined, the nucleotide Sequence of the antibody may be the art. manipulated using methods well known in the art for the manipulation of nucleotide Sequences, e.g., recombinant 0210. In a one embodiment, a polypeptide or a fusion DNA techniques, site directed mutagenesis, PCR, etc. (see, protein that immuno-specifically binds to an Eph receptor Or example, the techniques described in Current Protocols comprises an Eph receptor ligand or a fragment thereof in Molecular Biology, F. M. Ausubel et al., ed., John Wiley which immunospecifically binds to an Eph receptor fused to & Sons (Chichester, England, 1998); Molecular Cloning: A an Fc domain. It is specifically contemplated that the Fc Laboratory Manual, 3nd Edition, J. Sambrook et al., ed., domain of Said fusion protein comprises at least one high Cold Spring Harbor Laboratory Press (Cold Spring Harbor, effector function amino acid and/or Substitution as described N.Y., 2001); Antibodies: A Laboratory Manual, E. Harlow Supra. In a preferred embodiment, Said Fc domain is that of and D. Lane, ed., Cold Spring Harbor Laboratory Press an Fc variant of the present invention, the Fc domain of an (Cold Spring Harbor, N.Y., 1988); and Using Antibodies: A Fc variant is hereafter referred to as a variant Fc domain. Laboratory Manual, E. Harlow and D. Lane, ed., Cold Examples of an Eph receptor ligandinclude, but are not Spring Harbor Laboratory (Cold Spring Harbor, N.Y., 1999) limited to, GPI-membrane anchored ligands of the Ephrin-A which are incorporated by reference herein in their entire Subclass (e.g., A1, A2, A3, A4, A5) and transmembrane ties), to generate antibodies having a different amino acid domain-membrane anchored ligands of the Ephrin-B Sub Sequence by, for example, introducing deletions, and/or class (e.g., B1, B2, B3). An alignment of preferred Ephrin insertions into desired regions of the antibodies. molecules of the present invention is shown in FIG. 19. 0206. In a preferred embodiment, one or more substitu 0211. In another embodiment, a polypeptide or a fusion tions are made within the Fc region (e.g. Supra) of an protein that immunospecifically binds to an Eph receptor antibody able to immunospecifically bind an Eph receptor. comprises a bioactive molecule fused to a variant Fc domain In one embodiment, the amino acid Substitutions modify of the present invention. In accordance with these embodi binding to one or more Fc ligands (e.g., FcyRs, C1q) and ments, the bioactive molecule immunospecifically binds to alter ADCC and/or CDC activity. an Eph receptor. Bioactive molecules that immunospecifi cally bind to an Eph receptor include, but are not limited to, 0207. In a specific embodiment, one or more of the CDRs peptides, polypeptides, proteins, Small molecules, mimetic is inserted within framework regions using routine recom agents, Synthetic drugs, inorganic molecules, and organic binant DNA techniques. The framework regions may be molecules. In one embodiment, a bioactive molecule that naturally occurring or consensus framework regions, and immunospecifically binds to an Eph receptor is a polypep preferably human framework regions (see, e.g., Chothia et tide comprising at least 5, at least 10, at least 20, at least 30, al., 1998, J. Mol. Biol. 278: 457-479 for a listing of human at least 40, at least 50, at least 60, at least 70, at least 80, at framework regions). In one embodiment, the polynucleotide least 90 or at least 100 contiguous amino acid residues, and generated by the combination of the framework regions and is heterologous to the amino acid Sequence of the variant Fc CDRs encodes an antibody that immunospecifically binds to domain of the invention. In another embodiment, a bioactive an Eph receptor. In another embodiment, as discussed Supra, molecule that immunospecifically binds to an Eph receptor one or more amino acid Substitutions may be made within is a polypeptide comprising at least about 5, at least about the framework regions, and, in yet another embodiment, the 10, at least about 20, at least about 30, at least about 40, at amino acid Substitutions improve binding of the antibody to least about 50, at least about 60, at least about 70, at least its antigen. Additionally, Such methods may be used to make about 80, at least about 90 or at least about 100 contiguous US 2006/0039904 A1 Feb. 23, 2006 30 amino acid residues, and is heterologous to the amino acid 0216) The present invention further encompasses Sequence of the variant Fc domain of the invention. polypeptides and fusion proteins that immunospecifically bind to an Eph receptor fused to a variant Fc further 0212. In another embodiment, a peptide, a polypeptide or conjugated to a therapeutic moiety. A polypeptide or a fusion a fusion protein that immunospecifically binds to an Eph protein that immunospecifically binds to an Eph receptor receptor comprises a polypeptide having an amino acid may be conjugated to a therapeutic moiety Such as a cyto sequence that is at least 35%, at least 40%, at least 45%, at toxin, e.g., a cytostatic or cytocidal agent, an agent which least 50%, at least 55%, at least 60%, at least 65%, at least has a potential therapeutic benefit, or a radioactive metalion, 70%, at least 75%, at least 80%, at least 85%, at least 90%, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes at least 95%, or at least 99% identical to the amino acid any agent that is detrimental to cells. Examples of a thera Sequence of an Eph receptor ligand (e.g., Ephrin-A and/or-B peutic moieties and cytotoxin or cytotoxic agents are listed subclass, see FIG. 19) or a fragment thereof fused to a Supra (see Section 6.3 entitled "Antibody Conjugates And variant Fc domain of the present invention. In a further embodiment, a peptide, a polypeptide or a fusion protein that Derivatives”) immunospecifically binds to an Eph receptor comprises a 0217 Polypeptides, proteins and fusion proteins can be polypeptide having an amino acid Sequence that is at least produced by standard recombinant DNA techniques or by about 35%, at least about 40%, at least about 45%, at least protein Synthetic techniques, e.g., by use of a peptide about 50%, at least about 55%, at least about 60%, at least Synthesizer. For example, a nucleic acid molecule encoding about 65%, at least about 70%, at least about 75%, at least a peptide, polypeptide, protein or a fusion protein can be about 80%, at least about 85%, at least about 90%, at least Synthesized by conventional techniques including auto about 95%, or at least about 99% identical to the amino acid mated DNA synthesizers. Alternatively, PCR amplification Sequence of an Eph receptor ligand (e.g., Ephrin-A and/or-B of gene fragments can be carried out using anchor primers subclass, see FIG. 19) or a fragment thereof fused to a which give rise to complementary overhangs between two variant Fc domain of the present invention. consecutive gene fragments which can Subsequently be annealed and reamplified to generate a chimeric gene 0213 The present invention provides polypeptides or Sequence (see, e.g., Current Protocols in Molecular Biology, fusion proteins that immunospecifically bind to an Eph Ausubel et al., eds., John Wiley & Sons, 1992). Moreover, receptor comprising a variant Fc domain of the present a nucleic acid encoding a bioactive molecule can be cloned invention fused to a polypeptide encoded by a nucleic acid into an expression vector containing the variant Fc domain molecule that hybridizes to the nucleotide sequence encod or a fragment thereof Such that the bioactive molecule is ing an Eph receptor ligand (e.g., Ephrin-A and/or -B Sub linked in-frame to the variant Fc domain or variant Fc class, see FIG. 19), or a fragment thereof. domain fragment. 0214. In a specific embodiment, a polypeptide or a fusion 0218 Methods for fusing or conjugating polypeptides to protein that immunospecifically binds to an Eph receptor the constant regions of antibodies are known in the art. See, comprises a variant Fc domain of the present invention fused e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349, to a polypeptide encoded by a nucleic acid molecule that 053, 5,447.851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, hybridizes to the nucleotide Sequence encoding an Eph and 5,112,946, EP307434; EP 367,166; EP 394,827; Inter receptor ligand (e.g., Ephrin-A and/or -B Subclass, see FIG. national Publication Nos. WO 91/06570, WO 96/04388, 19) or a fragment thereof under Stringent conditions, e.g., WO 96/22024, WO 97/34631, and WO 99/04813; Ash hybridization to filter-bound DNA in 6x sodium chloride/ kenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535 sodium citrate (SSC) at about 45° C. followed by one or 10539; Traunecker et al., 1988, Nature, 331:84-86; Zheng et more washes in 0.2xSSC/0.1% SDS at about 50-65 C., al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, under highly Stringent conditions, e.g., hybridization to Proc. Natl. Acad. Sci. USA 89:11337-11341, which are filter-bound nucleic acid in 6xSSC at about 45 C. followed incorporated herein by reference in their entireties. by one or more washes in 0.1XSSC/0.2% SDS at about 68° C., or under other Stringent hybridization conditions which 0219. The nucleotide sequences encoding a bioactive are known to those of skill in the art (see, for example, molecule and an Fc domain or fragment thereof may be Ausubel, F. M. et al., eds., 1989, Current Protocols in obtained from any information available to those of skill in Molecular Biology, Vol. I, Green Publishing Associates, Inc. the art (i.e., from GenBank, the literature, or by routine and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 cloning). The nucleotide Sequences encoding Eph receptor ligands may be obtained from any available information, and 2.10.3). e.g., from GenBank, the literature or by routine cloning. See, 0215. The present invention also encompasses polypep e.g., FIG. 3. The nucleotide Sequence coding for a polypep tides and fusion proteins that immunospecifically bind to an tide a fusion protein can be inserted into an appropriate Eph receptor comprising of a variant Fc domain, fused to expression vector, i.e., a vector that contains the necessary marker Sequences, Such as but not limited to, a peptide, to elements for the transcription and translation of the inserted facilitate purification. In preferred embodiments, the marker protein-coding Sequence. A variety of host-vector Systems amino acid Sequence is a hexa-histidine peptide, Such as the may be utilized in the present invention to express the tag provided in a pCE vector (QIAGEN, Inc., 92.59 Eton protein-coding Sequence. These include but are not limited Avenue, Chatsworth, Calif., 91311), among others, many of to mammalian cell Systems infected with virus (e.g., vac which are commercially available. Other peptide tags useful cinia virus, adenovirus, etc.); insect cell Systems infected for purification include, but are not limited to, the hemag with virus (e.g., baculovirus); microorganisms. Such as yeast glutinin"HA' tag, which corresponds to an epitope derived containing yeast vectors, or bacteria transformed with bac from the influenza hemagglutinin protein (Wilson et al., teriophage, DNA, plasmid DNA, or cosmid DNA. The 1984, Cell 37:767) and the “flag” tag. expression elements of vectors vary in their Strengths and US 2006/0039904 A1 Feb. 23, 2006
Specificities. Depending on the host-vector System utilized, DNA, plasmid DNA or cosmid DNA expression vectors any one of a number of Suitable transcription and translation containing antibody or fusion protein coding Sequences, elements may be used. yeast (e.g., Saccharomyces Pichia) transformed with recom binant yeast expression vectors containing antibody or 6.6 Recombinant Expression of Antibodies and fusion protein coding Sequences; insect cell Systems infected Fusion Proteins with recombinant virus expression vectors (e.g., baculovi 0220 Recombinant expression of an Fc variant or fusion rus) containing antibody or fusion protein coding sequences; protein comprising a variant Fc domain (referred to herein as plant cell Systems infected with recombinant virus expres an “variant Fc fusion protein', or “variant Fc fusion”), Sion vectors (e.g., cauliflower mosaic virus, CaMV; tobacco derivative, analog or fragment thereof, (e.g., a heavy or light mosaic virus, TMV) or transformed with recombinant plas chain of an antibody of the invention or a portion thereof or mid expression vectors (e.g., Ti plasmid) containing anti a single chain antibody of the invention), requires construc body or fusion protein coding Sequences, or mammalian cell tion of an expression vector containing a polynucleotide that systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) encodes the antibody, or fusion protein. Once a polynucle harboring recombinant expression constructs containing otide encoding an antibody molecule or a heavy or light promoters derived from the genome of mammalian cells chain of an antibody, or fusion protein of the invention has (e.g., metallothionein promoter) or from mammalian viruses been obtained, the vector for the production of the antibody (e.g., the adenovirus late promoter; the vaccinia virus 7.5K or fusion protein molecule may be produced by recombinant promoter). In one embodiment, bacterial cells Such as DNA technology using techniques well known in the art. Escherichia coli, and in another embodiment, eukaryotic Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody or fusion protein cells, especially for the expression of whole recombinant encoding nucleotide Sequence are described herein. Methods antibody or fusion protein molecules, are used for the that are well known to those skilled in the art can be used to expression of a recombinant antibody or fusion protein construct expression vectors containing antibody or fusion molecules. For example, mammalian cells Such as Chinese protein coding Sequences and appropriate transcriptional hamster ovary cells (CHO), in conjunction with a vector and translational control Signals. These methods include, for Such as the major intermediate early gene promoter element example, in vitro recombinant DNA techniques, Synthetic from human cytomegalovirus is an effective expression techniques, and in Vivo genetic recombination. The inven system for antibodies (Foecking et al., 1986, Gene 45:101; tion, thus, provides replicable vectors comprising a nucle and Cockett et al., 1990, Bio/Technology 8:2). In a specific otide Sequence encoding an Fc variant or variant Fc fusion embodiment, the expression of nucleotide Sequences encod of the invention, operably linked to a promoter. Such vectors ing antibodies or fusion protein that bind to an Eph receptor may include the nucleotide Sequence encoding the constant is regulated by a constitutive promoter, inducible promoter region of the antibody molecule (see, e.g., International or tissue Specific promoter. Publication No. WO 86/05807; International Publication 0223) In bacterial systems, a number of expression vec No. WO 89/01036; and U.S. Pat. No. 5,122,464) and the tors may be advantageously Selected depending upon the use variable domain of the antibody, or a polypeptide for gen intended for the antibody or fusion protein molecule being erating an variant Fc fusion may be cloned into Such a vector expressed. For example, when a large quantity of Such a for expression of the full length antibody chain (e.g. heavy protein is to be produced, for the generation of pharmaceu or light chain), or complete variant Fc fusion protein. tical compositions of an antibody or fusion protein mol 0221) The expression vector is transferred to a host cell ecule, Vectors that direct the expression of high levels of by conventional techniques and the transfected cells are then fusion protein products that are readily purified may be cultured by conventional techniques to produce an Fc Vari desirable. Such vectors include, but are not limited to, the E. ant or variant Fc fusion protein of the invention. Thus, the coli expression vector puR278 (Ruther et al., 1983, EMBO invention includes host cells containing a polynucleotide 12:1791), in which the antibody or fusion protein coding encoding an Fc variant or variant Fc fusion protein of the Sequence may be ligated individually into the vector in invention or fragments thereof, or a heavy or light chain frame with the lac Z coding region So that a lac Z-fusion thereof, or portion thereof, or a Single chain antibody of the protein is produced; plN vectors (Inouye & Inouye, 1985, invention, operably linked to a heterologous promoter. In Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, preferred embodiments for the expression of double-chained 1989, J. Biol. Chem. 24:5503–5509); and the like. pCEX antibodies, vectors encoding both the heavy and light chains vectors may also be used to express foreign polypeptides as may be co-expressed in the host cell for expression of the fusion proteins with glutathione 5-transferase (GST). In entire immunoglobulin molecule, as detailed below. general, Such fusion proteins are Soluble and can easily be purified from lysed cells by adsorption and binding to matrix 0222. A variety of host-expression vector systems may be glutathione agarose beads followed by elution in the pres utilized to express the antibody or fusion protein molecules ence of free glutathione. The pGEX vectors are designed to of the invention (see, e.g., U.S. Pat. No. 5,807,715). Such include thrombin or factor Xa protease cleavage Sites So that host-expression Systems represent vehicles by which the the cloned target gene product can be released from the GST coding Sequences of interest may be produced and Subse moiety. quently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide 0224. In an insect System, Autographa Californica coding Sequences, express an antibody or fusion protein nuclear polyhedrosis virus (AcNPV) is used as a vector to molecule of the invention in situ. These include but are not express foreign genes. The virus grows in Spodoptera fru limited to microorganisms such as bacteria (e.g., E. coli and giperda cells. The antibody or fusion protein coding B. Subtilis) transformed with recombinant bacteriophage Sequence may be cloned individually into non-essential US 2006/0039904 A1 Feb. 23, 2006 32 regions (for example the polyhedrin gene) of the virus and active in lymphoid cells (Grosschedl et al., 1984, Cell placed under control of an AcNPV promoter (for example 38:647-658; Adames et al., 1985, Nature 318:533-538; the polyhedrin promoter). Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), 0225. In mammalian host cells, a number of viral-based mouse mammary tumor virus control region which is active expression Systems may be utilized. In cases where an in testicular, breast, lymphoid and mast cells (Leder et al., adenovirus is used as an expression vector, the antibody or 1986, Cell 45:485-495), albumin gene control region which fusion protein coding Sequence of interest may be ligated to is active in liver (Pinkert et al., 1987, Genes and Devel. an adenovirus transcription/translation control complex, 1:268-276), alpha-fetoprotein gene control region which is e.g., the late promoter and tripartite leader Sequence. This active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. chimeric gene may then be inserted in the adenovirus 5:1639-1648; Hammer et al., 1987, Science 235:53-58; genome by in vitro or in Vivo recombination. Insertion in a alpha 1-antitrypsin gene control region which is active in the non-essential region of the viral genome (e.g., region E1 or liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), E3) will result in a recombinant virus that is viable and beta-globin gene control region which is active in myeloid capable of expressing the antibody or fusion protein mol cells (Mogram et al., 1985, Nature 315:338-340; Kollias et ecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. al., 1986, Cell 46:89-94, myelin basic protein gene control Natl. Acad. Sci. USA 8 1:355-359). Specific initiation sig region which is active in oligodendrocyte cells in the brain nals may also be required for efficient translation of inserted (Readhead et al., 1987, Cell 48:703-712); myosin light antibody coding Sequences. These signals include the ATG chain-2 gene control region which is active in skeletal initiation codon and adjacent Sequences. Furthermore, the muscle (Sani, 1985, Nature 314:283-286); neuronal-specific initiation codon must be in phase with the reading frame of enolase (NSE) which is active in neuronal cells (Morelli et the desired coding Sequence to ensure translation of the al., 1999, Gen. Virol. 80:571-83); brain-derived neu entire insert. These exogenous translational control signals rotrophic factor (BDNF) gene control region which is active and initiation codons can be of a variety of origins, both in neuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. natural and Synthetic. The efficiency of expression may be Res. Com. 253:818-823); glial fibrillary acidic protein enhanced by the inclusion of appropriate transcription (GFAP) promoter which is active in astrocytes (Gomes et al., enhancer elements, transcription terminators, etc. (See, e.g., 1999, Braz. J Med Biol Res 32(5): 619–631; Morelli et al., Bittner et al., 1987, Methods in Enzymol. 153:516-544). 1999, Gen. Virol. 