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. GlinCAAATGAACAGCCTG.A.A.A.A. 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 ..