US010316092B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 ,316 ,092 B2 Yao et al. ( 45 ) Date of Patent : Jun . 11 , 2019 ( 54 ) ANTI- B7- H5 ANTIBODIES AND THEIR USES ( 56 ) References Cited (71 ) Applicants :MEDIMMUNE , LLC , Gaithersburg , U . S . PATENT DOCUMENTS MD (US ) ; THE JOHNS HOPKINS 2010/ 0041074 A1 * 2 /2010 Kimura . .. .. A61K 47/ 48415 UNIVERSITY , Baltimore , MD (US ) 435 / 7 .23 2017 /0306024 Al * 10/ 2017 Noelle .. .. .. C07K 16 / 2827 (72 ) Inventors : Sheng Yao , Columbia , MD (US ) ; Lieping Chen , Hampden , CT (US ) ; FOREIGN PATENT DOCUMENTS Linda Liu , Columbia , MD (US ) ; Solomon Langermann , Baltimore , MD WO 2011020024 A2 2 / 2011 (US ) OTHER PUBLICATIONS (73 ) Assignees : THE JOHN HOPKINS Ni et al. (2017 ) Mol Cancer Ther; 16 ( 7 ) : 1203 - 1211 . * UNIVERSITY , Baltimore , MD (US ) ; Zhao et al . Proc Natl Acad SciUS A . Jun . 11, 2013 ; 110 ( 24 ) :9879 MEDIMMUNE , LLC , Gaitherburgh , 9884 . * Zhu et al . Nature Communications vol . 4 , Article No . 2043 ( 2013 ) ; MD (US ) 12 pages. * Wang , L . et al. , Vista , a novel mouse Ig superfamily ligand that ( * ) Notice : Subject to any disclaimer, the term of this negatively regulates T cell responses , Nature Reviews Immunology , patent is extended or adjusted under 35 vol. 8 , No . 6 , Mar. 14 , 2011 , pp . 467 -492 . U . S . C . 154 (b ) by 454 days . Flies, D . B . et al . , " Cutting Edge : A Monoclonal Antibody Specific for the Programmed Death - 1 Homolog Prevents Graft- versus- Host Disease in Mouse Models ,” The Journal of Immunology , vol . 187 , (21 ) Appl . No . : 14 /893 , 463 No . 4 , Jul. 18 , 2011 , pp . 1537 - 1541. Moustafa , S . et al. , “ GI24 enhances tumor invasiveness by regulat ( 22 ) PCT Filed : May 27 , 2014 ing cell surface membrane - type 1 matrix metalloproteinase , ” Can cer Science , vol . 101, No . 11 , Jul. 13 , 2010 , pp . 2368 - 2374 ( 86 ) PCT No. : PCT/ US2014 / 039621 Linsley, P . et al. , “ The clinical utility of inhibiting CD28 -mediated costimulation ,” May 2009 , Immunological Reviews, vol. 229 , pp . $ 371 ( C ) ( 1 ), 307 - 321 . ( 2 ) Date : Nov . 23 , 2015 Flajnik et al. , “ Evolution of the B7 family : co - evolution of B7H6 and NKP30 , identification of a new B7 family member, B7H7, and of B7 ' s historical relationship with the MHC ” , Immunogenetics , (87 ) PCT Pub . No. : WO2014 /190356 Springer , Berlin , DE , vol. 64 , No . 8 , Apr. 11 , 2012 , pp . 571 -590 . PCT Pub . Date : Nov . 27 , 2014 Printed website entry for HHLA2 Gene ; from GeneCards Human Gene Database; Weizmann Institute of Science , LifeMap Sciences , printed from < http :/ /www .genecards .org /cgi -bin / carddisp . pl ? gener (65 ) Prior Publication Data HHLA2 & keywords = B7 - H5 > ; 16 pages . US 2016 /0096891 A1 Apr . 7 , 2016 English translation of second examination report in corresponding Japanese Application No . 2016 -515141 , dated Dec . 4 , 2018 , ( 5 pages) . Related U . S . Application Data * cited by examiner (60 ) Provisional application No .61 /827 ,216 , filed on May Primary Examiner — Ilia I Ouspenski 24 , 2013 . (74 ) Attorney , Agent, or Firm — Melissa Hunter- Ensor ; ( 51 ) Int . Ci. Leslie Serunian ; Greenberg Traurig , LLP COZK 16 / 28 (2006 . 