80:571-83) and gonadotropic releasing 0226. The expression of an antibody or a fusion protein hormone gene control region which is active in the hypo may be controlled by any promoter or enhancer element thalamus (Mason et al., 1986, Science 234:1372-1378). known in the art. Promoters which may be used to control 0227 Expression vectors containing inserts of a gene the expression of the gene encoding an antibody or fusion encoding an antibody or fusion protein can be identified by protein include, but are not limited to, the SV40 early three general approaches: (a) nucleic acid hybridization, (b) promoter region (Bernoist and Chambon, 1981, Nature presence or absence of “marker gene functions, and (c) 290:304-310), the promoter contained in the 3' long terminal expression of inserted Sequences. In the first approach, the repeat of Rous Sarcoma virus (Yamamoto, et al., 1980, Cell presence of a gene encoding a peptide, polypeptide, protein 22:787-797), the herpes thymidine kinase promoter (Wagner or a fusion protein in an expression vector can be detected et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441–1445), by nucleic acid hybridization using probes comprising the regulatory Sequences of the metallothionein gene (Brin Sequences that are homologous to an inserted gene encoding ster et al., 1982, Nature 296:39–42), the tetracycline (Tet) the peptide, polypeptide, protein or the fusion protein, promoter (Gossen et al., 1995, Proc. Nat. Acad. Sci. USA respectively. In the Second approach, the recombinant vec 89:5547-5551); prokaryotic expression vectors such as the tor/host System can be identified and Selected based upon the f3-lactamase promoter (Villa-Kamaroffet al., 1978, Proc. presence or absence of certain “marker gene functions (e.g., Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter thymidine kinase activity, resistance to antibiotics, transfor (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:21–25; mation phenotype, occlusion body formation in baculovirus, See also “Useful proteins from recombinant bacteria” in etc.) caused by the insertion of a nucleotide sequence Scientific American, 1980, 242:74–94); plant expression encoding an antibody or fusion protein in the vector. For vectors comprising the nopaline Synthetase promoter region example, if the nucleotide Sequence encoding the antibody (Herrera-Estrella et al., Nature 303:209-213) or the cauli or fusion protein is inserted within the marker gene Sequence flower mosaic virus 35S RNA promoter (Gardner et al., of the vector, recombinants containing the gene encoding the 1981, Nucl. Acids Res. 9:2871), and the promoter of the antibody or fusion protein insert can be identified by the photosynthetic enzyme ribulose biphosphate carboxylase absence of the marker gene function. In the third approach, (Herrera-Estrella et al., 1984, Nature 310:115-120); pro recombinant expression vectors can be identified by assay moter elements from yeast or other fungi Such as the Gal 4 ing the gene product (e.g., antibody or fusion protein) promoter, the ADC (alcohol dehydrogenase) promoter, PGK expressed by the recombinant. Such assays can be based, for (phosphoglycerol kinase) promoter, alkaline phosphatase example, on the physical or functional properties of the promoter, and the following animal transcriptional control fusion protein in in vitro assay Systems, e.g., binding with regions, which exhibit tissue Specificity and have been anti-bioactive molecule antibody. utilized in transgenic animals: elastase I gene control region 0228. In addition, a host cell strain may be chosen which which is active in pancreatic acinar cells (Swift et al., 1984, modulates the expression of the inserted Sequences, or Cell 38:639-646; Omitz et al., 1986, Cold Spring Harbor modifies and processes the gene product in the Specific Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepa fashion desired. Expression from certain promoters can be tology 7:425-515); insulin gene control region which is elevated in the presence of certain inducers; thus, expression active in pancreatic beta cells (Hanahan, 1985, Nature of the genetically engineered fusion protein may be con 315:115-122), immunoglobulin gene control region which is trolled. Furthermore, different host cells have characteristic US 2006/0039904 A1 Feb. 23, 2006 33 and Specific mechanisms for the translational and post midine kinase (Wigler et al., 1977, Cell 11:223), hypoxan translational processing and modification (e.g., glycosyla thine-guanine phosphoribosyltransferase (Szybalska & Szy tion, phosphorylation of proteins). Appropriate cell lines or balski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and host Systems can be chosen to ensure the desired modifica adenine phosphoribosyltransferase (Lowy et al., 1980, Cell tion and processing of the foreign protein expressed. For 22:817) genes can be employed in tk-, hgprt- or aprt-cells, example, expression in a bacterial System will produce an respectively. Also, antimetabolite resistance can be used as unglycosylated product and expression in yeast will produce the basis of selection for dhfr, which confers resistance to a glycosylated product. Eukaryotic host cells that possess methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA the cellular machinery for proper processing of the primary 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA transcript (e.g., glycosylation, and phosphorylation) of the 78:1527); gpt, which confers resistance to mycophenolic gene product may be used. Such mammalian host cells acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA include, but are not limited to, CHO, VERY, BHK, Hela, 78.2072); neo, which confers resistance to the aminoglyco COS, MDCK, 293, 3T3, WI38, NS0, and in particular, side G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. neuronal cell lines such as, for example, SK-N-AS, SK-N- 150:1); and hygro, which confers resistance to hygromycin FI, SK-N-DZhuman neuroblastomas (Sugimoto et al., 1984, (Santerre et al., 1984, Gene 30:147) genes. J. Natl. Cancer Inst. 73: 51-57), SK-N-SH human neuro blastoma (Biochim. Biophys. Acta, 1982, 704: 450-460), 0231. Once a peptide, polypeptide, protein or a fusion Daoy human cerebellar medulloblastoma (He et al., 1992, protein of the invention has been produced by recombinant Cancer Res. 52: 1144-1148) DBTRG-05MG glioblastoma expression, it may be purified by any method known in the cells (Kruse et al., 1992, In vitro Cell. Dev. Biol. 28A: art for purification of a protein, for example, by chromatog 609-614), IMR-32 human neuroblastoma (Cancer Res., raphy (e.g., ion exchange, affinity, particularly by affinity for 1970, 30: 2110-2118), 1321N1 human astrocytoma (Proc. the Specific antigen after Protein A, and sizing column Natl Acad. Sci. USA, 1977, 74: 4816), MOG-G-CCM chromatography), centrifugation, differential Solubility, or human astrocytoma (Br. J. Cancer, 1984, 49: 269), U87MG by any other Standard technique for the purification of human glioblastoma-astrocytoma (Acta Pathol. Microbiol. proteins. Scand., 1968, 74: 465-486), A172 human glioblastoma 0232 The expression levels of an antibody or fusion (Olopade et al., 1992, Cancer Res. 52: 2523-2529), C6 rat protein molecule can be increased by vector amplification glioma cells (Benda et al., 1968, Science 161: 370-371), (for a review, see Bebbington and Hentschel, The use of Neuro-2a mouse neuroblastoma (Proc. Natl. Acad. Sci. vectors based on gene amplification for the expression of USA, 1970, 65: 129-136), NB41A3 mouse neuroblastoma cloned genes in mammalian cells in DNA cloning, Vol.3. (Proc. Natl. Acad. Sci. USA, 1962,48: 1184-1190), SCP (Academic Press, New York, 1987)). When a marker in the sheep choroid plexus (Bolin et al., 1994, J. Virol. Methods vector System expressing an antibody or fusion protein is 48: 211-221), G355-5, PG-4 Cat normal astrocyte (Haapala amplifiable, increase in the level of inhibitor present in et al., 1985, J. Virol. 53: 827-833), Mpf ferret brain (Trow culture of host cell will increase the number of copies of the bridge et al., 1982, In vitro 18: 952-960), and normal cell marker gene. Since the amplified region is associated with lines such as, for example, CTX TNA2 rat normal cortex the antibody gene, production of the antibody or fusion brain (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89: protein will also increase (Crouse et al., 1983, Mol. Cell. 6467-6471) such as, for example, CRL7030 and Hss78Bst. Biol. 3:257). Furthermore, different vector/host expression Systems may effect processing reactions to different extents. 0233. The host cell may be co-transfected with two 0229. For long-term, high-yield production of recombi expression vectors of the invention. For example, the first nant proteins, Stable expression is often preferred. For vector encoding a heavy chain derived polypeptide and the example, cell lines which Stably express an antibody or Second vector encoding a light chain derived polypeptide. fusion protein may be engineered. Rather than using expres The two vectors may contain identical Selectable markers, Sion vectors that contain Viral origins of replication, host which enable equal expression of heavy and light chain cells can be transformed with DNA controlled by appropri polypeptides. Alternatively, a single vector may be used ate expression control elements (e.g., promoter, enhancer, which encodes, and is capable of expressing, a fusion Sequences, transcription terminators, polyadenylation sites, protein or both heavy and light chain polypeptides. The etc.), and a selectable marker. Following the introduction of coding Sequences for the fusion protein or heavy and light the foreign DNA, engineered cells may be allowed to grow chains may comprise cDNA or genomic DNA. for 1-2 days in an enriched medium, and then are Switched to a Selective medium. The Selectable marker in the recom 6.7 Antagonists of an Eph Receptor binant plasmid conferS resistance to the Selection and allows 0234. The invention specifically encompasses Fc vari cells to Stably integrate the plasmid into their chromosomes ants, or variant Fc fusions, of the invention that are antago and grow to form foci that in turn can be cloned and nists of at least one Eph receptor. The terms “antagonist' and expanded into cell lines. This method may advantageously “antagonists" when used herein refers to a to any protein, be used to engineer cell lines that express an Fc variant or polypeptide, peptide, peptidomimetic, glycoprotein, anti variant Fc fusion protein that Specifically binds to an Eph body, antibody fragment, carbohydrate, nucleic acid, organic receptor. Such engineered cell lines may be particularly molecule, inorganic molecule, large molecule, or Small useful in Screening and evaluation of compounds that affect molecule that blocks, inhibits, reduces or neutralizes the the activity of a polypeptide or a fusion protein that immu function, activity and/or expression of a target molecule, nospecifically binds to an Eph receptor. Such as an Eph receptor. In various embodiments, an antago 0230. A number of selection systems may be used, nist reduces the function, activity and/or expression of including but not limited to the herpes simplex virus thy another molecule by at least 10%, at least 15%, at least 20%, US 2006/0039904 A1 Feb. 23, 2006 34 at least 25%, at least 30%, at least 35%, at least 40%, at least but not limited to inflammatory diseases, autoimmune dis 45%, at least 50%, at least 55%, at least 60%, at least 65%, eases, bone metabolism related disorders, angiogenic related at least 70%, at least 75%, at least 80%, at least 85%, at least disorders, disorders related to aberrant expression and/or 90%, at least 95% or at least 99% relative to a control Such activity of an Eph receptor, and cancer. Such Fc variants as phosphate buffered saline (PBS). More specifically, an and/or variant Fc fusions can be used in the methods and antagonist of at least one Eph receptor inhibits, reduces or formulations of the present invention. neutralizes the function, activity and/or expression of at least one Eph receptor or inhibits or reduces at least one Eph 6.8 Antibody Agonists of an Eph Receptor receptor-mediated pathology. 0238 An “agonist' herein refers to to any protein, 0235 Antagonists may act by interfering with the binding polypeptide, peptide, peptidomimetic, glycoprotein, anti of a receptor to a ligand and Vice versa, by incapacitating or body, antibody fragment, carbohydrate, nucleic acid, organic killing cells which have been activated by a ligand, and/or molecule, inorganic molecule, large molecule, or Small by interfering with receptor or ligand activation (e.g. molecule which is capable of activating one or more of the tyrosine kinase activation) or signal transduction after ligand biological activities of a target molecule, Such as an Eph binding to a cellular receptor. The antagonist may com receptor. In various embodiments, an agonist activates the pletely block receptor-ligand interactions or may Substan function, activity and/or expression of another molecule by tially reduce Such interactions. All Such points of interven at least 10%, at least 15%, at least 20%, at least 25%, at least tion by an antagonist Shall be considered equivalent for 30%, at least 35%, at least 40%, at least 45%, at least 50%, purposes of this invention. Thus, included within the Scope at least 55%, at least 60%, at least 65%, at least 70%, at least of the invention are antagonists (e.g. Fc variants and/or 75%, at least 80%, at least 85%, at least 90%, at least 95% variant Fc fusion proteins (infra)) that bind to Eph receptor, or at least 99% relative to a control such as phosphate Ephligand or a complex of an Eph receptor and Ephligand; buffered saline (PBS). In further various embodiments, an Soluble Eph receptor or Soluble Ephligand fused to a variant agonist activates the function, activity and/or expression of Fc region of the invention, as well as Synthetic or native another molecule by at least about 10%, at least about 15%, Sequence peptides which bind to Eph receptor or Ephligand at least about 20%, at least about 25%, at least about 30%, fused to a variant Fc region of the invention. In addition, an at least about 35%, at least about 40%, at least about 45%, Eph receptor “antagonist' herein includes, but is not limited at least about 50%, at least about 55%, at least about 60%, to antibodies that antagonize at least one Eph receptor and at least about 65%, at least about 70%, at least about 75%, also inhibit cancer cell phenotype (e.g., colony formation in at least about 80%, at least about 85%, at least about 90%, Soft agar or tubular network formation in a three-dimen at least about 95% or at least about 99% relative to a control Sional basement membrane or extracellular matrix prepara such as phosphate buffered saline (PBS). More specifically, tion). An Eph receptor antagonist may or may not prefer an agonist of at least one Eph receptor activates the function, entially bind an Eph epitope that is exposed in a cancer cell activity and/or expression of at least one Eph receptor. It is relative to a non-cancer cell and may or may not have a low Specifically contemplated that the action of agonizing at Koff rate. least one Eph receptor will result in the inhibition or 0236. The invention also provides methods for screening reduction in at least one Eph receptor-mediated pathology. for antagonists of an Eph receptor. Said Screening methods 0239 Agonists may, for example, act by activating a include but are not limited to assays that monitor Eph target molecule and/or mediating Signal transduction. receptor activity (e.g., phosphorylation of the Eph receptor, Included within the scope of the invention are Fc variants degradation of the Eph receptor protein and downstream and/or variant Fc fusion proteins (infra) that bind to and Eph receptor mediated signaling events), ligand binding activate the Eph receptor, Ephligand or a complex of an Eph and/or plasma concentration. These and additional methods receptor and Eph ligand; Soluble Eph receptor or Soluble are further described infra (see section 6.9 entitled “Bio Eph ligand fused to a variant Fc region of the invention logical Assays”) and in PCT Publication Nos. WO which activate the Eph receptor, as well as Synthetic or 03/094859, WO 04/014292, WO 04/069264, WO native Sequence peptides, Eph receptor or Eph ligand fused 04/028551, WO 03/004057, U.S. Patent 5,795,734 and U.S. to a variant Fc region of the invention that bind to and patent application Ser. Nos. 10/863,729, 10/770,543; each of activate the Eph receptor, Eph ligand or a complex of Eph which is incorporated herein by reference in their entireties. receptor and Eph ligand. In addition, the invention provides for a method to manipu late both the ADCC activity and the binding affinities for 0240. In addition, an Eph receptor “agonist” herein FcyR of antibodies identified using Such Screening methods. includes, but is not limited to antibodies that agonize at least one Eph receptor and also inhibit cancer cell phenotype 0237) The Fc of antibodies identified from such screening (e.g., colony formation in Soft agar or tubular network methods can be substituted as described Supra to alter ADCC formation in a three-dimensional basement membrane or and/or CDC activity and to modify binding affinities for one or more Fc ligand (e.g., FcyRS, C1q). ). Other antagonistic extracellular matrix preparation). An Eph receptor agonist binding molecules (e.g., Eph receptor ligands and variants may or may not preferentially bind an Eph epitope that is thereof) identified from Such screening methods can be exposed in a cancer cell relative to a non-cancer cell and fused to a variant Fc domain of the invention. It is further may or may not have a low K rate. contemplated that the Fc variants of the newly identified Eph 0241 Thus, in one embodiment, the Eph antibodies (and receptor antagonistic antibodies and variant Fc fusions of the Fc variants thereof) of the invention agonize Eph receptor newly identified Eph receptor antagonists are useful for the (e.g., Epha2 and/or Epha-4) signaling and increase phospho prevention, management and treatment of Eph receptor rylation of an Eph receptor and/or the Eph-associated mediated and/or associated diseases and disorders, including polypeptides. US 2006/0039904 A1 Feb. 23, 2006 35
0242. The invention also provides methods for screening limited to, ELISA, Western Blot analysis, cell surface stain for antibody agonists of an Eph receptor. Said Screening ing, inhibition of ligand-receptor interactions, flow cytomet methods include but are not limited to assays that monitor ric analysis and those disclosed in International Publication Eph receptor activity (e.g., phosphorylation of the Eph Nos. WO 04/014292, WO 03/094859, WO 04/069264, WO receptor, degradation of the Eph receptor protein and down 04/028551, WO 03/004057, WO 03/040304, WO 00/78815, Stream Eph receptor mediated signaling events), ligand WO 02/070007 and WO 03/075957, U.S. Pat. Nos. 5,795, binding and/or plasma concentration. These and additional 734, 6,248,326 and 6,472,403 and U.S. patent applications methods are further described infra (see section 6.9 entitled Ser. Nos.10/770,543, and 10/863,729, Pecheur et al., 2002, “Biological Assays”) and in PCT Publication Nos. WO FASEB J. 16(10): 1266-1268; Almed et al., The Journal of 03/094859, WO 04/014292, WO 04/069264, WO Histochemistry & Cytochemistry 50:1371-1379 (2002), all 04/028551, WO 03/004057, U.S. Pat. No. 5,795,734 and of which are incorporated herein by reference. For example, U.S. patent application Ser. Nos. 10/863,729, 10/770,543; the binding affinity, specificity and the off-rate of an Fc each of which is incorporated herein by reference in their variant and/or variant Fc fusion protein can be determined entireties. In addition, the present invention provides for a by a competitive binding assay with the parental anti-Eph method to manipulate both the ADCC activity and the receptor antibody, by measuring the inhibitory activity of an binding affinities for FcyR of antibodies identified using Fc variant, or variant Fc fusion protein of the invention on Such Screening methods. an Eph receptor binding to Ephrin. One example of a competitive binding assay is a radioimmunoassay compris 0243 The Fc of antibodies identified from such screening ing the incubation of labeled Eph receptor (e.g., 3H or 125I) methods can be substituted as described Supra to alter ADCC with the Fc variant of interest in the presence of increasing and/or CDC activity and to modify binding affinities for one amounts of unlabeled Eph receptor, and the detection of the or more Fc ligand (e.g., FcyRs, C1q). Other agonistic monoclonal antibody bound to the labeled Eph receptor. The binding molecules (e.g., Eph receptor ligands and variants affinity of an Fc variant for an Eph receptor and the binding thereof) identified from Such screening methods can be off-rates can be determined from the data by Scatchard plot fused to a variant Fc domain of the invention. It is further analysis. Competition with a Second antibody can also be contemplated that the Fc variants of the newly identified Eph determined using radioimmunoassays. In this case, an Eph receptor agonistic antibodies and variant Fc fusions of the receptor is incubated with an Fc variant conjugated to a newly identified Eph receptor agonists are useful for the labeled compound (e.g., 3H or 125I) in the presence of prevention, management and treatment of Eph receptor increasing amounts of a Second unlabeled monoclonal anti mediated and/or associated diseases and disorders, including body. but not limited to inflammatory diseases, autoimmune dis eases, bone metabolism related disorders, angiogenic related 0246 The kinetic parameters of an Fc variant, or variant disorders, disorders related to aberrant expression and/or Fc fusion protein may also be determined using any Surface activity of an Eph receptor, and cancer. Such Fc variants plasmon resonance (SPR) based assays known in the art. For and/or variant Fc fusions can be used in the methods and a review of SPR-based technology see Mullet et al., 2000, formulations of the present invention. Methods 22: 77-91; Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et al., 1998, Current Opinion in 6.9 Biological Assays Biotechnology 9: 97-101; Rich et al., 2000, Current Opinion in Biotechnology 11: 54-61; all of which are incorporated 0244. The antagonistic and/or agonistic effect of one or herein by reference in their entirety. Additionally, any of the more Fc variant, or variant Fc fusion protein of the invention SPR instruments and SPR based methods for measuring on an Eph receptor activity can be determined by any protein-protein interactions described in U.S. Pat. Nos. method known in the art. Methods include but are not 6,373,577; 6,289.286; 5,322,798; 5,341,215; 6,268,125 are limited to those described infra and in PCT publications WO contemplated in the methods of the invention, all of which 04/014292, WO 03/094859, WO 04/069264, WO are incorporated herein by reference in their entirety. 04/028551, WO 03/004057, WO 03/040304, U.S. Pat. No. 5,795,734 and U.S. patent applications Ser. Nos. 10/770, 0247 The binding specificity of an Fc variant, or variant 543, and 10/863,729; each of which are incorporated herein Fc fusion protein of the invention to an Eph receptor can be by reference in their entireties. For example, the blockage of assessed by any method known in the art including but not an Eph receptor activity and/or the plasma concentration of limited to, measuring binding to an Eph receptor and its an Eph receptor can be assayed by any technique known in croSSreactivity to other Eph receptors. In addition, binding the art that measures the activity and/or expression of an Eph affinity and Specificity can be determined by a competitive receptor, including but not limited to, Western blot, Northern binding assay with the parental anti-Eph receptor antibody blot, RNase protection assays, enzymatic activity assays, in against an Eph receptor or by measuring the inhibitory Situ hybridization, immunohistochemistry, and immunocy activity of an Fc variant, or variant Fc fusion protein of the tochemistry. More specifically, the activity of an Fc variant invention on Eph receptor binding to its ligand, Ephrin. or variant Fc fusion protein can be determined by measuring 0248. The inhibitory, antagonistic and/or agonistic activ binding to an Eph receptor and its croSS-reactivity to other ity of an Fc variant, or variant Fc fusion protein of the Eph receptors, inhibition or Stimulation of Eph receptor invention can be tested by numerous assays known to one phosphorylation, Eph receptor degradation, Eph receptor skilled in the art including but not limited to, phosphoryla ligand (e.g., Ephrin) binding. tion assays (Koolpe et al., 2002, J Biol Chem 277:46974 and 0245. The binding specificity, affinity and functional Gu et al., 2001, Mol Cell Biol 21:4579), cell adhesion activity of an Fc variant, or variant Fc fusion protein of the (Lawrenson et al., 2002, J Cell Sci 115:1059 and Davy et al., invention can be characterized in various in vitro binding 2000, EMBO 19:5396), endothelial cell migration assays and cell adhesion assays known in the art, including but Such as the transwell cell migration assay (Choi et al., 1994, US 2006/0039904 A1 Feb. 23, 2006 36
J. Vascular Sur 19:125-134 and Leavesly et al., 1993, J Cell coated coverslips in 24-well dishes. Cells are allowed to Biol 121:163-170) and cell rounding assays (Miao et al., adhere for 48 hours, then treated with media with or without 2000, Nature Cell. Bio. 2:62) signal transduction assays an Fc variant and/or variant Fc fusion protein for 10 minutes. (Sharfe et al., 2002, Eur J. Immunol 32:3745; Zou et al., Plates or coverslips are washed, fixed and Stained and 1999, PNAS U.S.A. 96:13813) (all of which are incorpo visualized by microscopy. Cells treated with an Fc variant rated herein by reference in their entirety). The ability of an and/or variant Fc fusion protein show cell rounding relative Fc variant, or variant Fc fusion protein of the invention to to cells treated with media lacking an Fc variant and/or inhibit a cancer cell phenotype can be determined by in Vitro variant Fc fusion protein, indicating decreased attachment to assays including, but not limited to, colony formation in Soft the ECM matrix. agar, tubular network formation in a three-dimensional basement membrane or extracellular matrix preparation 0252) The ability of the antibodies of the invention to Such as MATRIGELTM. inhibit cancer cell formation in Soft agar may be assayed (Such assays may be carried out, e.g., as described in 0249 Phosphorylation/degradation assays can be per Zelinski et al., 2001, Cancer Res. 61:2301, incorporated by formed as described in U.S. Pat. No. 5,766,863 and PCT reference herein in its entirety). Briefly, cells are Suspended Publication Nos. WO 03/094859, WO 04/014292, and WO in soft agar for 7 days at 37 C. in the presence of an Fc 04/069264 (all of which are incorporated herein by reference variant and/or variant Fc fusion protein or control Solution in their entirety). Briefly, cells are incubated in the presence (PBS). Following incubation with an Fc variant and/or of an Fc variant and/or variant Fc fusion protein or control variant Fc fusion protein or PBS, cells are washed and molecule for at least 15 minutes at 37 C. Cell lysates are incubated with either an anti-Fc variant and/or anti-variant then immunoprecipitated with an appropriate anti-Eph anti Fc fusion protein Secondary monoclonal antibody (Second body (e.g., anti-Eph receptor antibodies are available from ary mab) or PBS. Colony formation is scored microscopi commercial Sources including Santa Cruz, Biotechnology, cally. Clusters containing at least three cells are Scored as a Inc.; Santa Cruz, Calif.) resolved by SDS-PAGE, and Sub positive. jected to Western blot analysis with a cocktail of the anti phosphotyrosine antibodies 4G10 (Upstate Biotechnology; 0253) Tumor cell behavior within a three-dimensional Lake Placid N.Y.) and PY20 (BD Transduction Laborato microenvironment, such as MATRIGELTM, can reliably ries, Franklin Lakes, N.J.). Increased Eph receptor phospho predict the differentiation State and aggressiveness of breast rylation following treatment with Fc variant and/or variant epithelial cells. Monolayer cultures of benign (MCF-10A) or Fc fusion protein, indicates that the Fc variant and/or variant malignant (MDA-MB-231) breast epithelial cells are incu Fc fusion protein agonize the Eph receptor and likely bated on MATRIGELTM in the presence of an Fc variant promote auto-phosphorylation, while a decrease in phoS and/or variant Fc fusion protein or control solution (PBS). phorylation is consistent with antagonistic activity of an Fc The behavior of cells on MATRIGELTM is analyzed as variant and/or variant Fc fusion protein. Western blot West described in Zelinski et al. (2001, Cancer Res. 61:2301). ern blot analyses and immunoprecipitations are performed Briefly, tissue culture dishes are coated with MATRIGELTM as described previously (Zantek et al., 1999, Cell Growth (Collaborative Biomedical Products, Bedford, Mass.) at 37 Diff. 10:629, which is incorporated by reference in its C. before adding 1x105 MDA-MB-231 or MCF-10A cells entirety). Briefly, detergent extracts of cell monolayers are previously incubated on ice for 1 hour with the Fc variant extracted in Tris-buffered saline containing 1% Triton X-100 and/or variant Fc fusion protein or control solution (PBS). (Sigma, St. Louis, Mo.). After measuring protein concen Cells are incubated on MATRIGELTM for 24 hours at 37°C., trations (BioPad, Hercules, Calif.), 1.5 mg of cell lysate is and cell behavior is assessed using an Olympus IX-70 immunoprecipitated, resolved by SDS-PAGE and trans inverted light microScope. All images are recorded onto 35 ferred to nitrocellulose (PROTRANTM, Schleicher and mm film (T-Max-400. Kodak, Rochester, N.Y.). Schuell, Keene, N.H.). Antibody binding is detected by 0254 Additional examples of in vitro assays, e.g., West enhanced chemiluminescence (Pierce, Rockford, Ill.) and ern blotting analysis, flow cytometric analysis, cell adhesion autoradiography (Kodak X-OMAT; Rochester, N.Y.). assay to cortical bone and extracellular matrix proteins, cell 0250 Cell adhesion and cell rounding assays can also be migration assay, cell invasion assay, and cell proliferation performed as described in Miao, et al. (Nature Cell Biol. assay, can be found in Pecheur et al., 2002, FASEB J. 2:62, 2000), which is incorporated by reference herein in its 16(10): 1266-1268, of which the entire text is incorporated entirety. To Study cell adhesion, briefly, cells are plated in herein by reference. triplicate onto 96-well plates previously coated with various ECM proteins or poly-L-lysine. Cells are plated at a density 0255 The anti-cancer activity of an Fc variant, or variant of 1x105 cells per well in the presence or absence of an Fc Fc fusion protein of the invention can be determined by variant and/or variant Fc fusion protein and allowed to using various experimental animal models for the Study of adhere for 30 minutes at 37 C. Non-adherent cells are cancer Such as the Scid mouse model or transgenic mice washed from the Wells, and adherent cells are fixed, stained, where a mouse Eph receptor is replaced with the human Eph and quantified by measuring absorbance on an enzyme receptor, nude mice with human Xenografts, animal models Such as hamsters, rabbits, etc. known in the art and described linked immunosorbent assay (ELISA) reader. Cells treated in Relevance of Tumor Models for Anticancer Drug Devel with an Fc variant and/or variant Fc fusion protein show opment (1999, eds. Fiebig and Burger); Contributions to decreases in attachment to ECM protein-treated wells rela Oncology (1999, Karger); The Nude Mouse in Oncology tive to control cells allowed to adhere in the absence of and Research (1991, eds. Boven and Winograd); and Anticancer Fc variant or variant Fc fusion protein of the invention. Drug Development Guide (1997 ed. Teicher), herein incor 0251 For cell rounding assays, briefly, cells are plated porated by reference in their entireties. For example the onto ECM protein coated six-well dishes, or ECM protein ability of the Fc variants an/or variant Fc fusion proteins of US 2006/0039904 A1 Feb. 23, 2006 37 the invention to inhibit tumor cancer growth in Vivo can be Many assays Standard in the art can be used to assess Such assayed as follows, MDA-MB-231 breast cancer cells are Survival and/or growth; for example, cell proliferation can implanted Subcutaneously into athymic mice. After the be assayed by measuring 3H-thymidine incorporation, by tumors have grown to an average Volume of 100 mm3, mice direct cell count, by detecting changes in transcriptional are administered an Fc variant an/or variant Fc fusion activity of known genes Such as proto-oncogenes (e.g., fos, protein or PBS control intraperitoneally twice a week for 3 myc) or cell cycle markers; cell viability can be assessed by weeks. Tumor growth is assessed and expressed as a ratio of trypan blue Staining, differentiation can be assessed visually the tumor volume divided by initial tumor volume (100 based on changes in morphology, etc. mm3). Tumor growth is allowed to proceed until tumor volume reaches 1000 mm3. Survival of the mice is assessed 0258 Prophylactic or therapeutic agents can be tested in by Scoring the percent of mice living each day post treat Suitable animal model Systems prior to testing in humans, ment. Similarly, animal models of colon cancer can be including but not limited to in rats, mice, chicken, cows, generated by passaging colon cancer cells in animals, e.g., monkeys, rabbits, hamsters, etc. nude mice, leading to the appearance of tumors in these 0259. The principle animal models for known in the art animals. An orthotopic transplant model of human colon and widely used are known and described in the art as cancer in nude mice has been described, for example, by described above. Wang et al., 1994, Cancer Research, 54: 4726 and Too et al., 0260 Further, any assays known to those skilled in the art 1995, Cancer Research, 55: 681. This model is based on the can be used to evaluate the prophylactic and/or therapeutic so-called “METAMOUS5" sold by AntiCancer, Inc., (San utility of the combinatorial therapies disclosed herein for Diego, Calif.). treatment or prevention of cancer. 0256 Various animal models known in the art that are relevant to a targeted disease or disorder, e.g., inflammatory 6.10 Prophylactic and Therapeutic Uses diseases, autoimmune diseases, diseases or disorders asso ciated with aberrant bone metabolism and/or aberrant angio 0261 AS discussed above, agents that immunospecifi genesis, cancers, disorders associated with aberrant Eph cally bind an Eph receptor can be utilized for the inhibition receptor expression and/or activity can be used, including of angiogenesis or the inhibition of other functions mediated but not limited to, growth factor or tumor-induced angio or influenced by an Eph receptor, including but not limited genesis in the chick chorioallantoic membrane (CAM) (see, to cell proliferation, cell attachment, cell migration, granu e.g., Ausprunk et al. (1980) Am. J. Pathol, 79:597-618; lation tissue development, and/or inflammation. Accord Ossonski et al. (1975) Cancer Res., 40:2300-2309; Brooks ingly, the present invention relates to the use of agents that et al. (1994) Science, 264:569-571 and Brooks et al., (1994), immunospecifically bind and preferably modulate the activ Cell, 79:1157-1164), Vx2 carcinoma cells in rabbits (see, ity of at least one Eph receptor for the prevention, manage e.g., Voelkel et al., (1975) Metabolism 24:973-86), tumors ment, treatment or amelioration of cancer or one or more induced in BALB/c nu/nu mice and SCID mice with Sub Symptoms thereof and/or the inhibition of angiogenesis. cutaneously implanted human bone fragments (SCID-hu 0262 Angiogenesis, also called neovascularization, is the man-bone model). Additional examples of tumor models can process where new blood vessels form from pre-existing be found in Teicher et al., Tumor Models in Cancer vessels within a tissue. AS described above, Eph receptors Research, (Humana Press, Totowa, N.J., 2001). are believed to play a role in this process this process. There 0257 The protocols and formualations of the invention are a variety of pathological conditions that require new are preferably tested in vitro, and then in vivo, for the blood vessel formation or tissue angiogenesis and inhibition desired therapeutic or prophylactic activity, prior to use in of this process inhibits the pathological condition. AS Such, humans. For example, in Vitro assays which can be used to pathological conditions that require angiogenesis for growth determine whether administration of a Specific therapeutic or maintenance may be considered to be Eph receptor protocol formulation or combination therapy of the inven mediated diseases. The extent of treatment, or reduction in tion is indicated, include in vitro cell culture assays in which Severity, of these diseases will therefore depend on the a patient tissue Sample is grown in culture, and exposed to extent of inhibition of angiogenesis. These Eph receptor or otherwise contacted with a formulation of the invention, mediated diseases include, for example, inflammatory dis and the effect of Such a formulation upon the tissue Sample orderS Such as immune and non-immune inflammation, is observed. The tissue sample can be obtained by biopsy thrombosis, acute ischemic Stroke, chronic articular rheu from the patient. This test allows the identification of the matism, pSoriasis, disorders associated with inappropriate or therapeutically most effective prophylactic or therapeutic inopportune invasion of vessels. Such as diabetic retinopathy, agent(s) for each individual patient. In various specific neovascular glaucoma and capillary proliferation in athero embodiments, in vitro assays can be carried out with rep Sclerotic plaques as well as cancer disorders. resentative cells of cell types involved in an autoimmune disorder, an inflammatory disorder, a disorder associated 0263 Such cancer disorders can include, for example, with aberrant expression and/or activity of at least one Eph Solid tumors, tumor metastasis, angiofibromas, angiosarco receptor, to determine if a formulation of the invention has mas, retrolental, fibroplasia, he mangiomas, Kaposi's Sar a desired effect upon Such cell types. A lower level of coma, carcinomas, carcinosarcomas, and other cancers proliferation or Survival of the contacted cells indicates that which require neovascularization to Support tumor growth. the composition of the invention is effective to treat the Additional diseases which are considered angiogenic condition in the patient. Alternatively, instead of culturing include psoriasis and rheumatoid arthritis as well as retinal cells from a patient, a formulation of the invention may be diseases Such as macular degeneration. Screened using cells of a tumor or malignant cell line, 0264. Further examples of such cancers include the fol Osteoclasts, endothelial cells or an endothelial cell line. lowing: leukemias, Such as but not limited to, acute leuke US 2006/0039904 A1 Feb. 23, 2006 38 mia, acute lymphocytic leukemia, acute myelocytic leuke dermoid carcinoma), adenocarcinoma, large-cell carcinoma mias, Such aS, myeloblastic, promyelocytic, and Small-cell lung cancer, testicular cancerS Such as but not myelomonocytic, monocytic, and erythroleukemia leuke limited to germinal tumor, Seminoma, anaplastic, classic mias and myelodysplastic Syndrome; chronic leukemias, (typical), Spermatocytic, nonseminoma, embryonal carci Such as but not limited to, chronic myelocytic (granulocytic) noma, teratoma carcinoma, choriocarcinoma (yolk-sac leukemia, chronic lymphocytic leukemia, hairy cell leuke tumor), prostate cancers Such as but not limited to, adeno mia; polycythemia Vera, lymphomas Such as but not limited carcinoma, leiomyosarcoma, and rhabdomyosarcoma, penal to Hodgkin's disease, non-Hodgkin's disease; multiple cancers, oral cancerS Such as but not limited to Squamous myelomas Such as but not limited to Smoldering multiple cell carcinoma, basal cancers, Salivary gland cancerS Such as myeloma, nonsecretory myeloma, Osteosclerotic myeloma, but not limited to adenocarcinoma, mucoepidermoid carci plasma cell leukemia, Solitary plasmacytoma and extramed noma, and adenoidcystic carcinoma, pharynx cancerS Such ullary plasmacytoma; WaldenStrom's macroglobulinemia; as but not limited to Squamous cell cancer, and Verrucous; monoclonal gammopathy of undetermined Significance; skin cancerS Such as but not limited to, basal cell carcinoma, benign monoclonal gammopathy; heavy chain disease, bone Squamous cell carcinoma and melanoma, Superficial spread and connective tissue Sarcomas Such as but not limited to ing melanoma, nodular melanoma, lentigo malignant mela bone Sarcoma, Osteosarcoma, chondrosarcoma, Ewing's Sar noma, acral lentiginous melanoma, kidney cancerS Such as coma, malignant giant cell tumor, fibrosarcoma of bone, but not limited to renal cell carcinoma, adenocarcinoma, chordoma, perioSteal Sarcoma, Soft-tissue Sarcomas, hypemephroma, fibrosarcoma, transitional cell cancer (renal angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's pelvis and/or uterer); Wilms tumor; bladder cancers such as Sarcoma, leiomyosarcoma, liposarcoma, lymphangiosar but not limited to transitional cell carcinoma, Squamous cell coma, neurilemmoma, rhabdomyosarcoma, Synovial Sar cancer, adenocarcinoma, carcinosarcoma. In addition, can coma; brain tumors Such as but not limited to, glioma, cers include myxosarcoma, Osteogenic Sarcoma, endothe astrocytoma, brain Stem glioma, ependymoma, oligodendro liosarcoma, lymphangioendotheliosarcoma, mesothelioma, glioma, nonglial tumor, acoustic neurinoma, craniopharyn Synovioma, he mangioblastoma, epithelial carcinoma, cysta gioma, medulloblastoma, meningioma, pineocytoma, pine denocarcinoma, bronchogenic carcinoma, Sweat gland car oblastoma, primary brain lymphoma; breast cancer cinoma, Sebaceous gland carcinoma, papillary carcinoma including but not limited to adenocarcinoma, lobular (Small and papillary adenocarcinomas (for a review of Such disor cell) carcinoma, intraductal carcinoma, medullary breast ders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. cancer, mucinous breast cancer, tubular breast cancer, pap Lippincott Co., Philadelphia and Murphy et al., 1997, illary breast cancer, Paget's disease, and inflammatory Informed Decisions: The Complete Book of Cancer Diag breast cancer, adrenal cancer Such as but not limited to nosis, Treatment, and Recovery, Viking Penguin, Penguin pheochromocytom and adrenocortical carcinoma, thyroid Books U.S.A., Inc., United States of America). cancer Such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic 0265 Accordingly, the present invention relates to the thyroid cancer; pancreatic cancer Such as but not limited to, use of agents that immunospecifically bind and preferably insulinoma, gastrinoma, glucagonoma, Vipoma, Somatosta modulate at least one Eph receptor activity for the preven tin-Secreting tumor, and carcinoid or islet cell tumor, pitu tion, management, treatment or amelioration of cancer, Solid itary cancerS Such as but limited to Cushing's disease, tumor metastasis, as well as, inflammatory diseaseS Such as prolactin-Secreting tumor, acromegaly, and diabetes insip rheumatoid arthritis and pSoriasis or one or more Symptoms ius, eye cancerS Such as but not limited to ocular melanoma thereof and/or the inhibition of angiogenesis or conditions Such as iris melanoma, choroidal melanoma, and cilliary asSociated there with body melanoma, and retinoblastoma; vaginal cancerS Such 0266. In one embodiment, the methods and formulations as Squamous cell carcinoma, adenocarcinoma, and mela of the invention are used for inhibiting angiogenesis. In a noma, Vulvar cancer Such as Squamous cell carcinoma, Specific embodiment, the methods and formulations of the melanoma, adenocarcinoma, basal cell carcinoma, Sarcoma, invention are used for inhibiting angiogenesis in a Solid and Paget’s disease; cervical cancerS Such as but not limited tumor. In another embodiment, the methods and formula to, Squamous cell carcinoma, and adenocarcinoma, uterine tions of the invention are used for inhibiting angiogenesis in cancerS Such as but not limited to endometrial carcinoma an inflamed, angiogenic tissue including but not limited to and uterine Sarcoma, Ovarian cancerS Such as but not limited retinal tissues and joint tissues. to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and Stromal tumor; esophageal cancerS Such as but 0267 Further, the present invention provides Fc variants not limited to, Squamous cancer, adenocarcinoma, adenoid that immunospecifically bind and preferably inhibit an Eph cyctic carcinoma, mucoepidermoid carcinoma, adenoSqua receptor which are useful for therapeutic purposes, more mous carcinoma, Sarcoma, melanoma, plasmacytoma, Ver Specifically, for the treatment, prevention, management or rucous carcinoma, and oat cell (Small cell) carcinoma; amelioration of cancer. Specific examples of cancers that can Stomach cancerS Such as but not limited to, adenocarcinoma, be prevented, managed, treated or ameliorated in accordance fungating (polypoid), ulcerating, Superficial spreading, dif with the invention include, but are not limited to, cancer of fusely spreading, malignant lymphoma, liposarcoma, fibro the head, neck, eye, mouth, throat, esophagus, chest, bone, Sarcoma, and carcinosarcoma; colon cancers, rectal cancers; lung, colon, rectum, colorectal or other gastrointestinal tract liver cancerS Such as but not limited to hepatocellular organs, Stomach, Spleen, renal, Skeletal muscle, Subcutane carcinoma and hepatoblastoma; gallbladder cancerS Such as ous tissue, metastatic melanoma, endometrial, prostate, adenocarcinoma, cholangiocarcinomas Such as but not lim breast, ovaries, testicles or other reproductive organs, skin, ited to pappillary, nodular, and diffuse; lung cancerS Such as thyroid, blood, lymph nodes, kidney, liver, pancreas, and non-Small cell lung cancer, Squamous cell carcinoma (epi brain or central nervous System. US 2006/0039904 A1 Feb. 23, 2006 39