01) (57 ) ABSTRACT A61K 39 / 395 ( 2006 . 01 ) The present invention relates to antibodies and their antigen A61K 39 /00 ( 2006 .01 ) binding fragments and to other molecules that are capable of (52 ) U . S . CI. immunospecifically binding to the B7 -H5 ligand of the CPC .. CO7K 16 / 2827 ( 2013 .01 ) ; A6IK 2039 /505 B7- H5 :CD28H pathway , and to the uses of such molecules (2013 .01 ) ; CO7K 2317 / 24 ( 2013 . 01 ) ; COOK in the treatment and diagnosis of autoimmune disease , 2317/ 565 ( 2013 .01 ) ; CO7K 2317 / 76 (2013 .01 ) transplant rejection and other inflammatory diseases . (58 ) Field of Classification Search None 12 Claims, 20 Drawing Sheets See application file for complete search history. Specification includes a Sequence Listing . atent Jun . 11 , 2019 Sheet 1 of 20 US 10, 316 ,092 B2 V DC , Macrophage W O VI T cell , NK cell , poC Constitutive in Constitutively expressed macrophages Down -regulated upon Inducible in DCs activation FIGppose & . 1 U . S . Patent Jun . 11, 2019 Sheet 2 of 20 US 10, 316 ,092 B2 HKLM OLZOLLOLQOL orm f TOLGOLZOLLOLOOKFOLZOLLOLOOL PolyI:C ILMULILLMLLIM ZOLLOLO . NEODN02 FOOL FIG.2 IFN-Y 100101102103104 WLLLWWLLLWW Ils . -ZH28!UV 401 201 LPS WHLLLLLLL BOLGOLZOLLOLLOLOAPOLGOLZOL.LOL00! YFOR FOOL 201 100101102103104100101102103104 ?uou . www VITYwerk . .. wwwwwwwwwwwwwwwwww Macrophage Cell Dendrictic U . S . Patent Jun . 11, 2019 Sheet 3 of 20 US 10 ,316 ,092 B2 0 . 4 umbullhoudendbubble 48 . 1 FL4-H:H6R t FL4-H:BiotinantiH6R/SAAPC * * * USASUKESKUS Lambodhlehelewding ?????????????????????????? 1716 1001175 . L TIMITTITUT13. 1 100 101 102 103 104 100 101mitmenin 102 103 22 . 1049 CD19 FL1- H : CD19 CD3 FL1- H : CD3 H7CR- FIG . 3A FIG . 3B 4 78 52. 1 9 . 04 ? FL4-H:BiotinantiH6R/SAAPC FL4-H:HOR 1011mendebaldellomlapotballLehmmmhund 9 h ?????? 100 36. 8NOTITI . 100 101 102 103 104 100 101 102 103 104 CD56 FL2- H :CD56 CD16 FL1- H : CD16 FIG . 3C FIG . 3D atent Jun . 11 , 2019 Sheet 4 of 20 US 10 ,316 ,092 B2 Count O + TTTTTTTTTTT 0 102 103 Tu 104 105 Comp- APC - A : a -mig Antibody 2D3 FIG . 4A Count O TO2 103 104 1165 Comp- APC - A : a -mig Antibody 1803 FIG . 4B U . S . Patent Jun . 11, 2019 Sheet 5 of 20 US 10 ,316 ,092 B2 .. Count Count . 04 o m niet om Trom 0 102 103 104 105 0 102 103 104 105 Comp -PE - A Comp - PE - A Antibody 2D3 Antibody 1803 FIG . 5A FIG . 5B Count 0 - cm Trimmisi 102 103 104 105 Comp - PE - A Control lg FIG . 5C atent Jun . 11 , 2019 Sheet 6 of 20 US 10, 316 ,092 B2 nebene Ctig in B7 - H71g Anti- human CD3 (ug /ml ) FIG . 6 IL - 2 IFN « y IL - 10 2002 um M Cti ig B7 -H7 lg Ctlig B7« H7 Wlg Ctl lg 37- H7 lg FIG . 7 atent Jun . 11 , 2019 Sheet 7 of 20 US 10, 316 ,092 B2 15 , ** * * ** * * CPMX104 : w : 1 . 1. iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii 1. 11111 FIG . 8 G + ???????????????? + iiiii W + IFN(pg/ml) + + + 20000 + iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii! +:. control 203 control 203 FIG . 