0268. In a specific embodiment, the methods and formu passes treatment protocols that enhance the prophylactic or lations of the invention are used for the prevention, man therapeutic effect of said Fc variants and/or variant Fc fusion agement, treatment or amelioration of a primary or Second proteins. ary cancer that expresses an Eph receptor. In another embodiment, the methods and formulations of the invention 0273. The invention provides methods for preventing, are used for the prevention, management, treatment or managing, treating or ameliorating cancer that has the poten amelioration of a primary or Secondary cancer that does not tial to metastasize or has metastasized to an organ or tissue express an Eph receptor. In a preferred embodiment, the (e.g., bone) or one or more Symptoms thereof, said methods methods and formulations are used for the prevention, comprising administering to a Subject in need thereof one or management, treatment or amelioration of a cancer that has more doses of a prophylactically or therapeutically amount the potential to metastasize or has metastasized to other of one or more Fc variants and/or variant Fc fusion protein tissues or organs (e.g., bone). In another preferred embodi of the invention. ment, the methods and formulations of the invention are used for the prevention, management, treatment or amelio 0274 The invention provides methods for preventing, ration of lung cancer, prostate cancer, Ovarian cancer, mela managing, treating or ameliorating cancer or one or more noma, bone cancer or breast cancer. Methods using agents Symptoms thereof, Said methods comprising administering that immunospecifically inhibit an Eph receptor include but to a Subject in need thereof one or more doses of a prophy are not limited to those disclosed in PCT publications WO lactically or therapeutically effective amount of one or more 03/094859 and WO 04/014292 and U.S. application Ser. No. Fc variants and/or variant Fc fusion protein with modified 10/863,729, each of which is herein incorporated by refer binding affinity to one or more Fc ligand (e.g., FcyRs, C1q) ence in its entirety. and altered ADCC and/or CDC activity fused or conjugated to a moiety (e.g., a therapeutic agent or drug). Examples of 0269. The invention provides methods for screening for a moiety that an Fc variant can be fused or conjugated to antibody and other antagonists and agonists of an Eph include, but are not limited to those disclosed in PCT receptor. Further, the invention provides for a method to publication WO 2003/075957 which is herein incorporated manipulate the ADCC and/or CDC activity and the binding by reference in its entirety. Examples of Fc variants and affinities for one or more Fc ligand (e.g., FcyR, C1q) of the variant Fc fusion proteins with modified binding affinity to antibodies and/or other antagonists or agonists identified one or more Fc ligand (e.g., FcyRs, C1q) and altered ADCC using Such Screening methods. The Eph receptor antagonists and/or CDC activity include, but are not limited to, those and agonists identified and manipulated utilizing Such meth variants disclosed Supra. ods can be used for the prevention, treatment, management or amelioration of Eph receptor-mediated and/or associated 0275. The present invention encompasses protocols for diseases and disorders or one or more Symptoms thereof, the prevention, management, treatment or amelioration of including but not limited to cancer, inflammatory and Ep receptor-mediated diseases and disorders or one or more autoimmune diseases either alone or in combination with Symptoms thereof, including but not limited to, cancer, other therapies. inflammatory and autoimmune diseases or one or more Symptoms thereof in which one or more Fc variants and/or 0270. The invention also provides variant Fc fusion pro variant Fc fusion protein with modified binding affinity to teins that immunospecifically bind to an Eph receptor. Said one or more Fc ligand (e.g., FcyRs, C1q) and altered ADCC variant Fc fusion proteins can be used for the prevention, and/or CDC activity that immunospecifically binds to an treatment, management or amelioration of Eph receptor Eph receptor is used in combination with the administration mediated and/or associated diseases and disorders or one or of a dosage of a prophylactically or therapeutically effective more Symptoms thereof, including but not limited to cancer, amount of one or more other therapies other than an Fc inflammatory and autoimmune diseases either alone or in variant and/or variant fusion protein. The invention is based, combination with other therapies. in part, on the recognition that the Fc variants and/or variant 0271 In a specific embodiment, Fc variants and/or Fc fusion proteins of the invention potentiate and Synergize variant fusion proteins of the invention that immunospecifi with, enhance the effectiveness of, improve the tolerance of, cally bind to an Eph receptor are used for the prevention, and/or reduce the Side effects caused by, other therapies, management, treatment or amelioration of cancer or one or including current Standard and experimental chemothera more Symptoms thereof. In a preferred embodiment, Fc pies. The combination therapies of the invention have addi variant antibodies and/or Fc variant fusion proteins of the tive potency, an additive therapeutic effect or a Synergistic invention used for the prevention, management, treatment or effect. The combination therapies of the invention enable amelioration of cancer or one or more Symptoms thereof are lower dosages of the therapy (e.g., prophylactic or thera antagonists of an Eph receptor. peutic agents) utilized in conjunction with Fc variants and/or variant Fc fusion proteins for the prevention, management, 0272. The invention also encompasses the use of Fc treatment or amelioration of Eph receptor-mediated diseases variants and/or variant Fc fusion proteins with modified and disorders or one or more Symptoms thereof, including binding affinity to one or more Fc ligand (e.g., FcyRs, C1q) but not limited to, cancer, inflammatory and autoimmune and altered ADCC and/or CDC activity that immunospe diseases and/or less frequent administration of Such prophy cifically bind to an Eph receptor conjugated or fused to a lactic or therapeutic agents to a Subject with an Eph receptor moiety (e.g., therapeutic agent or drug) for prevention, mediated diseases (e.g., cancer) to improve the quality of life treatment, management or amelioration of Integrin CB of Said Subject and/or to achieve a prophylactic or therapeu mediated diseases and disorders or one or more Symptoms tic effect. Further, the combination therapies of the invention thereof, including but not limited to cancer, inflammatory reduce or avoid unwanted or adverse side effects associated and autoimmune diseases. The invention further encom with the administration of current Single agent therapies US 2006/0039904 A1 Feb. 23, 2006 40 and/or existing combination therapies for diseases, Such as leukin-2, tumor necrosis factor-alpha, and melphalan), anti cancer, which in turn improves patient compliance with the inflammatory agents (e.g., non-steroidal anti-inflammatory treatment protocol. drugs (NSAIDs), Steroidal anti-inflammatory drugs, beta 0276. In one embodiment, the invention provides meth agonists, anticholingeric agents, and methyl Xanthines), ods for preventing, managing, treating or ameliorating an analgesics (e.g., NSAIDs, Salicylates, acetominophen, nar Eph receptor-mediated disease (e.g., cancer) or one or more cotics, and non-narcotic and anxiolytic combinations). Addi Symptoms thereof, Said methods comprising administering tional agents and therapies and their dosages, routes of to a Subject in need thereof a dose of a prophylactically or administration and recommended usage are known in the art therapeutically effective amount of an Fc variant and/or and have been described in such literature as the Physician's variant Fc fusion protein in combination with the adminis Desk Reference (57" ed., 2003). Additional agents and other tration of an Integrin antagonist, a Standard or experimental combination therapies are described in PCT applications chemotherapy, a hormonal therapy, a biological therapy/ WO 02/070007; WO 04/066956 WO 03/075741; and WO immunotherapy and/or a radiation therapy. In another 03/075957 each of which are incorporated herein in their embodiment, the invention provides methods for preventing, entireties. 10278J Further exemplary agents to be used in the managing, treating or ameliorating an Eph receptor-medi combination therapies described Supra include but are not ated disease (e.g., cancer) or one or more Symptoms thereof, limited to Examples of anti-cancer agents that can be used Said methods comprising administering to a Subject in need in the various embodiments of the invention, including thereof a dose of a prophylactically or therapeutically effec pharmaceutical compositions and dosage forms and kits of tive amount of an Fc variant and/or variant Fc fusion protein the invention, include, but are not limited to: acivicin, in combination with Surgery, alone or in further combination aclarubicin, acodazole hydrochloride, acronine, adoZelesin, with the administration of an Eph receptor antagonist and/or aldesleukin, altretamine, ambomycin, ametantrone acetate, Eph receptor agonist, a Standard or experimental chemo aminoglutethimide, amsacrine, anastrozole, anthramycin, therapy, a hormonal therapy, a biological therapy/immuno asparaginase, asperlin, azacitidine, azetepa, azotomycin, therapy and/or a radiation therapy. In accordance with these batimastat, benzodepa, bicalutamide, bisantrene hydrochlo embodiments, the Fc variant and/or variant Fc fusion protein ride, bisnafide dimeSylate, bizelesin, bleomycin Sulfate, bre utilized to prevent, manage, treat or ameliorate an Eph quinar Sodium, bropirimine, buSulfan, cactinomycin, calus receptor-mediated disease (e.g., cancer) or one or more terone, caracemide, carbetimer, carboplatin, carmustine, Symptoms thereof may or may not be conjugated or fused to carubicin hydrochloride, carZelesin, cedefingol, chloram a moiety (e.g., therapeutic agent or drug) and Said Fc bucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, variants and/or variant Fc fusion proteins are agonists or cyclophosphamide, cytarabine, dacarbazine, dactinomycin, antagonists that immunospecifically bind to an Eph receptor. daunorubicin hydrochloride, decarbazine, decitabine, dex 0277. Therapeutic or prophylactic agents include, but are ormaplatin, de Zaguanine, deZaguanine meSylate, diazi not limited to, Small molecules, Synthetic drugs, peptides, quone, docetaxel, doxorubicin, doxorubicin hydrochloride, polypeptides, proteins, nucleic acids (e.g., DNA and RNA droloxifene, droloxifene citrate, dromoStanolone propionate, nucleotides including, but not limited to, antisense nucle duaZomycin, edatrexate, eflornithine hydrochloride, otide Sequences, triple helices and nucleotide Sequences elsamitrucin, enloplatin, enpromate, epipropidine, epirubi encoding biologically active proteins, polypeptides or pep cin hydrochloride, erbulozole, eSorubicin hydrochloride, estramustine, estramustine phosphate Sodium, etanidazole, tides), antibodies, Synthetic or natural inorganic molecules, etoposide, etopoSide phosphate, etoprine, fadrozole hydro mimetic agents, and Synthetic or natural organic molecules. chloride, faZarabine, fenretinide, floxuridine, fludarabine Any agent which is known to be useful, or which has been phosphate, fluorouracil, flurocitabine, fosquidone, fostriecin used or is currently being used for the prevention, treatment Sodium, gemcitabine, gemcitabine hydrochloride, hydrox or amelioration of Eph receptor-mediated disease or disorder yurea, idarubicin hydrochloride, ifosfamide, ilmofoSine, including but not limited to cancer, inflammatory and interleukin 2 (including recombinant interleukin 2, or rIL2), autoimmune diseases or Symptom asSociated there with can interferon alpha 2a, interferon alpha2b, interferon alpha n1, be used in combination with an Fc variant and/or variant Fc interferon alpha n3, interferon beta I a, interferon gamma I fusion in accordance with the invention described herein. b, iproplatin, irinotecan hydrochloride, lanreotide acetate, 0278 Exemplary agents to be used in the combination letrozole, leuprolide acetate, liarozole hydrochloride, lom therapies described Supra include but are not limited to etrexol Sodium, lomustine, loSOXantrone hydrochloride, Integrin antagonists (e.g., RGD peptides and disintegrins), masoprocol, maytansine, mechlorethamine hydrochloride, Standard or experimental chemotherapy agents (e.g., doxo megestrol acetate, melengestrol acetate, melphalan, menog rubicin, epirubicin, cyclophosphamide, 5-fluorouracil, tax aril, mercaptopurine, methotrexate, methotrexate Sodium, anes Such as docetaxel and paclitaxel, leucovorin, levami metoprine, meturedepa, mitindomide, mitocarcin, mitocro Sole, irinotecan, estramustine, etoposide, vinblastine, min, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, dacarbazine, nitroSoureas Such as carmustine and lomustine, mitoxantrone hydrochloride, mycophenolic acid, Vinca alkaloids, platinum compounds, cisplatin, mitomycin, nitroSoureas, nocodazole, nogalamycin, Ormaplatin, OXisu Vinorelbine, gemcitabine, carboplatin, hexamethylmelamine ran, paclitaxel, pegaspargase, peliomycin, pentamustine, and/or topotecan), immunomodulatory agents (e.g., cytok peplomycin Sulfate, perfosfamide, pipobroman, pipOSulfan, ines, antibodies, interleukins and hemapoietic factors), bio piroXantrone hydrochloride, plicamycin, plomeStane, por logical therapies/immunotherapies (e.g., tamoxifen, LHRH fimer Sodium, porfiromycin, prednimustine, procarbazine agonists, non-Steroidal antiandrogens, Steroidal antiandro hydrochloride, puromycin, puromycin hydrochloride, pyra gens, estrogens, aminoglutethimide, hydrocortisone, fluta Zoflurin, riboprine, rogletimide, Safingol, Safingol hydrochlo mide withdrawal, progesterone, ketoconazole, prednisone, ride, Semustine, SimtraZene, Sparfosate Sodium, SparSomy interferon-alpha, interferon-beta, interferon-gamma, inter cin, Spirogermanium hydrochloride, Spiromustine, US 2006/0039904 A1 Feb. 23, 2006
Spiroplatin, Streptonigrin, Streptozocin, Sulofenur, tallisomy leinamycin, lenograstim, lentinan Sulfate, leptolstatin, letro cin, tecogalan Sodium, tegafur, teloxantrone hydrochloride, Zole, leukemia inhibiting factor, leukocyte alpha interferon, temoporfin, teniposide, teroxirone, testolactone, thiami leuprolide-estrogen-progesterone, leuprorelin, levamisole, prine, thioguanine, thiotepa, tiazofurin, tirapazamine, liarozole, linear polyamine analogue, lipophilic disaccharide toremifene citrate, treStolone acetate, triciribine phosphate, peptide, lipophilic platinum compounds, lisSoclinamide 7, trimetrexate, trimetrexate glucuronate, triptorelin, tubulo lobaplatin, lombricine, lometrexol, lonidamine, loSOX Zole hydrochloride, uracil mustard, uredepa, Vapreotide, antrone, lovastatin, loxoribine, lurtotecan, lutetium texaphy verteporfin, vinblastine Sulfate, Vincristine Sulfate, Vin rin, lySofylline, lytic peptides, maitansine, mannostatin A, desine, Vindesline Sulfate, Vinepidine Sulfate, Vinglycinate marimastat, maSoprocol, maspin, matrilysin inhibitors, Sulfate, Vinleurosine Sulfate, Vinorelbine tartrate, Vinrosidine matrix metalloproteinase inhibitors, menogaril, merbarone, Sulfate, Vinzolidine Sulfate, Vorozole, Zeniplatin, Zinostatin, meterelin, methioninase, metoclopramide, MIF inhibitor, Zorubicin hydrochloride. Other anti cancer drugs include, mifepristone, miltefosine, mirimoStim, mismatched double but are not limited to: 20 epi 1.25 dihydroxyvitamin D3, 5 Stranded RNA, mitoguaZone, mitolactol, mitomycin ana ethynyluracil, abiraterone, aclarubicin, acylfulvene, ade logues, mitonafide, mitotoxin fibroblast growth factor cypenol, adoZelesin, aldesleukin, ALL TKantagonists, altre Saporin, mitoxantrone, mofaroteine, molgramoStim, mono tamine, ambamustine, amidox, amifostine, aminolevulinic clonal antibody, human chorionic gonadotrophin, mono acid, amrubicin, amsacrine, anagrelide, anastroZole, phosphoryl lipid A+myobacterium cell wall Sk, mopidamol, andrographolide, angiogenesis inhibitors, antagonist D, multiple drug resistance gene inhibitor, multiple tumor Sup antagonist G, antarelix, anti dorsalizing morphogenetic pro preSSor I based therapy, mustard anticancer agent, my cap tein 1, antiandrogens, antiestrogens, antineoplaston, aphidi eroxide B, mycobacterial cell wall extract, myriaporone, N colin glycinate, apoptosis gene modulators, apoptosis regu acetyldinaline, N Substituted benzamides, nafarelin, lators, apurinic acid, ara CDP DL PTBA, arginine nagreStip, naloxone-pentazocine, napavin, naphterpin, nar deaminase, asulacrine, atameStane, atrimustine, axinastatin tograstim, medaplatin, memorubicin, neridronic acid, neutral 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azaty endopeptidase, nilutamide, nisamycin, nitric oxide modula rosine, baccatin III derivatives, balanol, batimastat, BCR/ tors, nitroxide antioxidant, nitrullyn, O6 benzylguanine, ABL antagonists, benzochlorins, benzoylstauroSporine, beta octreotide, okicenone, oligonucleotides, onapristone, lactam derivatives, beta alethine, betaclamycin B, betulinic Ondansetron, Ondansetron, oracin, oral cytokine inducer, acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridi ormaplatin, OSaterone, Oxaliplatin, oxaunomycin, paclitaxel, nylspermine, bisnafide, bistratene A, bizelesin, breflate, paclitaxel analogues, paclitaxel derivatives, palauamine, bropirimine, budotitane, buthionine Sulfoximine, calcipot palmitoylrhizoxin, pamidronic acid, panaxytriol, riol, calphostin C, camptothecin derivatives, canarypox IL2, panomifene, parabactin, paZelliptine, pegaspargase, capecitabine, carboxamide amino triazole, carboxyamidot peldesline, pentosan polysulfate Sodium, pentostatin, pentro riazole, CaRest M3, CARN 700, cartilage derived inhibitor, Zole, perflubron, perfosfamide, perillyl alcohol, phenazino carzelesin, casein kinase inhibitors (ICOS), castanosper mycin, phenylacetate, phosphatase inhibitors, picibanil, mine, cecropin B, cetrorelix, chloroquinoxaline Sulfona pilocarpine hydrochloride, pirarubicin, piritrexim, placetin mide, cicaprost, cis porphyrin, cladribine, clomifene ana A, placetin B, plasminogen activator inhibitor, platinum logues, clotrimazole, collismycin A, collismycin B, complex, platinum compounds, platinum triamine complex, combretastatin A4, combretastatin analogue, conagenin, porfimer Sodium, porfiromycin, prednisone, propyl bis acri crambescidin 816, crisinatol, cryptophycin 8, cryptophycin A done, prostaglandin J2, proteasome inhibitors, protein A derivatives, curacin A, cyclopentanthraquinones, cyclo based immune modulator, protein kinase C inhibitor, protein platam, cypemycin, cytarabine ocfoSfate, cytolytic factor, kinase C inhibitors, microalgal, protein tyrosine phosphatase cytostatin, dacliximab, decitabine, dehydrodidemnin B, inhibitors, purine nucleoside phosphorylase inhibitors, pur deslorelin, dexamethasone, dexifosfamide, deXraZOxane, purins, pyrazoloacridine, pyridoxylated hemoglobin poly deXVerapamil, diaziquone, didemnin B, didox, diethylnor oxyethylene conjugate, raf antagonists, raltitrexed, ramo Spermine, dihydro 5 azacytidine, dihydrotaXol, dioxamycin, Setron, ras farnesyl protein transferase inhibitors, ras diphenyl Spiromustine, docetaxel, docOSanol, dolasetron, inhibitors, ras GAP inhibitor, retelliptine demethylated, rhe doxifluridine, droloxifene, dronabinol, duocarmycin SA, nium Re 186 etidronate, rhizoxin, ribozymes, RII retina ebSelen, ecomustine, edelfosine, edrecolomab, eflornithine, mide, rogletimide, rohitukine, romurtide, roquinimex, elemene, emitefur, epirubicin, epristeride, estramustine ana rubiginone B1, ruboxyl, Safingol, Saintopin, SarCNU, Sar logue, estrogen agonists, estrogen antagonists, etanidazole, cophytol A, SargramoStim, Sdi 1 mimetics, Semustine, Senes etoposide phosphate, exemestane, fadrozole, faZarabine, cence derived inhibitor 1, Sense oligonucleotides, Signal fenretinide, filgrastim, finasteride, flavopiridol, fleZelastine, transduction inhibitors, Signal transduction modulators, fluiasterone, fludarabine, fluorodaunorunicin hydrochloride, Single chain antigen binding protein, sizofiran, Sobu Zoxane, forfenimex, formeStane, fostriecin, fotemustine, gadolinium Sodium borocaptate, Sodium phenylacetate, Solverol, teXaphyrin, gallium nitrate, galocitabine, ganirelix, gelati Somatomedin binding protein, Sonermin, Sparfosic acid, nase inhibitors, gemcitabine, glutathione inhibitors, hepSul Spicamycin D, Spiromustine, Splenopentin, Spongistatin 1, fam, heregulin, hexamethylene bisacetamide, hypericin, Squalamine, Stem cell inhibitor, Stem cell division inhibitors, ibandronic acid, idarubicin, idoxifene, idramantone, ilmo Stipiamide, Stromelysin inhibitors, Sulfinosine, Superactive fosine, ilomastat, imidazoacridones, imiquimod, immuno vasoactive intestinal peptide antagonist, Suradista, Suramin, Stimulant peptides, insulin like growth factor 1 receptor Swainsonine, Synthetic glycosaminoglycans, tallimustine, inhibitor, interferon agonists, interferons, interleukins, tamoxifen methiodide, tauromustine, taxol, tazarotene, tec iobenguane, iododoxorubicin, ipomeanol, iroplact, irSogla ogalan Sodium, tegafur, tellurapyrylium, telomerase inhibi dine, isobengaZole, isohomohalicondrin B, itasetron, jas tors, temoporfin, temozolomide, teniposide, tetrachlorode plakinolide, kahalalide F, lamellarin N triacetate, lanreotide, caoxide, tetraZomine, thaliblastine, thalidomide, US 2006/0039904 A1 Feb. 23, 2006 42 thiocoraline, thioguanine, thrombopoietin, thrombopoietin the invention” and “formulation(s)"). In a specific embodi mimetic, thymalfasin, thymopoietin receptor agonist, thy ment, the liquid formulations are Substantially free of Sur motrinan, thyroid Stimulating hormone, tin ethyl etiopurpu factant and/or inorganic Salts. In another Specific embodi rin, tirapazamine, titanocene bichloride, topSentin, ment, the liquid formulations have a pH ranging from about toremifene, totipotent Stem cell factor, translation inhibitors, 5.0 to about 7.0, about 5.5 to about 6.5, about 5.8 to about tretinoin, triacetyluridine, triciribine, trimetrexate, triptore 6.2, and about 6.0. In a further specific embodiment, the lin, tropisetron, turosteride, tyrosine kinase inhibitors, tyr liquid formulations have a pH ranging from 5.0 to 7.0, 5.5 phoStins, UBC inhibitors, ubenimex, urogenital Sinus to about 6.5, 5.8 to 6.2, and 6.0. In yet another specific derived growth inhibitory factor, urokinase receptor antago embodiment, the liquid formulations comprise histidine at a nists, Vapreotide, variolin B, vector System, erythrocyte gene concentration ranging from about 1 mM to about 100 mM, therapy, VelareSol, Veramine, Verdins, verteporfin, Vinorel from about 5 mM to about 50 mM, about 10 mM to about bine, VinXaltine, Vitaxin, Vorozole, Zanoterone, Zeniplatin, 25 mM. In still another specific embodiment, the liquid Zilascorb, and Zinostatin stimalamer. Additional anti-cancer formulations comprise histidine at a concentration ranging drugs are 5-fluorouracil and leucovorin. (02791. The meth from 1 mM to 100 mM, from 5 mM to 50 mM, 10 mM to ods and formulations of the invention are useful in prevent 25 mM. ing, managing, treating or ameliorating cancers, including, 0282. In a preferred embodiment, the liquid formulations but not limited to, cancer of the head, neck, eye, mouth, have a concentration of one or more Fc variants and/or Fc throat, esophagus, chest, bone, lung, colon, rectum, colorec variant fusions is about 50 mg/ml, about 75 mg/ml, about tal or other gastrointestinal tract organs, Stomach, Spleen, 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 175 renal, Skeletal muscle, Subcutaneous tissue, metastatic mela mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 noma, endometrial, proState, breast, Ovaries, testicles or mg/ml, about 275 mg/ml, or about 300 mg/ml. In one other reproductive organs, skin, thyroid, blood, lymph embodiment, the liquid formulations should exhibit one or nodes, kidney, liver, pancreas, and brain or central nervous more of the following characteristics, Stability, low to unde System. In a Specific embodiment, the methods and formu tectable levels of antibody fragmentation and/or aggrega lations of the invention are used for the prevention, man tion, very little to no loss of the biological activities of the agement, treatment or amelioration of a primary or Second antibodies or antibody fragments during manufacture, ary cancer that expresses an Eph receptor. In another preparation, transportation, and Storage. In other embodi embodiment, the methods and formulations of the invention ments the liquid formulations lose less than 50%, less than are used for the prevention, management, treatment or 30%, less than 20%, less than 10% or even less than 5% or amelioration of a primary or Secondary cancer that does not 1% of the antibody activity within 1 year storage under express an Eph receptor. suitable conditions at or about 4 C. In yet other embodi 0279 The methods and formulations of the invention are ments the liquid formulations lose less than about 50%, less useful not only in untreated cancer patients but are also than about 30%, less than about 20%, less than about 10% useful in the management or treatment of cancer patients or even less than about 5% or about 1% of the antibody partially or completely refractory to current Standard and activity within 1 year Storage under Suitable conditions at or experimental cancer therapies, including, but not limited to, about 4 C. The activity of an antibody can be determined chemotherapies, hormonal therapies, biological therapies, by a Suitable antigen-binding or effector function assay for radiation therapies, and/or Surgery. the respective antibody. In yet another preferred embodi ment, the liquid formulations are of low Visocisty and 6.11 Formulations and Administration turbidity. In a particular embodiment, the liquid formula tions have a viscosity of less than 10.00 cp or about 10.00 0280 AS described above, the present invention relates to cPat any temperature in the range of 1 C. to 26 C. or about the use of agents that immunospecifically bind and prefer between about 1 C. to about 26 C. Viscosity can be ably inhibit one or more Eph receptor activity for the determined by numerous method well known in the art. For prevention, management, treatment or amelioration of an example, the Viscosity of a polypeptide Solution can be Eph receptor-mediated disease (e.g., cancer) or one or more measured using a ViscoLab 4000 Viscometer System (Cam Symptoms thereof and/or the inhibition of angiogenesis. bridge Applied Systems) equipped with a ViscoLab Piston Accordingly, the present invention provides formulations (SN:7497, 0.3055", 1-20 cP) and S6S Reference Standard (e.g., a pharmaceutical composition) comprising one or (Koehler Instrument Company, Inc.) and connected to a more Fc variants and/or variant Fc fusion protein with water bath to regulate the temperature of the Samples being modified binding affinity to one or more Fc ligand (e.g., analyzed. The Sample is loaded into the chamber at a desired FcyRs, C1q) and altered ADCC and/or CDC activity that Starting temperature (e.g., 2 C.) and the piston lowered into immunospecifically bind to an Eph receptor (also referred to the Sample. After Sample was equilibrated to the temperature herein as “formulation(s) of the invention” or simply “for of the chamber, measurement is initiated. The temperature is mulation(s)). In a specific embodiment, said Fc variants increased at a desired rate to the desired final temperature and/or Fc variant fusions are antagonists of one or more Eph (e.g., 225 C.). And the Viscosity over time is recorded. receptor. In another specific embodiment, Said Fc variants 0283. It is contemplated that the liquid formulations may and/or Fc variant fusions are agonists of one or more Eph further comprise one or more excipients Such as a Saccha receptor. ride, an amino acid (e.g. arginine, lysine, and methionine) 0281. In one embodiment, formulations (e.g., a pharma and a polyol. Additional descriptions and methods of pre ceutical composition) comprising one or more Fc variants paring and analyzed liquid formulations can be found, for and/or Fc variant fusions are liquid formulations (referred to example, in PCT publications WO 03/106644; WO herein as “liquid formulation(s)” which are specifically 04/066957; WO 04/091658 each of which is herein incor encompassed by the more generic terms “formulation(s) of porated by reference in its entirety. US 2006/0039904 A1 Feb. 23, 2006 43
0284. In one embodiment the formulations (e.g., liquid 0286) Depending on the condition, the formulations can formulations) of the invention are pyrogen-free formulations be administered orally, parenterally, intramuscularly, intra which are substantially free of endotoxins and/or related nasally, vaginally, rectally, lingually, Sublingually, buccally, pyrogenic Substances. Endotoxins include toxins that are intrabuccally, intravenously, cutaneously, Subcutaneously confined inside a microorganism and are released when the and/or transdermally to the patient. microorganisms are broken down or die. Pyrogenic Sub stances also include fever-inducing, thermostable Substances 0287. Accordingly, formulations designed for oral, (glycoproteins) from the outer membrane of bacteria and parenteral, intramuscular, intranasal, Vaginal, rectal, lingual, other microorganisms. Both of these Substances can cause Sublingual, buccal, intrabuccal, intravenous, cutaneous, Sub fever, hypotension and Shock if administered to humans. cutaneous and/or transdermal administration can be made Due to the potential harmful effects, it is advantageous to without undue experimentation by means well known in the remove even low amounts of endotoxins from intravenously art, for example, with an inert diluent or with an edible administered pharmaceutical drug Solutions. The Food & carrier. The formulations may be enclosed in gelatin cap Drug Administration (“FDA") has set an upper limit of 5 Sules or compressed into tablets. For the purpose of oral endotoxin units (EU) per dose per kilogram body weight in therapeutic administration, the formulations of the present a single one hour period for intravenous drug applications invention may be incorporated with excipients and used in (The United States Pharmacopeial Convention, Pharma the form of tablets, troches, capsules, elixirs, Suspensions, copeial Forum 26 (1):223 (2000)). When therapeutic pro Syrups, wafers, chewing gums, and the like. teins are administered in amounts of Several hundred or thousand milligrams per kilogram body weight, as can be the 0288 Tablets, pills, capsules, troches and the like may case with monoclonal antibodies, it is advantageous to also contain binders, recipients, disintegrating agent, lubri remove even trace amounts of endotoxin. In one embodi cants, Sweetening agents, and/or flavoring agents. Some ment, endotoxin and pyrogen levels in the composition are examples of binders include microcrystalline cellulose, gum less then 10 EU/mg, or less then 5 EU/mg, or less then 1 tragacanth and gelatin. Examples of excipients include EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or Starch and lactose. Some examples of disintegrating agents less then 0.001 EU/mg. In another embodiment, endotoxin include alginic acid, cornstarch, and the like. Examples of and pyrogen levels in the composition are less then about 10 lubricants include magnesium Stearate and potassium Stear EU/mg, or less then about 5 EU/mg, or less then about 1 ate. An example of a glidant is colloidal Silicon dioxide. EU/mg, or less then about 0.1 EU/mg, or less then about Some examples of Sweetening agents include Sucrose, Sac 0.01 EU/mg, or less then about 0.001 EU/mg. charin, and the like. Examples of flavoring agents include peppermint, methyl Salicylate, orange flavoring, and the like. 0285. It will be apparent to one skilled in the art that a Materials used in preparing these various formulations formulation comprising one or more Fc variants and/or Fc should be pharmaceutically pure and non-toxic in the variant fusions to be administered to a Subject (e.g., a amounts used. human) in need thereof should be formulated in a pharma ceutically-acceptable excipient. Examples of formulations, 0289. The pharmaceutical formulations of the present pharmaceutical compositions in particular, of the invention invention can be administered parenterally, Such as, for include but are not limited to those disclosed in PCT example, by intravenous, intramuscular, intrathecal and/or publications WO 02/070007; WO 04/066957 and WO Subcutaneous injection. Parenteral administration can be 03/075957 each of which is herein incorporated by reference accomplished by incorporating the formulations of the in its entirety. Briefly, the excipient that is included with the present invention into a Solution or Suspension. Such Solu Fc variants and/or variant Fc fusion protein of the present tions or Suspensions may also include Sterile diluents, Such invention in these formulations (e.g., liquid formulations) as water for injection, Saline Solution, fixed oils, polyethyl can be Selected based on the expected route of administra ene glycols, glycerine, propylene glycol and/or other Syn tion of the composition in therapeutic applications. The thetic Solvents. Parenteral formulations may also include route of administration of the composition depends on the antibacterial agents, Such as, for example, benzyl alcohol condition to be treated. For example, intravenous injection and/or methyl parabens, antioxidants, Such as, for example, may be preferred for treatment of a Systemic disorder Such ascorbic acid and/or Sodium bisulfite, and chelating agents, as a lymphatic cancer or a tumor which has metastasized. Such as EDTA. Buffers, Such as acetates, citrates and phos The dosage of the compositions to be administered can be phates, and agents for the adjustment of tonicity, Such as determined by the skilled artisan without undue experimen Sodium chloride and dextrose, may also be added. The tation in conjunction with Standard dose-response Studies. parenteral preparation can be enclosed in ampules, dispos Relevant circumstances to be considered in making those able Syringes and/or multiple dose Vials made of glass or determinations include the condition or conditions to be plastic. Rectal administration includes administering the treated, the choice of composition to be administered, the formulation into the rectum and/or large intestine. This can age, weight, and response of the individual patient, and the be accomplished using Suppositories and/or enemas. Sup Severity of the patient's Symptoms. For example, the actual pository formulations can be made by methods known in the patient body weight may be used to calculate the dose of the art. Transdermal administration includes percutaneous Fc variants and/or variant Fc fusion of the present invention absorption of the formulation through the skin. Transdermal in these formulations in milliliters (mL) to be administered. formulations include patches, ointments, creams, gels, There may be no downward adjustment to “ideal weight. In Salves, and the like. The formulations of the present inven Such a situation, an appropriate dose may be calculated by tion can be administered nasally to a patient. AS used herein, the following formula: nasally administering or nasal administration includes Dose (mL)=patient weight (kg)xdose level (mg/kg), administering the formulations to the mucous membranes of drug concentration (mg/mL) the nasal passage and/or nasal cavity of the patient. US 2006/0039904 A1 Feb. 23, 2006 44
0290. In further embodiments, the formulations (e.g., (e.g., about 0.1 to about 100 ug/ml), which continuously liquid formulations) are administered to the mammal by binds to an Eph receptor. In a Specific embodiment, the Subcutaneous (i.e., beneath the skin) administration. For plasma concentration of the Said Fc variants an/or variant Fc Such purposes, the formulations may be injected using a fusion proteins is maintained at 10 ug/ml, 15 lug/ml, 20 Syringe. However, other devices for administration of the tug/ml, 25 ug/ml, 30 ug/ml, 35ug/ml, 40 lig/ml, 45 lug/ml or formulations are available Such as injection devices (e.g. the 50 ug/ml. In a specific embodiment, Said effective amount of Inject-ease and Genject devices), injector pens (Such as the Fc variant and/or variant Fc fusion protein to be adminis GenPen"); auto-injector devices, needleless devices (e.g., tered is between at least 1 mg/kg and 100 mg/kg per dose. Medijector and BioJector); and subcutaneous patch delivery In another specific embodiment, Said effective amount of Fc Systems. variant and/or variant Fc fusion to be administered is between at least 1 mg/kg and 20 mg/kg per dose. In another 0291. In another aspect of the invention there is provided Specific embodiment, Said effective amount of Fc variant a slow release formulations. In a Specific embodiment, a and/or variant Fc fusion protein to be administered is Slow release formulation comprises a liquid formulation. between at least 4 mg/kg and 10 mg/kg per dose. In yet Slow release formulations may be formulated from a num another specific embodiment, Said effective amount of Fc ber of agents including, but not limited to, polymeric nano variant and/or variant Fc fusion protein to be administered is or microparticles and gels (e.g., a hyaluronic acid gel). between 50 mg and 250 mg per dose. In still another specific Besides convenience, Slow release formulations offer other embodiment, said effective amount of Fc variant and/or advantages for delivery of protein drugs including protecting variant Fc fusion protein to be administered is between 100 the protein (e.g., Fc variant and/or variant Fc fusion) over an mg and 200 mg per dose. extended period from degradation or elimination, and the ability to deliver the protein locally to a particular Site or 0295) The present invention provides kits comprising one body compartment thereby lowering overall Systemic expo or more Fc variants and/or variant Fc fusions with modified SUC. binding affinity to one or more Fc ligand (e.g., FcyRs, C1q) and altered ADCC and/or CDC activity that immunospe 0292. The present invention, for example, also contem cifically bind to an Eph receptor conjugated or fused to a plates injectable depot formulations in which the protein detectable agent, therapeutic agent or drug, in one or more (e.g., Fc variant and/or variant Fc fusion) is embedded in a containers, for use in the prevention, treatment, manage biodegradable polymeric matrix. Polymers that may be used ment, amelioration, detection, monitoring or diagnosis of include, but are not limited to, the homo- and co-polymers cancer, inflammatory and autoimmune diseases, in particular of lactic and glycolic acid (PLGA). PLGA degrades by an Eph receptor-mediated disease. hydrolysis to ultimately give the acid monomers and is chemically unreactive under the conditions used to prepare, 0296. The invention also provides kits comprising one or for example, microSpheres and thus does not modify the more Fc variants and/or variant Fc fusions with modified protein. After Subcutaneous or intramuscular injection, the binding affinity to one or more Fc ligand (e.g., FcyRs, C1q) protein is released by a combination of diffusion and poly and altered ADCC and/or CDC activity that immunospe mer degradation. By using polymers of different composi cifically bind to an Eph receptor in a first vial and one or tion and molecular weight, the hydrolysis rate can be varied more prophylactic or therapeutic agents, other than Fc thereby allowing release to last from days to months. In a variants that immunospecifically bind to an Eph receptor, in further aspect the present invention provides a nasal Spray a Second vial for use in the prevention, treatment, manage formulation. In a Specific embodiment, a nasal Spray for ment, amelioration, detection, monitoring or diagnosis of mulation comprises the liquid formulation of the present cancer, inflammatory and autoimmune diseases, in particular invention. an Eph receptor-mediated disease. The invention also pro vides kits comprising one or more Fc variants and/or variant 0293. The formulations of the invention may be used in Fc fusions with modified binding affinity to one or more Fc accordance with the methods of the invention for the pre ligand (e.g., FcyRs, C1q) and altered ADCC and/or CDC vention, management, treatment or amelioration of cancer, activity that immunospecifically bind to an Eph receptor inflammatory and autoimmune diseases or one or more conjugated or fused to a therapeutic agent or drug in a first Symptoms thereof. In one embodiment, the formulations of Vial and one or more prophylactic or therapeutic agents, the invention are Sterile and in Suitable form for a particular other than an Fc variant an/or variant Fc fusion protein that method of administration to a Subject with cancer, inflam immunospecifically binds an Eph receptor, in a Second vial matory and autoimmune diseases, in particular an Eph for use in the prevention, treatment, management, amelio receptor-mediated disease. ration, detection, monitoring or diagnosis of cancer, inflam 0294 The invention provides methods for preventing, matory and autoimmune diseases, in particular an Eph managing, treating or ameliorating cancer, inflammatory and receptor-mediated disease. The kits may further comprise autoimmune diseases (in particular an Eph receptor-medi packaging materials and/or instructions. ated disease) or one or more Symptoms thereof, said method comprising: (a) administering to a Subject in need thereof a 7. EXAMPLES dose of a prophylactically or therapeutically effective 0297. The invention is now described with reference to amount of a formulation comprising one or more Fc variants the following examples. These examples are provided for and/or variant Fc fusion proteins, that immunospecifically the purpose of illustration only and the invention should in bind to an Eph receptor and (b) administering one or more no way be construed as being limited to these examples but Subsequent doses of Said Fc variants an/or variant Fc fusion rather should be construed to encompass any and all varia proteins, to maintain a plasma concentration of the Said Fc tions which become evident as a result of the teachings variants an/or variant Fc fusion proteins at a desirable level provided herein. US 2006/0039904 A1 Feb. 23, 2006 45
7.1 Example 1 the amplification and library construction are listed in table 5. The IgG1 of antibody VitaxinTM, converted into scFv-Fc Construction and Expression of Novel Fc Variants of Anti format, was used as the model for this study. The DNA and bodies corresponding amino acid Sequences of the variable regions 0298 Based on the structural information available for of the Vitaxin E) heavy and light chains used to generate the the Fc-FcyRIIIB complex, each of the putative FcyR contact scFv-Fc are shown in FIG. 1 (panels A and B, respectively). residues of the IgG1 Fc portion was randomly mutated by The scFv-Fc was then harnessed as the template to build using degenerated oligonucleotides incorporating all poS three Fc mutant libraries containing Single mutations in the Sible Single mutations. The contact residues were divided Fc region. Library I contains all Single mutations in the RI into four regions (RI: Leu", Leu°35, Gly23, Gly27, Pro?, region; library II covers the RII and RIII regions; and library Ser239; RII: Asp25, Ser7, Glu; RIII: Ser; and RIV: III covers the RIV region. Overlapping PCR approach was Ala’, Leu, Pro’, Ala, and Ile). Primers used for used to Synthesize entire Fc region containing mutations.
TABLE 5
Primers
SEQ Primer Sequence Notes D MDAD-1 CCG TGC CCA GCA CCT GAA NNK CTG GGG GGA CCG contact Region 1 TCA GTC
MDAD-2 CCG TGC CCA GCA CCT GAA CTC NNK GGG GGA CCG contact Region 2 TCA GTC TTC
MDAD-3 CCG TGC CCA GCA CCT GAA CTC CTG NNK GGA CCG contact Region 3 TCA GTC TTC CTC
MDAD-4 CCG TGC CCA GCA CCT GAA CTC CTG GGG NNK CCG contact Region 4 TCA GTC TTC CTC TTC
MDAD-5 CCC TGG CCA GCA CGT GAA CTC GTG GGG GGA NNK contact Region 5 TCA GTC TTC CTC TTC COC
MDAD-6 CCG TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG contact Region 6 NNK GTC TTC CTC TTG CCC. CCA
MDAD-7 GTC ACA TGC GTG GTG NNK GTG AGC CAC GAA contact Region II 7 GAC CCT
MDAD-8 GTC ACA TGC GTG GTG GTG GAC GTG NNK CAC GAA contact Region II 8 GAC CCT GAG GTC
MDAD-9 GTC ACA TGC GTG GTG GAC GTG AGC CAC NNK contact Region II 9 GAC CCT GAG AAG TTC
MDAD-10 CGG GAG GAG CAG TAC AAC NNK ACG TAC CGT GTG contact Region III 20 GTC AGC
MDAD-11 TGC AAG GTC TCC AAC AAA NNK CTC CCA GCC CCC contact Region IV 21 ATC GAG
MDAD-12 TGC AAG GTC TCC AAC AAA GCC NNK CCA GCC CCC contact Region IV 22 ATC GAG AAA
MDAD-13 TGC AAG GTC TCC AAC AAA GCC CTC NNK GCC CCC contact Region IV 23 ATC GAG AAA ACC
MDAD-1 4 TGC AAG GTC TCC AAC AAA GCC CTC CCA NNK CCC contact Region IV 24 ATC GAG AAA ACC ATC
MDAD-15 TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC CCC contact Region IV 25 NNK GAG AAA ACC ATC. TCC AAA
MDAD-16 ACT CAC ACA CCA CCG TGC CCA GCA CCT GAA Fo N-terminus 26
MDAD-17 CAC CAC CAC GCA TGT, GAC RII primer 27
MDAD-18 GTT GTA CTG CTC CTC CCG RIII primer 28
MDAD-19 TTT GTT GGA GAC CTT, GCA RIV primer 29
MDAD-2O AAC CTC TAC TGT. GGT ATG GCT Fo C- terminus 30
US 2006/0039904 A1 Feb. 23, 2006
TABLE 5-continued
Primers
SEQ Primer Sequence Notes ID Oligo 20 CTGGGGGGACCG gac GTCTTCCTCTTC S239D 60 Oligo 21 AAAGCCCTCCCA citg CCC gagGAGAAA A330L/I332E 61