9 atent Jun . 11 , 2019 Sheet 8 of 20 US 10 ,316 ,092 B2 Spleen %ofCD45RO+TCells wwwwww CD3 CD4 CD & Figure 10A PBL %ofCD45RO+TCells ??? CD4 ??? Figure 10B OPBS Anti- B7- H7 Antibody atent Jun . 11 , 2019 Sheet 9 of 20 US 10, 316 ,092 B2 Spleen %ofCD45RO-TCells CD3 CD4 CD8 Figure 11A PBL www %ofCD45RO-TCells minn CD3 CD4 CD Figure 11B O PBS Anti -B7 - H7 Antibody U . S . Patent Jun . 11 , 2019 Sheet 10 of 20 US 10 ,316 ,092 B2 Count Purified Anti -Human B7 -H7 Antibody 2D3 Oooppipomognethyggelige O 102 103 104 105 APC - A : : 2D3 + a -mlg FIG . 12A Count Recombinant Anti- Human B7 -H7 Chimeric Antibody O - run raportohet 1 Freiburg 2D3 (hlgG4 ) 102 103 104 105 PE - A :: 2D3 sup + a -hlg FIG . 12B Count Recombinant Anti- Human B7 -H7 Chimeric Antibody 0 - tama apa rati 18C3 (hlgG4 ) PE0 -102 A :: 18C3103 sup104 + a - hlg105 FIG . 120 atent Jun . 11 , 2019 Sheet 11 of 20 US 10 ,316 ,092 B2 Counts Counts illHwwwww617701411Nhaniwimm shew 0 102 103 104 105 0 102 103 104 105 PE - A :: Control+ H7CR -Biotin + SA PE- A : 2D3+ H7CR -Biotin + SA Panel A Panel B Control Antibody 2D3 . I+* # ten SA -PE * * * H7CR -Biotin + SA -PE Counts *#IES .. Anti -B7 -H7 + H7CR - Biotin + SA - PE # * * 0 104 104 104 105 PE- A :: 18C3 + H7CR -Biotin + SA Panel C Antibody 18C3 FIG . 13 atent Jun . 11 , 2019 Sheet 12 of 20 US 10 ,316 ,092 B2 B7H7 IgV only Absorbance450 0 . 00 2 $ 32 mAb concentration (ng /mL ) Antibody 2D3 FIG . 14A B7H7 IgV only 0 . 121 0 . 10 Absorbance450 0 .08 0 . 06 0 .04 0 2 § 32 mAb concentration (ng /mL ) Antibody 18C3 FIG . 14B CHO. H7CR . %ofMax . w w 0 1000 10000 1 x 105 B7 -H7 ( IgV only )- IgG + anti- lg PE FIG . 14C U . S . Patent Jun . 11 , 2019 Sheet 13 of 20 US 10 ,316 ,092 B2 CHO .HB7 -H7 FL © 203 2D3MFI 18C3MFI ST www ity 0.001 0 .01 1001000 * (Ab ) (nM ) 008 11111FIG . 15 U . S . Patent Jun . 11 , 2019 Sheet 14 of 20 US 10 ,316 ,092 B2 % 13 . 4 % FL1-H:BrdU% Panel A : Control % % 86 . 6 100 101 102 103 104 BrdU FL4 - H : CD3 CD3 — > FL1-H:BrdU Panel B : Antibody 2D3 .: : . 10010 100 101 102 103 104 BrdU FL4 -H : CD3 CD3 - > FIG . 16A atent Jun . 11 , 2019 Sheet 15 of 20 US 10 ,316 ,092 B2 Panel A : Cytokine juubd Levels IFN -Y L -5 1 - 10 V . 02 BrdU-positivecells(x103) Panel B : BrdU - Positive * * * * * Control FIG . 16B atent Jun . 11 , 2019 Sheet 16 of 20 US 10 ,316 ,092 B2 2D3Control 203 10 W ) % ( cells T + CD8 + 67- Ki ) %( cells T + CD4 + 67- Ki 12.3 57.8 . 1111111 '. 2D3 FL4-H:CD3 FL4-H:CD3 EL 101102 Whytyytytytetyn FIG.17A-B . .. .. .. 10VHompanie100 wewe 100 1040 101 1040 10010 67- Ki : H - FL2 10010104 67-Ki : H -FL2 6.25 22.17 77.9 TIT 1001011021031041 www FL4-H:CD3 FL4-H:CD3 Control wwwwwwwww Tommy 100101 "CD3 CD3 A10050 010 67-Ki : H - FL2 67-Ki 67- Ki : H- FL2 ' b- K CD8+ CD4+ U . S . Patent Jun . 11 , 2019 Sheet 17 of 20 US 10 ,316 ,092 B2 w KD 0 5 10 30 60 5 10 30 60 5 10 30 60 wp - AKT WWW * * * * * * * * FIG . 18 atent Jun . 11 , 2019 Sheet 18 of 20 US 10 , 316 ,092 B2 CTL: 624Mel000000 - B7 -17 624Mel- B7 + - 624Mel- B7 H7 + H7CRIN 0 0. 11 0 .3 : 1 1: 1 3: 1 10 :1 E : T ratio FIG .