7.1.1 Materials and Methods anti-human IgG (Pierce) at a 1:60000 dilution. The signals were detected by TME substrate (Pierce) and read by an 0299 Construction of Fc Libraries: For constructing Fc ELISA reader at 450 nm. Purified parental VitaxinTM scFv library I, primers MDAD-16, equimolar mixture of MAD-2 Fc expressed in a pMI vector was employed as a Standard (at to -6, and MDAD-20 were used in the PCR reaction. The serial dilutions of 0.003 ug-10 ug/ml). PCR products were gel purified and digested by restriction enzymes Not I/Pci I, and ligated into the expression vector 7.2 Example 2 pMI under the control of the CMV promoter. For construct ing Fc library II, two PCR products incorporating RII and Construction and Expression of the Extracellular Domains RIII mutations were mixed at 3:1 molar ratio for cloning into of FeyRIIIA and FeyRIIB pMI vector. Primers MDAD-16, MDAD-17, equimolar mix 0304) To facilitate the binding studies of the Fc variants ture of MDAD-7 to -9, and MDAD-20 were used to amplify to FcyRs the extracellular domains of FcyRIIIA and FcyRIIB Fc region to incorporate RII mutations, and primers MDAD were Subcloned for expression as Strepavidin fusion proteins 16, -18, -10, and -20 were used to amplify Fc region to in E. coli and for expression in mammalian cells. The incorporate RIII mutations. For Fc library III, primers FcyRIIIA prepared for analysis is the low affinity (F158) MDAD-16, MDAD-19, equimolar mixture of MDAD-11 to allotype. Two forms of FcyRIIIA and FcyRIIB were pre -15, and MDAD-20 were used in the PCR reaction. pared, a “tetramer' form, generated as as Strepavidin fusion, 0300 Transfection: The plasmids of three Fc libraries (I, and a "monomer form generated as a Flag-tagged. II, and III) were linearized by Sal I, ethanol precipitated and resuspended in H.O. 50 tug of each linearized library DNA 7.2.1 Materials and Methods was individually transfected into 107 NSO cells by elec 0305 Construction and Bacterial Expression of the troporation. After electroporation, the cells were transferred Extracellular Domains of FcyRIIIA- and FcyRIIB-Strepavi to a tube containing 30 ml of growth medium (Glutamine din Fusion Proteins (Tetramer): Primer pairs SA1/SA2, free IMDM, 1xGS supplement and 2 mM L-glutamine) and A1/A2, and B1/B2 (see primer list, Table 5) were used to seeded in 96-well plates (50 ul/well) at variable dilutions. PCR amplify streptavidin and the extracellular domains of The cells were cultured at 37 C. in humid air containing 5% FcyR IIIA and FcyR IIB, respectively. The cDNA library of CO. human bone marrow (Clontech) was used as a template for 0301 Selection of Stable Transfectants: The selection of FcyR IIIA and FcyR IIB amplification, and the genomic stably transfected NS0 cells expressing Scriv-Fc mutants was DNA of Streptomyces avidinii was used as the template for started 18-24 hours after transfection by converting to the amplification of Streptavidin. Overlapping PCR was Selection medium (same as growth medium but without used to assemble fusion genes of FcyR IIIA-streptavidin and glutamine). The medium was changed twice a week at one FcyR IIB-strepavidin. The fusion genes were digested by the half of the total volume. After 2-3 weeks of incubation, the restriction enzymes Nco I/Nhe I and cloned into the expres culture Supernatants were collected for Screening of anti Sion vector p T-28a. The fusion proteins were expressed as body expression. inclusion bodies and refolded by dialysis to slowly remove urea as described by C. Gao, et al. (1997, PNAS USA 0302) Purification of scFv-Fc Variants: The culture Super 94:11777-82). The refolded fusion proteins were then puri natants containing ScFV-Fc mutants were purified by using a fied by an immunobiotin column (PIERCE) according to Protein Aspin chromatography kit following manufacturer's manufacturers instructions. protocol (Pierce). The bound scFv-Fc mutants were eluted with 0.1 M citrate buffer and then dialyzed in PBS. All 0306 Construction and Mammalian Expression of the proteins were analyzed by SDS-polyacrylamide gel electro Extracellular Domains of FcyRIIIA and FcyRIlB (Mono phoresis and were applied to quantitative ELISA using mer): The extracellular domains of FcyR IIIA and FcyR IIB were PCR amplified from the cDNA library of human bone anti-human IgG assay plates (Becton Dickson) or BCA kits marrow (Clontech) with primers EA1/EA2 and EB1/EB2, (PIERCE) to determine scFv-Fc concentrations. respectively (see primer list, Table 5). The PCR products 0303 Antibody Ouantitation by ELISA: To determine the were digested by Xba I/Not I and cloned into the mammalian expression level of the Fc variants, anti-human IgG-coated cell expression vectorpMI226 under the control of the CMV microtiter plates (Becton Dickson) were used. The culture promoter to generate proteins in which the extracellular Supernatants were added to the wells at dilutions of 1:10 and domains of FcyR IIIA and FcyR IIB are tagged with His6-tag 1:100. After a 1 hour incubation at room temperature, the followed by FLAG tag at the C-terminal end. The plasmid plates were washed with PBST (PBS+0.1% Tween 20) and DNA was transiently transfected into 293H cells by Lipo incubated at room temperature for an additional hour with fectamine 2000 Transfection Reagent (Invitrogen). After US 2006/0039904 A1 Feb. 23, 2006 48 three collections within 9 days, the proteins were purified by passing the culture Supernatant through anti-FLAG M2 TABLE 6 agarose columns (Sigma). The FLAG-tagged FcyRIIIA/IIB Binding Constants (KD) of wild type antibodies and Fc variants to proteins were eluted from the column and dialyzed against FeyRIA PBS. Run RUs KD KD Fold increase 7.3 Example 3 Antibody # Immobilized Isotherm Scatchard over WT Vitaxin (R) 1. 9608 3.47 uM 3.26 uM Characterization of the Fc Variants Vitaxin-1M 1. 9331 458 nM 458 nM 6.5 Vitaxin (R) 2 9434 8.9 uM 7.6 uM Vitaxin-1M 2 9.383 1.28 uM 1.22 uM 7.0 0307 After mutagenesis of the Fc domain (see example Vitaxin-3M 2 8284 114 nM 113 nM 78.O 1 Supra) Fc variants, in the scFV-Fc fusion format, were 3F2 3 8568 15.6 uM 14.2 uM screened for enhanced binding to FcyRIIIA tetramer by 3F2-1M 3 7718 1.77 uM 1.68 uM 8.8 ELISA as detailed below. The results for several clones are 3F2-3M 3 7809 158 nM 162 nM 99 shown in FIG. 5. In addition, the ADCC activity of these "calculated using Isoltherm values clones was determined against M21 cells. The results for several clones are shown in FIG. 6. Based on these studies 0311. The Vitaxin-1M Fc variant was further character three substitutions were chosen for further study, S239D, ized in ADCC assays against M21 cells. First, the ratio of A330L and I332E. These Substitutions were introduced into target to effector cells was kept constant at 50:1 and the concentration of the two antibodies was varied from 0.4 to the Fc region of the intact Vitaxin(R) IgG1 heavy chain and 1000 ng/ml (FIG. 9). Next, the concentration of antibody coexpressed with Vitaxin(R) light chain to produce full length was varied for several different ratios of target to effector cell Vitaxin(R) Fc variant IgG1 molecules. The Vitaxin(R) Fc (6.25:1, 12.5:1, 25:1 and 50:1) (FIG. 10). In both assays the variant having the I332E substitution was designated ADCC activity of the Vitaxin-1M Fc (I332E) variant was Vitaxin(R)-1M, the Vitaxin(R) Fc variant having the S239D, approximately 3 fold higher than that of the parent Vitaxin(R) A330L, I332L triple substitution was designated Vitaxin(R)- antibody. 3M. 0312 The Vitaxin-3M Fc variant was also characterized 0308) A panel of Vitaxin(R) Fc variants, in IgG format, in ADCC assays against a target cells expressing differing levels of Integrin CVB3 (FIG. 11). The target cell lines used was generated in which each of the Standard 20 amino acids were M21 (a high expressor), DU145 (a low expressor), was substituted at position 332. These variants were char A498 and ACHN (moderate expressors). The assays were acterized. FIG. 7A shows the relative binding to FcyRIIIA performed using two different ratios of target to effector cell of these Fc variants, as determined by ELISA. It can be seen (50:1 and 25:1) and antibody concentrations ranging from 4 that under these conditions Several Substitutions showed to 400 ng per well. In all cases the ADCC activity of the enhanced binding including I332T, I332L, I332F and most Vitaxin-3M Fc variant was seen to be higher than wild type dramatically, I332E. However, as shown in FIG. 7B, only Vitaxin(E). Vitaxin-3M Fc variant was also shown to have the I332E Substitution showed a similar increase in ADCC higher ADCC activity compared to the wild type Vitaxin(R) activity. antibody against SKMEL28 target cells which express Inte 0309 Representative binding curves for Vitaxin(R) and grin CVB3 (FIG. 18). one Fc variant of Vitaxin(R) (I332E.; Vitaxin-1M) to FcyRIIIA and FcyRIIB are shown in FIGS. 8A and 8B respectively. 7.3.1. Materials and Methods Vitaxin(R) was prepared from two cell sources, NSO and 0313 ELISA Receptor Binding Assay: Microtiter plates HEK293 cells, no difference in binding was observed were coated with protein A/G (PIERCE) solution (0.25 between these two sources of Vitaxin. The Vitaxin(E) Fc Aug/ml) and incubated at 4 C. overnight. Any remaining variant was then prepared from HEK293 cells. The Vitaxin(R) binding sites were blocked with 4% skim milk. Approxi Fc variant showed approximately a 2.5 fold increase in mately 25 ul perwell of mutant antibody solution was added binding affinity to FcyRIIIA (FIG. 8A) with no correspond to each well and incubated for 1 h at 37 C. After washing, ing change in binding to FcyRIIB as determined by ELISA FcyRIIIA-streptavidin or FcyRIIB-streptavidin fusion pro tein (in 1% BSA) was added for 1 hour at 37° C., followed (FIG. 8B). by washing and biotin-conjugated HRP for 30 min. Detec 0310. The binding of Vitaxin(R) and the Vitaxin(R) Fic tion was carried out by adding 30 ul of tetramethylbenzidine variants to FcyRIIIA was further analyzed by BIAcore substrate (Pierce) followed by neutralization with 30 ul of analysis. The binding of Vitaxin(R) and the Vitaxin(R) Fc 0.2 MH2SO. The absorbance was read at 450 nm variants were analyzed with the receptor Soluble and the 0314 Generation of 332 Amino Acid Substitutions: antibody immobile (see methods below). The Vitaxin-1M Fc QuikChange(R) II XL site-directed mutagenesis kit (Strat variant was shown to have a roughly 7 fold increase in agene, San Diego) was used to generate all the amino acid binding affinity to Fcy IIIA as compared to that of the Substitutions at position 332 of the gene encoding the heavy parental wild type Vitaxin antibody. The interaction of the chain of wild type Vitaxin(R) in the plasmid pMI331 (see Vitaxin-3M Fc variant to FcyRIIIA was also analyzed by FIG. 4). Oligos 1 to 19 (see Table 5) were applied to change BIAcore and found to have a binding affinity of ~114 nM, the Isoleucine to all other 19 different amino acids at the nearly 80 time better than that of the parental wild type position 332, using Vitaxin as the template. The mutation Vitaxin antibody. The results are summarized in Table 6. was further confirmed by DNA sequencing. US 2006/0039904 A1 Feb. 23, 2006 49
0315. The plasmid DNA containing antibody genes was concentration were made. All binding experiments were transiently transfected into 293H cells by Lipofectamine performed at 25 C., and at a flow rate of 10 u/min. Binding 2000 Transfection Reagent (Invitrogen). After three collec was monitored for 25 min. Following each injection of tions within 9 days, the culture Supernatants containing FcyRIIIA, the IgG surfaces were regenerated with a 30 sec. antibody were affinity purified by using a pre-packed Protein pulse of 5 mM HCl. FcyRIIIA was also passed over a blank A column (Amersham BioSciences, now belongs to GE reference cell which is connected, in Series, to the IgG healthcare). The bound antibody were eluted with elution containing flow cells. The Steady-state binding curves were buffer (100 mM Glycine, pH3.2), neutralized by 1M Tris also corrected for injection artifacts by Subtraction of buffer buffer (pH 8.0) and then dialyzed in PBS. All purified injections. This doubly-corrected data was then fit to a antibodies were analyzed by SDS-polyacrylamide gel elec Steady-state isotherm provided by the instrument manufac trophoresis and were applied to quantitative ELISA using turer (Pharmacia Biosensor, Uppsala, Sweden) to derive the anti-human IgG assay plates (Becton Dickson) or BCA kits respective equilibrium binding constants (KD). Separately, a (PIERCE) to determine IgG concentrations. Scatchard plot of the Req data from each IgG Surface was 0316 Generation of Vitaxin(R)-1M and 3M Fc variants: constructed to confirm the results of the binding isotherms. The I332E substitution was generated by site directed 0319 Antibody-Dependent Cell-Mediated Catotoxicity mutagenesis (as described above) of the gene encoding the (ADCC) Assay: Antibody-dependent cell cytotoxicity heavy chain of wild type Vitaxin(R) in the plasmid pMI331 (ADCC) was assayed in a four-hour non-radioactive lactate (see FIG. 4). The mutant I332E was designated as Vitaxin dehydrogenase (LDH) release assay (Promega Corporation, 1M. The Vitaxin 3M was further generated by two sequential Madison, Wis.). Briefly, M21, A549, or H358 target cells Site directed mutagenesis (as described above), using oligo were distributed into 96-well U-bottomed plates (1x10"/50 20 and 21 (see Table 5) as primers and Vitaxin 1M as ul) and pre-incubated with serial dilution of antibodies (50 template. Expression and purification of the 1M and 3M ul) for 20 min at 37° C. Human effector cells (100 ul) were Vitaxin(E) Fc variants was the same as described above. then added at effector to target ratios of 50:1 and 25:1. 0317 Kinetic Analysis via BIAcore: for Run 1 the inter Human effector cells were peripheral blood mononuclear action of FcyRIIIA with immobilized Vitaxine) and cells (PBMC) purified from healthy donors using Lympho Vitaxin(R) Fc variant IgGs were monitored by Surface plas cyte Separation Medium (MP Biomedicals, Irvine, Calif.). mon resonance detection using a BIAcore 3000 instrument After a 4-h incubation at 37 C., plates were centrifuged, and (Pharmacia Biosensor, Uppsala, Sweden). Vitaxin(R) and cell death was analyzed by measuring the release of LDH Vitaxin(R) Fc variant IgGs were coupled to the dextran matrix into the cell supernatant with a 30-minute coupled enzy of a CM5 sensor chip (Pharmacia Biosensor) using an matic assay. The percentage of Specific lysis was calculated Amine Coupling Kit, as described (Johnsson et al., 1992, according to the formula: % specific lysis=100x(E-E- Anal Biochem 198:268-277), at a surface density of between T)/(T-T) where E. represents the release from 7700 and 9400 RUs (see Table 6). FcyRIIIA was serially experimental wells, E spon is the Spontaneous release of diluted in 0.01 M HEPES pH 7.4 containing 0.15 M NaCl, effector cells alone, T is spontaneous release of target 3 mM EDTA and 0.005% P20, at concentrations ranging cells alone, and T is the maximum release from lysed from 2 uM down to 7.8 nM. Duplicate injections of each target cells. concentration were made. All binding experiments were 0320. The cell lines used for the ADCC studies included performed at 25 C., and at a flow rate of 10 ul/min. Binding the following: A498 and ACHN renal cell carcinomas with was monitored for 25 min. Following each injection of moderate expression of Integrin CVB3, M21 a melanoma FcyRIIIA, the IgG surfaces were regenerated with a 30 sec. cell line with high Integrin CVB3 expression, DU145 a pulse of 5 mM HCl. FcyRIIIA was also passed over a blank prostate cancer cell line with low levels of Integrin C.VB3, reference cell which is connected, in Series, to the IgG SKMEL28 a human melanoma expressing Integrin C.VB3 containing flow cells. The Steady-state binding curves were but little or no human EphA2. also corrected for injection artifacts by subtraction of buffer injections. This doubly-corrected data was then fit to a 7.4 Example 4 Steady-state isotherm provided by the instrument manufac turer (Pharmacia Biosensor, Uppsala, Sweden) to derive the Fc Variants of Antibodies Recognizing Other Epitopes respective equilibrium binding constants (KD). Separately, a 0321) Given the remarkable improvement in ADCC Scatchard plot of the Req data from each IgG Surface was activity of the Vitaxin(R) Fc (I332E) variant the (I332E) constructed to confirm the results of the binding isotherms. Substitution was made in two other antibodies designated 0318 For Run 2 the interaction of The interaction of 12G3H11 (abbreviated 12G3) and 3F2, both of which bind FcyRIIIA with immobilized Vitaxin(R) and Vitaxin(R) Fc vari the EphA2 tyrosine receptor kinase. The variable regions of ant IgGs were monitored by Surface plasmon resonance 12G3 (FIG. 2A) and 3F2 (FIG.3A) heavy chain were fused detection using a BIAcore 3000 instrument (Pharmacia to the wt and variant Fc domains generated above (see Biosensor, Uppsala, Sweden). Vitaxin(R) and Vitaxin(R) Fc sections 7.1 and 7.3). The variable region of the light chain variant IgGs were coupled to the dextran matrix of a CM5 of Vitaxins was replaced with the corresponding light chain Sensor chip (Pharmacia Biosensor) using an Amine Cou variable region (i.e., 12G3 or 3F2, see FIGS. 2B and 3B, pling Kit, as described (Johnsson et al., 1992, Anal Biochem resectively) such that an intact 12G3 or 3F2 antibody was 198:268-277), at a surface density of between approximately encoded by the plasmid (see FIG. 4 for a map of the plasmid 8200 and 9400 RUs (see Table 6). FcyRIIIA was serially encoding Vitaxin E). The antibodies containing the single diluted in 0.01 M HEPES pH 7.4 containing 0.15 M NaCl, substitutions were designated 12G3-1M and 3F2-1M, 3 mM EDTA and 0.005% P20, at concentrations ranging respectively. In addition, the S239D, A330L, I332L triple from 16 uM down to 7.8 nM. Duplicate injections of each substitution was generated in 3F2, designated 3F2-3M. US 2006/0039904 A1 Feb. 23, 2006 50
0322 The binding characteristics of the 3F2-wt, 3F2-1M seen to be higher than wild type 3F2. The activity of the and 3F2-3M Fc variants to several Fc ligands were exam 3F2-1M Fc variant was also higher than the 3F2-wt. ined in vitro by ELISA (FIG. 12). Representative binding curves for 3F2 and the Fc variants of 3F2 (3F2-1M and 7.4.1 Materials and Methods 3F2-3M) to FcyRIIIA tetramers (FIG. 12, top panel), FcyRIIIA monomers (FIG. 12, middle panel) and C1q (FIG. 0326 Generation of 12G3 and 3F2 Fc variants: To gen 12, bottom panel). From these data it can be seen that both erate the 12G3 and 3F2 Fc variants, the DNA sequences the 3F2 Fc variants have improved binding to the mono encoding the variable region of Vitaxin(R) 1M or 3M heavy meric and tetrameric forms of FcyRIIIA. In contrast both the chain (VH) was replaced with the variable region of 12G3 3F2 Fc variants have reduced C1q binding with 3F2-3M or 3F2 heavy chain to create 12G3-1M, 3F2-1M and 3F2 having the largest reduction in C1q binding (FIG. 12, 3M Fc variants using Xba I/Apa I restriction sites (see bottom panel). 103231 The binding of the 3F2 and the 3F2 plasmid map, FIG. 4). The DNA sequences encoding the Fc variants to FcyRIIIA was further analyzed by BIAcore variable region of Vitaxin E) light chain were also replaced analysis. The binding of 3F2 and the 3F2 Fc variant was with the variable region of 12G3 or 3F2 light chain using analyzed with the receptor Soluble and the antibody immo SmaI/BsiWI restriction sites (see plasmid map, FIG. 4). The bile (see methods below). The data obtained for 3F2 and the nucleotide Sequence of the 12G3 heavy and light chain 3F2 Fc variants (Run 3) is similar to that obtained for variable regions are listed as SEQ ID NO.: 62 and 63 Vitaxin(R) and the Vitaxin(R) Fc variants (Runs 1 & 2) with respectively. The amino acid Sequence of the 12G3 heavy improvements in binding of about 7 fold and 80 fold for the and light chain variable regions are listed as SEQ ID NO.: Vitaxin(R) 1M and 3M Fc variants, respectively, and about 9 64 and 65 respectively. The nucleotide sequence of the 3F2 fold and 100 fold for the 3F2-1M and 3M Fc variants, heavy and light chain variable regions are listed as SEQ ID respectively. The small differences between these numbers NO.: 66 and 67 respectively. The amino acid sequence of the may reflect Subtle differences in glycosylation between 3F2 heavy and light chain variable regions are listed as SEQ antibody produced in 293H cells vs NSO cells (Vitaxin ID NO.: 68 and 69 respectively. antibodies and 3F2 antibodies, respectively) as the variable domain is generally not thought to affect FcyRIIIA binding. 0327. The plasmid DNA containing the 12G3 antibody The results are Summarized in Table 6. genes was stably transfected into 293H cells by Lipo fectamine 2000 Transfection Reagent (Invitrogen). The plas 0323) The binding of 3F2-wt, 3F2-1M and 3F2-3M Fc mid DNA containing the 3F2 antibody genes was stably variants to the Surface of cells via Fc ligand interactions was transfected into NSO by electroporation. Antibodies were examined. Two cell types were utilized, THP-1 cells and NK purified from cell culture Supernatants by using a pre-packed cells. To determine which Fc ligands were present on the Protein A column (Amersham BioSciences, now belongs to Surface both cell types were Stained with antibodies recog GE healthcare). The bound antibody were eluted with elu nizing CD32 (FcyRII); CD64 (FcyRI) or CD16 (FcyIII) and tion buffer (100 mM Glycine, pH3.2), neutralized by 1M analyzed by FACS. The percent of cell staining positive for Tris buffer (pH 8.0) and then dialyzed in PBS. All purified each Fc ligand are plotted in FIG. 13. As can be seen in FIG. antibodies were analyzed by SDS-polyacrylamide gel elec 14 panel A, THP-1 cells predominantly express CD32 with trophoresis and were applied to quantitative ELISA using a small amount of CD64 present on the cell surface. In contrast NK cells express CD16 almost exclusively (FIG. anti-human IgG assay plates (Becton Dickson) or BCA kits 13, panel B). All three versions of 3F2 (wt, 1M and 3M) (PIERCE) to determine IgG concentrations. bound to a similar degree to THP-1 cells (FIG. 13, panel C). 0328 Kinetic Analysis via BIAcore: for Run 3 the inter However, the two Fc variants (3F2-1M and 3F2-3M) were action of FcyRIIIA with immobilized Vitaxin(R) and seen to bind to a greater extent to NK cells, with the 3F2-3M Vitaxin(R) Fc variant IgGs were monitored by Surface plas Fc variant showing the largest increase in binding (FIG. 13, mon resonance detection using a BIAcore 3000 instrument panel D). (Pharmacia Biosensor, Uppsala, Sweden). Vitaxin(R) and 0324. The ADCC activity of all the variants was exam Vitaxin(R) Fc variant IgGs were coupled to the dextran matrix ined. Shown in FIGS. 14A and 14B are ADCC assays of a CM5 sensor chip (Pharmacia Biosensor) using an performed using the 12G3H11-Fc (I332E) variant and the Amine Coupling Kit, as described (Johnsson et al., 1992, parental 12G3H11 antibody against A549 target cells using Anal Biochem 198:268-277), at a surface density of between effector cells from two donors. The assays were performed approximately 7700 and 9400 RUs (see Table 6). FcyRIIIA using two different ratios of target to effector cell (50:1 and was serially diluted in 0.01M HEPES pH 7.4 containing 0.15 25:1) and antibody concentrations ranging from 4 to 400 ng M NaCl, 3 mM EDTA and 0.005% P20, at concentrations per well. Remarkably, a 10 fold increase in ADCC activity ranging from 16 uM down to 7.8 nM. Duplicate injections is seen for the 12G3H11-Fc (I332E) variant compared to the of each concentration were made. All binding experiments parent antibody. were performed at 25 C., and at a flow rate of 10 ul/min. Binding was monitored for 25 min. Following each injection 0325 FIGS. 15, 16 and 17 are ADCC assays comparing of FcyRIIIA, the IgG surfaces were regenerated with a 30 the activity of 3F2-wt and the 3F2 Fc variants against target Sec. pulse of 5 mM HCl. FcyRIIIA was also passed over a cells expressing different levels of EphA2. The target cell blank reference cell which is connected, in Series, to the lines used were T23,1 A549 and Hey8 (high expressors), IgG-containing flow cells. The Steady-state binding curves SKOV3 (a moderate expressor), A498 and SKMEL28 (low were also corrected for injection artifacts by Subtraction of expressors). The assays were performed using three different buffer injections. This doubly-corrected data was then fit to ratios of target to effector cell (between 12.5:1 and 100:1) a steady-state isotherm provided by the instrument manu and antibody concentrations ranging from 0.02 to 2 ug/ml. facturer (Pharmacia Biosensor, Uppsala, Sweden) to derive In all cases the ADCC activity of the 3F2-3M Fc variant was the respective equilibrium binding constants (KD). Sepa US 2006/0039904 A1 Feb. 23, 2006
rately, a Scatchard plot of the Req data from each IgG each well and incubated for 30 min at 37 C. Detection was Surface was constructed to confirm the results of the binding carried out by adding 30 ul of tetramethylbenzidine (TMB) isotherms. substrate (Pierce) followed by neutralization with 30 ul of 0329 Cell Surface Binding: NK cells were isolated from 0.2 MHSO. The absorbance was read at 450 nm. healthy donor by using NK cell isolation kit from Milteny 0334 ELISA for C1q Bindini: Microtiter plates were biotec (Catil 130-091-152) THP-1: early passage of THP-1 coated with 50 ul of test antibody at concentration range cells were used. For FACS staining of FcyRs, either THP-1 from 20 tug/ml to 0.0019 tug/ml and incubated at 4 C. or human NK cells were resuspended in FACS buffer (1% overnight. The plate was then blocked with 5% nonfat BSA in PBS, pH 7.2) at 1x10 cells/ml and 0.5 ml of the powdered milk for 60 min at 37 C. 50 ul of 5 lug/ml human cells were transfered into 96 deep well plate, 10 ul of the C1q complement protein (Quidal, SanDiego) was added to anti-CD32-PE (Immunotech), anti-CD16-FITC (Pharmin each well and inclubated for 60 min at 37° C.50 ul of 1:1000 gen) or anti-CD64-FITC (PharMingen) was added to the dilution of anti-complement C1q antibody (Biodesign) was tubes. The samples were incubated at 40C for 30 min. After added to each well and incubated for 60 min at 37 C. 50 ul incubation the cells were washed with FACS buffer. The of 1:1000 dilution of donkey anti-sheep/goat antibody Samples were analyzed by using Guava EasyCyte conjugated HRP (PIERCE) was added to each well and 0330 For binding of antibody 3F2 to Human NK cell incubated for 60 min at 37 C. Detection was carried out by surface (FcyRIIIA), 10ul of the antibody dilution(10 ug/ml adding 30 ul of tetramethylbenzidine (TMB) substrate or 1 tug/ml) was added to the cells and incubated at 4°C. for (Pierce) followed by neutralization with 30 ul of 0.2 M 30 min. The cells were washed with FACS buffer, then HSO. The absorbance was read at 450 nm. stained with goat ant-human IgG(H+L)-FITC(Pierce) for 30 0335) Antibody-Dependent Cell-Mediated Cytotoxicity min at 4 C. The cells were washed and analyzed by Guava (ADCC) Assay: Antibody-dependent cell cytotoxicity EasyCyte. (ADCC) was assayed as described above in section 7.3.1 0331) For binding of antibody 3F2 to THP-1 cell surface using different target cells. The target cell lines used for (FcyRI and FcyRII), 10ul of the antibody dilution(10 ug/ml these assays are A549 a human non-Small cell lung adeno or 1 tug/ml) were added to the cells, incubatee at 4 C. for 30 carcinoma cell line expressing high levels of human EphA2, min. The cells were washed with FACS buffer, then stained T231 a more metastatic variant of MDA-MB-231 human with goat ant-human IgG(H+L)-FITC(Pierce) for 30 min at breast adenocarcinoma cell line obtained from collaborator 4 C. The cells were washed and analyzed by Guava Kathy Miller at Indiana University Medical Center express EasyCyte. ing high levels of human EphA2, HeyA8 a human ovarian carcinoma expressing high levels of human EphA2, SKOV3 0332 ELISA for FcyRIIIA Tetramer Binding: Microtiter a human ovarian adenocarcinoma derived from ascites plates were coated with protein A/G (PIERCE) solution expressing moderate levels of human EphA2, A498 a human (0.25 ug/ml) and incubated at 4 C. overnight. The plates renal cell carcinoma expressing low levels of human EphA2, were then washed with PBS/0.1% Tween and any remaining SKMEL28 a human melanoma expressing Integrin C.VB3 binding sites were blocked with 1% BSA. 50 ul of test but little or no human EphA2. antibody at 1:1 dilution (from 5000 ng/ml to 4.9 ng/ml), was added to each well and incubated for 60 min at 37 C. 50 ul 0336 Whereas, particular embodiments of the invention of 1:500 dilution of the Fcy tetramer was added to each well have been described above for purposes of description, it and incubated for 60 min at 37 C. followed by washing. 50 will be appreciated by those skilled in the art that numerous ul of 1:1000 dilution of biotin-conjugated HRP (PIERCE) variations of the details may be made without departing was added to each well and incubated for 30 min at 37 C. from the invention as described in the appended claims. Detection was carried out by adding 30 ul of tetramethyl 0337 All publications, patents and patent applications benzidine (TMB) substrate (Pierce) followed by neutraliza mentioned in this specification are herein incorporated by tion with 30 ul of 0.2 MHSO. The absorbance was read reference into the Specification to the same extent as if each at 450 nm. individual publication, patent or patent application was 0333 ELISA for FcyRIIIA Monomer Binding: Microtiter Specifically and individually indicated to be incorporated plates were coated with 50 ul to test antibody at concentra herein by reference. In addition, U.S. Provisional Patent tion range from 20 tug/ml to 0.0019 tug/ml and incubated at Application Nos.: 60/601,634, filed, Aug. 16, 2004 and 4 C. overnight. 50 ul of 10 ug/ml FcyRIIIA-flag protein was 60/608,852, filed, Sep. 13, 2004, and U.S. patent application added to each well and incubated for 60 min at 37 C. 50 ul entitled “Integrin Antagonists With Enhanced Antibody of 2.5 lug/ml anti-flag-ME-biotin (Sigma) was added to each Dependent Cell-Mediated Cytotoxicity Activity,” Attorney well and incubated for 30 min at 37° C. 50 ul of 1:1000 Docket No.: AE701 US, filed Aug. 15, 2005, are incorpo diulation of avidin-conjugated HRP (PIERCE) was added to rated by reference in their entirety
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS : 101
<210> SEQ ID NO 1 &2 11s LENGTH 351 US 2006/0039904 A1 Feb. 23, 2006 52
-continued
&212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 1 Caggtgcago toggtggagtc. togggggaggc gttgttgcagc Ctggalagg to CCtgagactic 60 to citgtgcag cctotggatt caccittcagt agctatoaca totcittgggit togcc aggct 120 cc.gggcaagg gtctggagtg g g togcaaaa gttagtag to gtggtggtag caccitacitat 18O ttagacactg tdcaggg.ccg attcaccatc. tccagagaca atagtaagaa caccotatac 240 citgcaaatga actictotgag agcc.gaggac acago.cgtgt attactgtgc aag acatctg 3OO catggcagtt ttgcttcttg g g gccaaggg act acagtga citgtttctag t 351
<210> SEQ ID NO 2 <211& LENGTH 321 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 2 gagattgttgc taactcagtc. tccagocacc ctdtctotca gcc caggaga aaggg.cg act 60 citttcc tocc aggc.ca.gc.ca aagtattagc aactitcc tac actggitatca acaaaggcct 120 ggtoaa.gc.cc caaggottct catcc.gctat cqttcc.cagt coatctotgg gatcc.ccgc.c 18O aggttcagtg gcagtggatc agggacagat titcaccotca citatcto cag totggagcct 240 gaagattittg cagtct atta citgtcaa.cag agtgg cagot goc citctgac gttcggaggg 3OO gggaccalagg toggaaattaa g 321
<210> SEQ ID NO 3 &2 11s LENGTH 117 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 3 Glin Val Glin Leu Val Glu Ser Gly Gly Gly Val Val Glin Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 2O 25 30 Asp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu Glu Trp Val 35 40 45 Ala Lys Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Leu Asp Thr Val 50 55 60 Glin Gly Arg Phe Thr Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu. Tyr 65 70 75 8O Leu Gln Met Asn. Ser Lieu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Leu. His Gly Ser Phe Ala Ser Trp Gly Glin Gly Thr Thr 100 105 110
Wall Thr Wal Ser Ser 115 US 2006/0039904 A1 Feb. 23, 2006 53
-continued <210> SEQ ID NO 4 &2 11s LENGTH 107 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 4 Glu Ile Val Leu Thr Glin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Lieu Ser Cys Glin Ala Ser Glin Ser Ile Ser Asn. Phe 2O 25 30 Lieu. His Trp Tyr Glin Glin Arg Pro Gly Glin Ala Pro Arg Lieu Lieu. Ile 35 40 45 Arg Tyr Arg Ser Glin Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thir Ile Ser Ser Leu Glu Pro 65 70 75 8O Glu Asp Phe Ala Val Tyr Tyr Cys Glin Glin Ser Gly Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
<210 SEQ ID NO 5 &2 11s LENGTH 5 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 5 Asp Tyr Ser Met Asn 1 5
<210> SEQ ID NO 6 &2 11s LENGTH 19 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 6 Phe Ile Arg Asn Lys Ala Asn Asp Tyr Thir Thr Glu Tyr Ala Asp Ser 1 5 10 15 Wall Lys Gly
<210 SEQ ID NO 7 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 7 Tyr Pro Arg His His Ala Met Asp Ser 1 5
<210 SEQ ID NO 8 <211& LENGTH: 11 &212> TYPE PRT <213> ORGANISM: Artificial US 2006/0039904 A1 Feb. 23, 2006 54
-continued
&220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 8 Arg Ala Ser Glin Ser Ile Ser Asn. Asn Lieu. His 1 5 10
<210 SEQ ID NO 9 &2 11s LENGTH 7 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 9 Tyr Ala Phe Glin Ser Ile Ser 1 5
<210> SEQ ID NO 10 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 10 Gln Glin Ala Asn Ser Trp Pro Leu Thr 1 5
<210> SEQ ID NO 11 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (19) . . (20) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 11 cc.gtgcc.cag cacctgaann kctgggggga cc.gtoagtc 39
<210> SEQ ID NO 12 <211& LENGTH 42 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (22). ... (23) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 12 cc.gtgcc.cag cacctgaact cinnkggggga cc.gtoagtct tc 42
<210> SEQ ID NO 13 &2 11s LENGTH 45 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (25) . . (26) US 2006/0039904 A1 Feb. 23, 2006 55
-continued
OTHER INFORMATION: n is a 9. or it <400 SEQUENCE: 13 cc.gtgcc.cag cacctgaact cotgninkgga cc.gtoagtct tcc to 45
SEQ ID NO 14 LENGTH 48 TYPE DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: synthetic primer FEATURE: NAME/KEY: misc feature LOCATION: (28) . . (29) OTHER INFORMATION: n is a 9. or it <400 SEQUENCE: 14 cc.gtgcc.cag cacctgaact cotgggginnik cc.gtoagtct tcc tottc 48
SEQ ID NO 15 LENGTH 51 TYPE DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: synthetic primer FEATURE: NAME/KEY: misc feature LOCATION: (31) . . (32) OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 15 cc.gtgcc.cag cacctgaact cotgggggga innktcagtct tcc tott.ccc c 51
SEQ ID NO 16 LENGTH 54 TYPE DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: synthetic primer FEATURE: NAME/KEY: misc feature LOCATION: (34) . . (35) OTHER INFORMATION: n is a C 9. or it <400 SEQUENCE: 16 cc.gtgcc.cag cacctgaact cotgggggga ccginnikgtot to citctt.ccc ccca 54
SEQ ID NO 17 LENGTH 39 TYPE DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: synthetic primer FEATURE: NAME/KEY: misc feature LOCATION: (19) . . (20) OTHER INFORMATION: n is a 9. or it <400 SEQUENCE: 17 gtoacatgcg toggtggtgnin kgtgagccac galagaccct 39
SEQ ID NO 18 LENGTH 45 TYPE DNA ORGANISM: Artificial FEATURE: US 2006/0039904 A1 Feb. 23, 2006 56
-continued <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (25) . . (26) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 18 gtoacatgcg toggtggtgga C gtgninkcac galagaccctd aggto 45
<210 SEQ ID NO 19 &2 11s LENGTH 51 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (31) . . (32) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 19 gtoacatgcg toggtggtgga C gtgagccac innkigacic ctd aggtoaagtt c 51
<210> SEQ ID NO 20 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (19) . . (20) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 20 cgg gaggagc agtacaa.cnn kac gtaccgt gtggtoagc 39
<210> SEQ ID NO 21 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (19) . . (20) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 21 tgcaaggtot coaacaaann kict cocagoc cccatcgag 39
<210> SEQ ID NO 22 <211& LENGTH 42 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (22). ... (23) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 22 tgcaaggtot coaacaaagc cinnkccagoc cccatc.gaga aa 42
<210> SEQ ID NO 23 US 2006/0039904 A1 Feb. 23, 2006 57
-continued
&2 11s LENGTH 45 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (25) . . (26) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 23 tgcaaggtot coaacaaagc cct cinnkgcc cccatc.gaga aaacc 45
<210> SEQ ID NO 24 &2 11s LENGTH 48 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (28) ... (29) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 24 tgcaaggtot coaacaaagc cct cocannik cccatc.gaga aaaccatc 48
<210> SEQ ID NO 25 &2 11s LENGTH 54 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (34) . . (35) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 25 tgcaaggtot coaacaaagc cct cocagoc cc.cnnikgaga aaacCatcto caaa 54
<210> SEQ ID NO 26 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 26 actcacacat gtccaccgtg cccago acct gaa 33
<210 SEQ ID NO 27 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 27 caccaccacg catgtgac 18
<210> SEQ ID NO 28 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE US 2006/0039904 A1 Feb. 23, 2006 58
-continued <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 28 gttgtactg.c toctoccg 18
<210 SEQ ID NO 29 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 29 tttgttggag accittgca 18
<210 SEQ ID NO 30 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 30 aaccitctaca aatgtggitat ggct 24
<210> SEQ ID NO 31 <211& LENGTH 42 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OOTHER INFORMATION: synthetic primer <400 SEQUENCE: 31 aagctt.cggit cogccaccat ggcaactgaa gatctoccaa ag 42
<210> SEQ ID NO 32 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OOTHER INFORMATION: synthetic primer <400 SEQUENCE: 32 gtotg.ccgaa cc.gctgcctg. ccaaaccttg agtgatggit 39
<210 SEQ ID NO 33 &2 11s LENGTH 48 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OOTHER INFORMATION: synthetic primer <400 SEQUENCE: 33 agctitcgg to cqccaccatg gotgtgct at toctogcago tocco cala 48
<210> SEQ ID NO 34 <211& LENGTH 42 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 34 US 2006/0039904 A1 Feb. 23, 2006 59
-continued gtotg.ccgaa cc.gctg.cccc ccatcggtga agagctdgga gc 42
<210 SEQ ID NO 35 &2 11s LENGTH 30 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 35 ggcagoggitt cqgcag acco citccaaggac 30
<210 SEQ ID NO 36 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 36 Caggggctag Cttact.gctg aacgg.cgt.cg agcgg 35
<210 SEQ ID NO 37 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 37 to cacaggtg to cacticcc.g. gactogaagat citc.ccaaag 39
<210 SEQ ID NO 38 &2 11s LENGTH 91. &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 38 gggaga attc cqcggcc.gct tatttgtcat cqtcatctitt gtagt catgg togatggtgat 60 ggtgtgcgcc toccaaacct to agtgatgg t 91
<210 SEQ ID NO 39 &2 11s LENGTH 48 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 39 to cacaggtg to cacticcgc tigtgctatto citggcagotc ccc.caaag 48
<210> SEQ ID NO 40 &2 11s LENGTH 94. &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 40 gggaga attc cqcggcc.gct tatttgtcat cqtcatctitt gtagt catgg togatggtgat 60 US 2006/0039904 A1 Feb. 23, 2006 60
-continued ggtgtgcgcc CCC catcggit galaga.gctgg gag C 94
<210> SEQ ID NO 41 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 41 gcc.citcc.cag ccc.ccgagga gaaaaccatc. tcc 33
<210> SEQ ID NO 42 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 42 gcc.citcc.cag ccc.cccagga gaaaaccatc. tcc 33
<210> SEQ ID NO 43 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 43 gcc.citcc.cag ccc.ccggcga gaaaaccatc. tcc 33
<210> SEQ ID NO 44 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 44 gcc.citcc.cag ccc.ccgc.cga gaaaaccatc. tcc 33
<210> SEQ ID NO 45 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 45 gcc.citcc.cag ccc.ccitacga gaaaaccatc. tcc 33
<210> SEQ ID NO 46 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 46 gcc.citcc.cag ccc.ccgacga gaaaaccatc. tcc 33
<210> SEQ ID NO 47 US 2006/0039904 A1 Feb. 23, 2006 61
-continued
&2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 47 gcc.citcc.cag ccc.ccaacga gaaaaccatc. tcc 33
<210> SEQ ID NO 48 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 48 gcc.citcc.cag ccc.ccgtgga gaaaaccatc. tcc 33
<210 SEQ ID NO 49 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 49 gccCtcCCag CCCCCtggga gaaaaccatc. tcc 33
<210 SEQ ID NO 50 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 50 gcc.citcc.cag ccc.ccc.gcga gaaaaccatc. tcc 33
<210 SEQ ID NO 51 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 51 gcc.citcc.cag ccc.ccagoga gaaaaccatc. tcc 33
<210> SEQ ID NO 52 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 52 gcc.citcc.cag ccc.ccaagga gaaaaccatc. tcc 33
<210 SEQ ID NO 53 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE US 2006/0039904 A1 Feb. 23, 2006 62
-continued <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 53 gcc.citcc.cag ccc.ccatgga gaaaaccatc. tcc 33
<210> SEQ ID NO 54 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 54 gcc.citcc.cag ccc.ccaccga gaaaaccatc. tcc 33
<210 SEQ ID NO 55 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 55 gcc.citcc.cag ccc.cctg.cga gaaaaccatc. tcc 33
<210 SEQ ID NO 56 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 56 gcc.citcc.cag ccc.ccctgga gaaaaccatc. tcc 33
<210 SEQ ID NO 57 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 57 gcc.citcc.cag ccc.cctitcga gaaaaccatc. tcc 33
<210 SEQ ID NO 58 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 58 gcc.citcc.cag ccc.cccacga gaaaaccatc. tcc 33
<210 SEQ ID NO 59 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 59 US 2006/0039904 A1 Feb. 23, 2006 63
-continued gcc.citcc.cag ccc.ccc.ctga gaaaaccatc. tcc 33
<210 SEQ ID NO 60 &2 11s LENGTH 27 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 60 citggggggac cqgacgtc.tt cottctitc 27
<210> SEQ ID NO 61 &2 11s LENGTH 27 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: synthetic primer <400 SEQUENCE: 61 aaag.ccctico cactgc.ccga ggagaaa 27
<210> SEQ ID NO 62 &2 11s LENGTH 360 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 62 caaatgcago toggtgcagtc. toggcctgag gtgaagaagc citggg acctic agtgaaggto 60 to citgcaagg cittctggatt caccitttgac gattact coa togaactgggit gcgacaggct 120 cgtggacaac gocttgagtg gataggattt attagaaa.ca aagctaatga citacacaa.ca 18O gagtacgctg actctgtgaa gogtagagtc accattacca gggacatgtc. cac gag caca 240 gcctacatgg agctgagcag cotgagatcc gaggacacgg cc.gtgtatta citgtgc.gaga 3OO tacccitaggc atcatgctat ggacticcitgg ggccaaggaa cctoggtoac cqtct cotca 360
<210 SEQ ID NO 63 <211& LENGTH 321 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 63 gacatccaga tigacccagtc. tccatcctico citgtctgcat citgtaggaga cagagtcacc 60 atcacttgca gggccago.ca aagtattagc aacaacctac actggitatca gcagaalacca 120 gggaaag.ccc ctaagctcct gatcaagtat gcc titccagt coatctotgg g g toccatca 18O aggttcagtg gaagtggatc toggacagat titt actittca ccatcagoag cotgcagoct 240 gaagattittg caa.cat atta citgtcaa.cag gocaa.cagot goccgct cac gttcggcgga 3OO gggaccalagg toggagatcaa a 321
<210> SEQ ID NO 64 <211& LENGTH: 120 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE US 2006/0039904 A1 Feb. 23, 2006 64
-continued <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 64 Gln Met Gln Leu Val Glin Ser Gly Pro Glu Val Lys Lys Pro Gly. Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Asp Asp Tyr 2O 25 30 Ser Met Asn Trp Val Arg Glin Ala Arg Gly Glin Arg Lieu Glu Trp Ile 35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr 65 70 75 8O Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Tyr Pro Arg His His Ala Met Asp Ser Trp Gly Glin 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
<210 SEQ ID NO 65 &2 11s LENGTH 107 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 65 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Ile Ser Asn. Asn 2O 25 30 Lieu. His Trp Tyr Glin Gln Lys Pro Gly Lys Ala Pro Lys Lieu Lieu. Ile 35 40 45 Lys Tyr Ala Phe Glin Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 8O Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin Ala Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
<210 SEQ ID NO 66 &2 11s LENGTH 361 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 66 gaggtgcago toggtggagtc. togggggaggt gtggtacggC Ctggggggtc. CCtgagactic 60 to citgtgcag cctotgggitt caccgtoagt gattact coa togaactgggit cogcc aggct 120 ccagggaagg gcc toggagtg gattgggttt attagaaa.ca aagctaatgc ctacacaa.ca 18O gagtacagtg catctgtgaa gogtagatto accatctoaa gagatgattic aaaaaac acg 240 US 2006/0039904 A1 Feb. 23, 2006 65
-continued citgitatctgc aaatgaacag cotgaaaacc gaggacacag cc.gtgtatta citgtaccaca 3OO tacccitaggt atcatgctat ggacticcitgg ggc.cagggca ccatggtoac cqtct cotca 360 g 361
<210 SEQ ID NO 67 <211& LENGTH 321 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 67 gccatccagt to acto agtc. tccatcctico citgtctgcat citgtaggaga cagagtcacc 60 atcacttgca gggccago.ca aagtattagc aacaacctac actggtacct gcagaagcca 120 gggcagtc.tc. cacagotcct gatct attat ggctt.ccagt coatctotgg g g toccatca 18O aggttcagtg gcagtggatc toggacagat titcactcitca ccatcagoag totgcaacct 240 gaagattittg caacttacta citgtcaa.cag gocaa.cagot goccgct cac gttcggcgga 3OO gggacCaagc tiggagatcaa a 321
<210 SEQ ID NO 68 <211& LENGTH: 120 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 68 Glu Val Glin Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Lieu Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 2O 25 30 Ser Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu Glu Trp Ile 35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 8O Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Tyr Pro Arg Tyr His Ala Met Asp Ser Trp Gly Glin 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120
<210 SEQ ID NO 69 &2 11s LENGTH 107 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody variable region <400 SEQUENCE: 69 Ala Ile Gln Leu Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 US 2006/0039904 A1 Feb. 23, 2006 66
-continued Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Ile Ser Asn. Asn 2O 25 30 Lieu. His Trp Tyr Lieu Gln Lys Pro Gly Glin Ser Pro Glin Leu Lieu. Ile 35 40 45 Tyr Tyr Gly Phe Glin Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thir Ile Ser Ser Leu Gln Pro 65 70 75 8O Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin Ala Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Lieu Glu Ile Lys 100 105
<210 SEQ ID NO 70 &2 11s LENGTH 5 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 70 Asp Tyr Ser Met Asn 1 5
<210> SEQ ID NO 71 &2 11s LENGTH 19 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 71 Phe Ile Arg Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser 1 5 10 15 Wall Lys Gly
<210 SEQ ID NO 72 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 72 Tyr Pro Arg Tyr His Ala Met Asp Ser 1 5
<210 SEQ ID NO 73 <211& LENGTH: 11 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 73 Arg Ala Ser Glin Ser Ile Ser Asn. Asn Lieu. His 1 5 10
<210> SEQ ID NO 74 &2 11s LENGTH 7 &212> TYPE PRT US 2006/0039904 A1 Feb. 23, 2006 67
-continued <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 74 Tyr Gly Phe Glin Ser Ile Ser 1 5
<210 SEQ ID NO 75 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: recombinant antibody CDR <400 SEQUENCE: 75 Gln Glin Ala Asn Ser Trp Pro Leu Thr 1 5
<210 SEQ ID NO 76 &2 11s LENGTH 976 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 76 Met Glu Arg Arg Trp Pro Leu Gly Lieu Gly Lieu Val Lieu Lleu Lieu. Cys 1 5 10 15 Ala Pro Leu Pro Pro Gly Ala Arg Ala Lys Glu Val Thr Lieu Met Asp 2O 25 30 Thir Ser Lys Ala Glin Gly Glu Lieu Gly Trp Lieu Lleu. Asp Pro Pro Lys 35 40 45 Asp Gly Trp Ser Glu Glin Glin Glin Ile Lieu. Asn Gly Thr Pro Leu Tyr 50 55 60 Met Tyr Glin Asp Cys Pro Met Glin Gly Arg Arg Asp Thr Asp His Trp 65 70 75 8O Leu Arg Ser Asn Trp Ile Tyr Arg Gly Glu Glu Ala Ser Arg Val His 85 90 95 Val Glu Leu Glin Phe Thr Val Arg Asp Cys Lys Ser Phe Pro Gly Gly 100 105 110 Ala Gly Pro Leu Gly Cys Lys Glu Thir Phe Asn Lieu Lleu Tyr Met Glu 115 120 125 Ser Asp Glin Asp Val Gly Ile Glin Leu Arg Arg Pro Leu Phe Glin Lys 130 135 1 4 0 Val Thr Thr Val Ala Ala Asp Glin Ser Phe Thir Ile Arg Asp Leu Ala 145 15 O 155 160 Ser Gly Ser Wall Lys Lieu. Asn Val Glu Arg Cys Ser Lieu Gly Arg Lieu 1.65 170 175 Thr Arg Arg Gly Lieu. Tyr Lieu Ala Phe His Asn. Pro Gly Ala Cys Wal 18O 185 19 O Ala Leu Val Ser Val Arg Val Phe Tyr Glin Arg Cys Pro Glu Thr Leu 195 200 2O5 Asn Gly Lieu Ala Glin Phe Pro Asp Thr Lieu Pro Gly Pro Ala Gly Lieu 210 215 220 Val Glu Val Ala Gly. Thir Cys Lieu Pro His Ala Arg Ala Ser Pro Arg 225 230 235 240 Pro Ser Gly Ala Pro Arg Met His Cys Ser Pro Asp Gly Glu Trp Leu US 2006/0039904 A1 Feb. 23, 2006 68
-continued
245 250 255 Val Pro Val Gly Arg Cys His Cys Glu Pro Gly Tyr Glu Glu Gly Gly 260 265 27 O Ser Gly Glu Ala Cys Val Ala Cys Pro Ser Gly Ser Tyr Arg Met Asp 275 280 285 Met Asp Thr Pro His Cys Leu Thr Cys Pro Gln Gln Ser Thr Ala Glu 29 O 295 3OO Ser Glu Gly Ala Thr Ile Cys Thr Cys Glu Ser Gly His Tyr Arg Ala 305 310 315 320 Pro Gly Glu Gly Pro Glin Val Ala Cys Thr Gly Pro Pro Ser Ala Pro 325 330 335 Arg Asn Lieu Ser Phe Ser Ala Ser Gly Thr Glin Leu Ser Lieu Arg Trip 340 345 35 O Glu Pro Pro Ala Asp Thr Gly Gly Arg Glin Asp Val Arg Tyr Ser Val 355 360 365 Arg Cys Ser Glin Cys Glin Gly Thr Ala Glin Asp Gly Gly Pro Cys Glin 370 375 38O Pro Cys Gly Val Gly Val His Phe Ser Pro Gly Ala Arg Ala Leu Thr 385 390 395 400 Thr Pro Ala Val His Val Asn Gly Leu Glu Pro Tyr Ala Asn Tyr Thr 405 410 415 Phe Asin Val Glu Ala Glin Asn Gly Val Ser Gly Leu Gly Ser Ser Gly 420 425 43 O His Ala Ser Thr Ser Val Ser Ile Ser Met Gly His Ala Glu Ser Leu 435 4 40 4 45 Ser Gly Lieu Ser Leu Arg Lieu Val Lys Lys Glu Pro Arg Glin Leu Glu 450 455 460 Lieu. Thir Trp Ala Gly Ser Arg Pro Arg Ser Pro Gly Ala Asn Lieu. Thr 465 470 475 480 Tyr Glu Lieu. His Val Lieu. Asn Glin Asp Glu Glu Arg Tyr Glin Met Val 485 490 495 Leu Glu Pro Arg Val Leu Leu Thr Glu Leu Gln Pro Asp Thr Thr Tyr 5 OO 505 51O. Ile Val Arg Val Arg Met Leu Thr Pro Leu Gly Pro Gly Pro Phe Ser 515 52O 525 Pro Asp His Glu Phe Arg Thr Ser Pro Pro Val Ser Arg Gly Leu Thr 530 535 540 Gly Gly Glu Ile Val Ala Val Ile Phe Gly Lieu Lleu Lleu Gly Ala Ala 545 550 555 560 Leu Lleu Lieu Gly Ile Leu Val Phe Arg Ser Arg Arg Ala Glin Arg Glin 565 570 575 Arg Glin Glin Arg Glin Arg Asp Arg Ala Thr Asp Wall Asp Arg Glu Asp 58O 585 59 O Lys Lieu Trp Lieu Lys Pro Tyr Val Asp Leu Glin Ala Tyr Glu Asp Pro 595 600 605 Ala Glin Gly Ala Lieu. Asp Phe Thr Arg Glu Lieu. Asp Pro Ala Trp Lieu 610 615 62O Met Val Asp Thr Val Ile Gly Glu Gly Glu Phe Gly Glu Val Tyr Arg 625 630 635 640 Gly Thr Lieu Arg Lieu Pro Ser Glin Asp Cys Lys Thr Val Ala Ile Lys 645 650 655 US 2006/0039904 A1 Feb. 23, 2006 69
-continued
Thr Lieu Lys Asp Thr Ser Pro Gly Gly Glin Trp Trp Asn. Phe Leu Arg 660 665 67 O Glu Ala Thr Ile Met Gly Glin Phe Ser His Pro His Ile Leu. His Leu 675 680 685 Glu Gly Val Val Thr Lys Arg Llys Pro Ile Met Ile Ile Thr Glu Phe 69 O. 695 7 OO Met Glu Asn Gly Ala Lieu. Asp Ala Phe Leu Arg Glu Arg Glu Asp Glin 705 710 715 720 Leu Val Pro Gly Glin Lieu Val Ala Met Leu Glin Gly Ile Ala Ser Gly 725 730 735 Met Asn Tyr Lieu Ser Asn His Asn Tyr Val His Arg Asp Leu Ala Ala 740 745 750 Arg Asn. Ile Leu Val Asn Glin Asn Lieu. Cys Cys Lys Wal Ser Asp Phe 755 760 765 Gly Lieu. Thir Arg Lieu Lieu. Asp Asp Phe Asp Gly Thr Tyr Glu Thr Glin 770 775 78O Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala His 785 790 795 8OO Arg Ile Phe Thr Thr Ala Ser Asp Val Trp Ser Phe Gly Ile Val Met 805 810 815 Trp Glu Val Leu Ser Phe Gly Asp Llys Pro Tyr Gly Glu Met Ser Asn 820 825 83O Gln Glu Val Met Lys Ser Ile Glu Asp Gly Tyr Arg Leu Pro Pro Pro 835 840 845 Val Asp Cys Pro Ala Pro Leu Tyr Glu Lieu Met Lys Asn. Cys Trp Ala 85 O 855 860 Tyr Asp Arg Ala Arg Arg Pro His Phe Glin Lys Lieu Glin Ala His Lieu 865 870 875 88O Glu Gln Leu Lieu Ala Asn Pro His Ser Lieu Arg Thr Ile Ala Asn. Phe 885 890 895 Asp Pro Arg Val Thr Lieu Arg Lieu Pro Ser Lieu Ser Gly Ser Asp Gly 9 OO 905 910 Ile Pro Tyr Arg Thr Val Ser Glu Trp Leu Glu Ser Ile Arg Met Lys 915 920 925 Arg Tyr Ile Leu. His Phe His Ser Ala Gly Leu Asp Thr Met Glu Cys 930 935 940 Val Leu Glu Leu Thr Ala Glu Asp Leu Thr Gln Met Gly Ile Thr Leu 945 950 955 96.O Pro Gly. His Glin Lys Arg Ile Lieu. Cys Ser Ile Glin Gly Phe Lys Asp 965 970 975
<210 SEQ ID NO 77 &2 11s LENGTH 976 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 77 Met Glu Lieu Glin Ala Ala Arg Ala Cys Phe Ala Lieu Lleu Trp Gly Cys 1 5 10 15 Ala Lieu Ala Ala Ala Ala Ala Ala Glin Gly Lys Glu Val Val Lieu Lieu 2O 25 30 Asp Phe Ala Ala Ala Gly Gly Glu Lieu Gly Trp Lieu. Thir His Pro Tyr US 2006/0039904 A1 Feb. 23, 2006 70
-continued
35 40 45 Gly Lys Gly Trp Asp Leu Met Glin Asn. Ile Met Asn Asp Met Pro Ile 50 55 60 Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp 65 70 75 8O Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile 85 90 95 Glu Lieu Lys Phe Thr Val Arg Asp Cys Asn. Ser Phe Pro Gly Gly Ala 100 105 110 Ser Ser Cys Lys Glu Thr Phe Asn Lieu. Tyr Tyr Ala Glu Ser Asp Lieu 115 120 125 Asp Tyr Gly Thr Asn. Phe Glin Lys Arg Lieu Phe Thr Lys Ile Asp Thr 130 135 1 4 0 Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His 145 15 O 155 160 Wall Lys Lieu. Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thir Arg Lys 1.65 170 175 Gly Phe Tyr Lieu Ala Phe Glin Asp Ile Gly Ala Cys Val Ala Lieu Lieu 18O 185 19 O Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Lieu Lieu Glin Gly Lieu 195 200 2O5 Ala His Phe Pro Glu Thir Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala 210 215 220 Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly 225 230 235 240 Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro 245 250 255 Ile Gly Glin Cys Lieu. Cys Glin Ala Gly Tyr Glu Lys Val Glu Asp Ala 260 265 27 O Cys Glin Ala Cys Ser Pro Gly Phe Phe Llys Phe Glu Ala Ser Glu Ser 275 280 285 Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala 29 O 295 3OO Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Glin Asp Pro 305 310 315 320 Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr 325 330 335 Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Glin 340 345 35 O Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Glin 355 360 365 Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg 370 375 38O Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser 385 390 395 400 Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn 405 410 415 Gly Val Ser Gly Leu Val Thir Ser Arg Ser Phe Arg Thr Ala Ser Val 420 425 43 O Ser Ile Asn Glin Thr Glu Pro Pro Llys Val Arg Lieu Glu Gly Arg Ser 435 4 40 4 45 US 2006/0039904 A1 Feb. 23, 2006 71
-continued
Thir Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Glin Glin Ser 450 455 460 Arg Val Trp Llys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn 465 470 475 480 Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp 485 490 495 Leu Ala Pro Asp Thr Thr Tyr Leu Val Glin Val Glin Ala Leu Thr Glin 5 OO 505 51O. Glu Gly Glin Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu Ser 515 52O 525 Pro Glu Gly Ser Gly Asn Lieu Ala Val Ile Gly Gly Val Ala Val Gly 530 535 540 Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg 545 550 555 560 Arg Arg Lys Asn Glin Arg Ala Arg Glin Ser Pro Glu Asp Val Tyr Phe 565 570 575 Ser Lys Ser Glu Glin Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His 58O 585 59 O Thr Tyr Glu Asp Pro Asn Glin Ala Val Leu Lys Phe Thr Thr Glu Ile 595 600 605 His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe 610 615 62O Gly Glu Val Tyr Lys Gly Met Leu Lys Thir Ser Ser Gly Lys Lys Glu 625 630 635 640 Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Glin 645 650 655 Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Glin Phe Ser His 660 665 67 O His Asn. Ile Ile Arg Lieu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met 675 680 685 Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Lieu. Asp Llys Phe Lieu 69 O. 695 7 OO Arg Glu Lys Asp Gly Glu Phe Ser Val Lieu Glin Lieu Val Gly Met Lieu 705 710 715 720 Arg Gly Ile Ala Ala Gly Met Lys Tyr Lieu Ala Asn Met Asn Tyr Val 725 730 735 His Arg Asp Leu Ala Ala Arg Asn. Ile Leu Val Asn. Ser Asn Lieu Val 740 745 750 Cys Lys Val Ser Asp Phe Gly Lieu Ser Arg Val Lieu Glu Asp Asp Pro 755 760 765 Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr 770 775 78O Ala Pro Glu Ala Ile Ser Tyr Arg Llys Phe Thr Ser Ala Ser Asp Val 785 790 795 8OO Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg 805 810 815 Pro Tyr Trp Glu Lieu Ser Asn His Glu Wal Met Lys Ala Ile Asn Asp 820 825 83O Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Glin 835 840 845 US 2006/0039904 A1 Feb. 23, 2006 72
-continued Leu Met Met Glin Cys Trp Glin Glin Glu Arg Ala Arg Arg Pro Llys Phe 85 O 855 860 Ala Asp Ile Val Ser Ile Leu Asp Llys Lieu. Ile Arg Ala Pro Asp Ser 865 870 875 88O Leu Lys Thr Lieu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Lieu Pro 885 890 895 Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp 9 OO 905 910 Leu Glu Ser Ile Lys Met Gln Glin Tyr Thr Glu His Phe Met Ala Ala 915 920 925 Gly Tyr Thr Ala Ile Glu Lys Val Val Glin Met Thr Asn Asp Asp Ile 930 935 940 Lys Arg Ile Gly Val Arg Lieu Pro Gly. His Glin Lys Arg Ile Ala Tyr 945 950 955 96.O Ser Lieu Lieu Gly Lieu Lys Asp Glin Val Asn Thr Val Gly Ile Pro Ile 965 970 975
<210 SEQ ID NO 78 &2 11s LENGTH 983 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 78 Met Asp Cys Gln Leu Ser Ile Leu Leu Leu Leu Ser Cys Ser Val Leu 1 5 10 15 Asp Ser Phe Gly Glu Lieu. Ile Pro Glin Pro Ser Asn. Glu Val Asn Lieu 2O 25 30 Leu Asp Ser Lys Thr Ile Glin Gly Glu Lieu Gly Trp Ile Ser Tyr Pro 35 40 45 Ser His Gly Trp Glu Glu Ile Ser Gly Val Asp Glu His Tyr Thr Pro 50 55 60 Ile Arg Thr Tyr Glin Val Cys Asn Val Met Asp His Ser Glin Asn Asn 65 70 75 8O Trp Lieu Arg Thr Asn Trp Val Pro Arg Asn. Ser Ala Glin Lys Ile Tyr 85 90 95 Val Glu Lieu Lys Phe Thr Lieu Arg Asp Cys Asn. Ser Ile Pro Leu Val 100 105 110 Leu Gly. Thr Cys Lys Glu Thir Phe Asn Leu Tyr Tyr Met Glu Ser Asp 115 120 125 Asp Asp His Gly Wall Lys Phe Arg Glu His Glin Phe Thr Lys Ile Asp 130 135 1 4 0 Thir Ile Ala Ala Asp Glu Ser Phe Thr Gln Met Asp Lieu Gly Asp Arg 145 15 O 155 160 Ile Leu Lys Lieu. Asn Thr Glu Ile Arg Glu Val Gly Pro Val Asn Lys 1.65 170 175 Lys Gly Phe Tyr Lieu Ala Phe Glin Asp Val Gly Ala Cys Val Ala Lieu 18O 185 19 O Val Ser Val Arg Val Tyr Phe Lys Lys Cys Pro Phe Thr Val Lys Asn 195 200 2O5 Leu Ala Met Phe Pro Asp Thr Val Pro Met Asp Ser Glin Ser Leu Val 210 215 220 Glu Val Arg Gly Ser Cys Wall Asn. Asn. Ser Lys Glu Glu Asp Pro Pro 225 230 235 240 US 2006/0039904 A1 Feb. 23, 2006 73
-continued
Arg Met Tyr Cys Ser Thr Glu Gly Glu Trp Leu Val Pro Ile Gly Lys 245 250 255 Cys Ser Cys Asn Ala Gly Tyr Glu Glu Arg Gly Phe Met Cys Glin Ala 260 265 27 O Cys Arg Pro Gly Phe Tyr Lys Ala Lieu. Asp Gly Asn Met Lys Cys Ala 275 280 285 Lys Cys Pro Pro His Ser Ser Thr Glin Glu Asp Gly Ser Met Asin Cys 29 O 295 3OO Arg Cys Glu Asn. Asn Tyr Phe Arg Ala Asp Lys Asp Pro Pro Ser Met 305 310 315 320 Ala Cys Thr Arg Pro Pro Ser Ser Pro Arg Asn Val Ile Ser Asn Ile 325 330 335 Asn Glu Thir Ser Val Ile Leu Asp Trp Ser Trp Pro Leu Asp Thr Gly 340 345 35 O Gly Arg Lys Asp Val Thr Phe Asn. Ile Ile Cys Lys Lys Cys Gly Trp 355 360 365 Asn. Ile Lys Glin Cys Glu Pro Cys Ser Pro Asn Val Arg Phe Lieu Pro 370 375 38O Arg Glin Phe Gly Leu Thr Asn Thr Thr Val Thr Val Thr Asp Leu Leu 385 390 395 400 Ala His Thr Asn Tyr Thr Phe Glu Ile Asp Ala Val Asn Gly Val Ser 405 410 415 Glu Leu Ser Ser Pro Pro Arg Glin Phe Ala Ala Val Ser Ile Thr Thr 420 425 43 O Asn Glin Ala Ala Pro Ser Pro Val Lieu. Thir Ile Lys Lys Asp Arg Thr 435 4 40 4 45 Ser Arg Asn Ser Ile Ser Leu Ser Trp Glin Glu Pro Glu His Pro Asn 450 455 460 Gly Ile Ile Leu Asp Tyr Glu Val Lys Tyr Tyr Glu Lys Glin Glu Glin 465 470 475 480 Glu Thir Ser Tyr Thr Ile Leu Arg Ala Arg Gly Thr Asn Val Thr Ile 485 490 495 Ser Ser Leu Lys Pro Asp Thr Ile Tyr Val Phe Glin Ile Arg Ala Arg 5 OO 505 51O. Thr Ala Ala Gly Tyr Gly. Thir Asn Ser Arg Llys Phe Glu Phe Glu Thr 515 52O 525 Ser Pro Asp Ser Phe Ser Ile Ser Gly Glu Ser Ser Glin Val Val Met 530 535 540
Ile Ala Ile Ser Ala Ala Wall Ala Ile Ile Leu Lleu. Thir Wal Wall Ile 545 550 555 560 Tyr Val Lieu. Ile Gly Arg Phe Cys Gly Tyr Lys Ser Lys His Gly Ala 565 570 575 Asp Glu Lys Arg Lieu. His Phe Gly Asn Gly His Lieu Lys Lieu Pro Gly 58O 585 59 O Leu Arg Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro Thr Glin Ala 595 600 605 Val His Glu Phe Ala Lys Glu Lieu. Asp Ala Thr Asn. Ile Ser Ile Asp 610 615 62O Lys Val Val Gly Ala Gly Glu Phe Gly Glu Val Cys Ser Gly Arg Lieu 625 630 635 640 US 2006/0039904 A1 Feb. 23, 2006 74
-continued Lys Lieu Pro Ser Lys Lys Glu Ile Ser Val Ala Ile Lys Thr Lieu Lys 645 650 655 Val Gly Tyr Thr Glu Lys Glin Arg Arg Asp Phe Leu Gly Glu Ala Ser 660 665 67 O Ile Met Gly Glin Phe Asp His Pro Asn. Ile Ile Arg Lieu Glu Gly Val 675 680 685 Val Thr Lys Ser Lys Pro Val Met Ile Val Thr Glu Tyr Met Glu Asn 69 O. 695 7 OO Gly Ser Lieu. Asp Ser Phe Leu Arg Lys His Asp Ala Glin Phe Thr Val 705 710 715 720 Ile Glin Leu Val Gly Met Leu Arg Gly Ile Ala Ser Gly Met Lys Tyr 725 730 735 Leu Ser Asp Met Gly Tyr Val His Arg Asp Leu Ala Ala Arg Asn. Ile 740 745 750 Lieu. Ile Asn. Ser Asn Lieu Val Cys Llys Val Ser Asp Phe Gly Lieu Ser 755 760 765 Arg Val Lieu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg Gly Gly 770 775 78O Lys Ile Pro Ile Arg Trp Thir Ser Pro Glu Ala Ile Ala Tyr Arg Lys 785 790 795 8OO Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Leu Trp Glu 805 810 815 Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Glu Met Ser Asn Glin Asp 820 825 83O Val Ile Lys Ala Wall Asp Glu Gly Tyr Arg Lieu Pro Pro Pro Met Asp 835 840 845 Cys Pro Ala Ala Leu Tyr Glin Leu Met Lieu. Asp Cys Trp Gln Lys Asp 85 O 855 860 Arg Asn. Asn Arg Pro Llys Phe Glu Glin Ile Val Ser Ile Leu Asp Lys 865 870 875 88O Lieu. Ile Arg Asn. Pro Gly Ser Lieu Lys Ile Ile Thr Ser Ala Ala Ala 885 890 895 Arg Pro Ser Asn Lieu Lleu Lleu. Asp Glin Ser Asn. Wall Asp Ile Thir Thr 9 OO 905 910 Phe Arg Thr Thr Gly Asp Trp Leu Asn Gly Val Trp Thr Ala His Cys 915 920 925 Lys Glu Ile Phe Thr Gly Val Glu Tyr Ser Ser Cys Asp Thr Ile Ala 930 935 940 Lys Ile Ser Thr Asp Asp Met Lys Llys Val Gly Val Thr Val Val Gly 945 950 955 96.O Pro Gln Lys Lys Ile Ile Ser Ser Ile Lys Ala Leu Glu Thr Glin Ser 965 970 975 Lys Asn Gly Pro Val Pro Val 98O
<210 SEQ ID NO 79 &2 11s LENGTH 539 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 79 Met Asp Cys Glin Leu Ser Ile Leu Lleu Lleu Lleu Ser Cys Ser Val Lieu 1 5 10 15 US 2006/0039904 A1 Feb. 23, 2006 75
-continued
Asp Ser Phe Gly Glu Lieu. Ile Pro Glin Pro Ser Asn. Glu Val Asn Lieu 2O 25 30 Leu Asp Ser Lys Thr Ile Glin Gly Glu Lieu Gly Trp Ile Ser Tyr Pro 35 40 45 Ser His Gly Trp Glu Glu Ile Ser Gly Val Asp Glu His Tyr Thr Pro 50 55 60 Ile Arg Thr Tyr Glin Val Cys Asn Val Met Asp His Ser Glin Asn Asn 65 70 75 8O Trp Lieu Arg Thr Asn Trp Val Pro Arg Asn. Ser Ala Glin Lys Ile Tyr 85 90 95 Val Glu Lieu Lys Phe Thr Lieu Arg Asp Cys Asn. Ser Ile Pro Leu Val 100 105 110 Leu Gly. Thr Cys Lys Glu Thir Phe Asn Leu Tyr Tyr Met Glu Ser Asp 115 120 125 Asp Asp His Gly Wall Lys Phe Arg Glu His Glin Phe Thr Lys Ile Asp 130 135 1 4 0 Thir Ile Ala Ala Asp Glu Ser Phe Thr Gln Met Asp Lieu Gly Asp Arg 145 15 O 155 160 Ile Leu Lys Lieu. Asn Thr Glu Ile Arg Glu Val Gly Pro Val Asn Lys 1.65 170 175 Lys Gly Phe Tyr Lieu Ala Phe Glin Asp Val Gly Ala Cys Val Ala Lieu 18O 185 19 O Val Ser Val Arg Val Tyr Phe Lys Lys Cys Pro Phe Thr Val Lys Asn 195 200 2O5 Leu Ala Met Phe Pro Asp Thr Val Pro Met Asp Ser Glin Ser Leu Val 210 215 220 Glu Val Arg Gly Ser Cys Wall Asn. Asn. Ser Lys Glu Glu Asp Pro Pro 225 230 235 240 Arg Met Tyr Cys Ser Thr Glu Gly Glu Trp Leu Val Pro Ile Gly Lys 245 250 255 Cys Ser Cys Asn Ala Gly Tyr Glu Glu Arg Gly Phe Met Cys Glin Ala 260 265 27 O Cys Arg Pro Gly Phe Tyr Lys Ala Lieu. Asp Gly Asn Met Lys Cys Ala 275 280 285 Lys Cys Pro Pro His Ser Ser Thr Glin Glu Asp Gly Ser Met Asin Cys 29 O 295 3OO Arg Cys Glu Asn. Asn Tyr Phe Arg Ala Asp Lys Asp Pro Pro Ser Met 305 310 315 320 Ala Cys Thr Arg Pro Pro Ser Ser Pro Arg Asn Val Ile Ser Asn Ile 325 330 335 Asn Glu Thir Ser Val Ile Leu Asp Trp Ser Trp Pro Leu Asp Thr Gly 340 345 35 O Gly Arg Lys Asp Val Thr Phe Asn. Ile Ile Cys Lys Lys Cys Gly Trp 355 360 365 Asn. Ile Lys Glin Cys Glu Pro Cys Ser Pro Asn Val Arg Phe Lieu Pro 370 375 38O Arg Glin Phe Gly Leu Thr Asn Thr Thr Val Thr Val Thr Asp Leu Leu 385 390 395 400 Ala His Thr Asn Tyr Thr Phe Glu Ile Asp Ala Val Asn Gly Val Ser 405 410 415 US 2006/0039904 A1 Feb. 23, 2006 76
-continued Glu Leu Ser Ser Pro Pro Arg Glin Phe Ala Ala Val Ser Ile Thr Thr 420 425 43 O Asn Glin Ala Ala Pro Ser Pro Val Lieu. Thir Ile Lys Lys Asp Arg Thr 435 4 40 4 45 Ser Arg Asn Ser Ile Ser Leu Ser Trp Glin Glu Pro Glu His Pro Asn 450 455 460 Gly Ile Ile Leu Asp Tyr Glu Val Lys Tyr Tyr Glu Lys Glin Glu Glin 465 470 475 480 Glu Thir Ser Tyr Thr Ile Leu Arg Ala Arg Gly Thr Asn Val Thr Ile 485 490 495 Ser Ser Leu Lys Pro Asp Thr Ile Tyr Val Phe Glin Ile Arg Ala Arg 5 OO 505 51O. Thr Ala Ala Gly Tyr Gly. Thir Asn Ser Arg Llys Phe Glu Phe Glu Thr 515 52O 525 Ser Pro Asp Cys Met Tyr Tyr Phe Asn Ala Val 530 535
<210 SEQ ID NO 80 &2 11s LENGTH 986 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 80 Met Ala Gly Ile Phe Tyr Phe Ala Leu Phe Ser Cys Leu Phe Gly Ile 1 5 10 15 Cys Asp Ala Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr 2O 25 30 Leu Lieu. Asp Ser Arg Ser Val Glin Gly Glu Lieu Gly Trp Ile Ala Ser 35 40 45 Pro Leu Glu Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn 50 55 60 Thr Pro Ile Arg Thr Tyr Glin Val Cys Asn Val Met Glu Pro Ser Glin 65 70 75 8O Asn Asn Trp Lieu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Glin Arg 85 90 95 Val Tyr Ile Glu Ile Lys Phe Thr Lieu Arg Asp Cys Asn. Ser Lieu Pro 100 105 110 Gly Val Met Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu 115 120 125 Ser Asp Asn Asp Lys Glu Arg Phe Ile Arg Glu Asn Glin Phe Wall Lys 130 135 1 4 0 Ile Asp Thir Ile Ala Ala Asp Glu Ser Phe Thr Glin Val Asp Ile Gly 145 15 O 155 160 Asp Arg Ile Met Lys Lieu. Asn. Thr Glu Ile Arg Asp Val Gly Pro Leu 1.65 170 175 Ser Lys Lys Gly Phe Tyr Lieu Ala Phe Glin Asp Val Gly Ala Cys Ile 18O 185 19 O Ala Leu Val Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val 195 200 2O5 Arg Asn Lieu Ala Glin Phe Pro Asp Thir Ile Thr Gly Ala Asp Thir Ser 210 215 220 Ser Lieu Val Glu Val Arg Gly Ser Cys Val Asn. Asn. Ser Glu Glu Lys 225 230 235 240 US 2006/0039904 A1 Feb. 23, 2006 77
-continued
Asp Val Pro Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Lieu Val Pro 245 250 255 Ile Gly Asn. Cys Lieu. Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu 260 265 27 O Cys Glin Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala 275 280 285 Thr Cys Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala 29 O 295 3OO Thir Ser Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala 305 310 315 320 Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile 325 330 335 Ser Asn Val Asin Glu Thir Ser Val Asn Leu Glu Trp Ser Ser Pro Glin 340 345 35 O Asn Thr Gly Gly Arg Glin Asp Ile Ser Tyr Asn Val Val Cys Lys Lys 355 360 365 Cys Gly Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val 370 375 38O His Tyr Thr Pro Gln Glin Asn Gly Leu Lys Thr Thr Lys Val Ser Ile 385 390 395 400 Thr Asp Leu Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val 405 410 415 Asn Gly Val Ser Lys Tyr Asn Pro Asn Pro Asp Gln Ser Val Ser Val 420 425 43 O
Thr Wall Thir Thr Asn Glin Ala Ala Pro Ser Ser Ile Ala Leu Wall Glin 435 4 40 4 45 Ala Lys Glu Val Thr Arg Tyr Ser Val Ala Lieu Ala Trp Leu Glu Pro 450 455 460 Asp Arg Pro Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu 465 470 475 480 Lys Asp Glin Asn. Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg 485 490 495 Asn Thr Asp Ile Lys Gly Leu Asn Pro Leu Thir Ser Tyr Val Phe His 5 OO 505 51O. Val Arg Ala Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu 515 52O 525 Glu Val Thr Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala 530 535 540 Asn Ser Thr Val Leu Leu Val Ser Val Ser Gly Ser Val Val Leu Val 545 550 555 560 Val Ile Lieu. Ile Ala Ala Phe Val Ile Ser Arg Arg Arg Ser Lys Tyr 565 570 575 Ser Lys Ala Lys Glin Glu Ala Asp Glu Glu Lys His Lieu. Asn. Glin Gly 58O 585 59 O Val Arg Thr Tyr Val Asp Pro Phe Thr Tyr Glu Asp Pro Asn Glin Ala 595 600 605 Val Arg Glu Phe Ala Lys Glu Ile Asp Ala Ser Cys Ile Lys Ile Glu 610 615 62O Lys Val Ile Gly Val Gly Glu Phe Gly Glu Val Cys Ser Gly Arg Lieu 625 630 635 640