THI MONGOLUS009739772B2MULTUMITUR (12 ) United States Patent ( 10 ) Patent No. : US 9 , 739 ,772 B2 Ryu et al. ( 45 ) Date of Patent: Aug. 22, 2017 ( 54 ) METHOD OF ANALYZING BINDING (51 ) Int. CI. ASPECT OF MEMBRANE PROTEIN IN A GOIN 33 /554 ( 2006 . 01 ) LIVING CELL GOIN 33 /557 (2006 .01 ) (71 ) Applicant: POSTECH ACADEMY- INDUSTRY (52 ) U . S . CI. FOUNDATION , Gyeongsangbuk - do CPC . .. GOIN 33/ 557 ( 2013 .01 ) ; GOIN 33/ 554 (2013 .01 ) (KR ) (58 ) Field of Classification Search ( 72 ) Inventors : Sung Ho Ryu , Gyeongsangbuk -do None (KR ) ; Dohyeon Kim , Seoul ( KR ) ; Nam See application file for complete search history. Ki Lee , Gyeongsangbuk -do (KR ) ; Dong Kyun Kim , Gyeongsangbuk - do (KR ) ; Soyeon Park , Seoul (KR ) ; Yonghoon ( 56 ) References Cited Kwon , Seoul (KR ) ; Kai Zhou , Gyeongsangbuk - do (KR ) PUBLICATIONS ( 73 ) Assignee : Postech Academy - Industry Stewart et al. Biochem J 1991 vol. 275 , p . 569 - 573. * Foundation , Gyeongsangbuk -Do (KR ) Daumas et al. Biophysical J . 2003 vol. 84 , p . 356 - 366 . * ( * ) Notice : Subject to any disclaimer , the term of this Jin et al . Biophysical J . 2007 vol. 93 , p . 1079 - 1088 . * patent is extended or adjusted under 35 Saxton Biophysical J . 1997 vol. 72, p . 1744 - 1753. * U . S . C . 154 (b ) by 0 days . * cited by examiner (21 ) Appl . No. : 14 / 891 , 555 Primary Examiner — Jacob Cheu (22 ) PCT Filed : May 16 , 2014 (74 ) Attorney , Agent, or Firm — Lathrop & Gage LLP ( 86 ) PCT No . : PCT /KR2014 / 004426 (57 ) ABSTRACT $ 371 ( C ) ( 1 ) , ( 2 ) Date : Nov. 16 , 2015 The present invention relates to a method for analyzing the pattern of live intercellular membrane protein binding . The ( 87) PCT Pub . No. : WO2014 /185752 method for analyzing the pattern according to the present invention can analyze accurately , sensitively, quickly , and PCT Pub . Date : Nov . 20 , 2014 readily the binding pattern of a targetmembrane protein and a candidate substance to be specifically bound therewith (65 ) Prior Publication Data without tagging to a ligand , and thus measure directly and US 2016 /0116468 A1 Apr. 28 , 2016 accurately the position and quantitative information of the Related U . S . Application Data binding of the membrane protein and the target substance . Such effects make it possible to apply the method for various (63 ) Continuation of application No . uses such as dissociation constant, mutant study, complex PCT/ KR2013 /011002 , filed on Nov. 29 , 2013 . formation , and signal transduction . Moreover , it is expected to use the method for searching out undiscovered membrane (30 ) Foreign Application Priority Data proteins and target substances . May 16 , 2013 (KR ) .. .. .. .. .. .. .. .. 10 - 2013 -0056008 May 16 , 2014 (KR ) . .. .. .. 10 - 2014 -0059027 19 Claims, 19 Drawing Sheets U . S . Patent Aug . 22, 2017 Sheet 1 of 19 US 9 ,739 , 772 B2 ( FIGM . 1 ] extracellular binding intracellular extracellular rotation total INTERNAL reflection licht intracellular92 . 5 lipid bilayer lioand O membrane protein a fluorescent protein [ FIG . 2 ] cell growth time (cycle ) medium rrrrrrrrr viw imw* voisnuocoefficient 1 2 3 4 coefficientinsinglecell sonrad Condition 1 time ( cycle Multiple temporal uojanjoubur? particle average average mahu wwwwwwwwwwwwwww tracking * ** ** ** * * * ** * ** * * * * ** ** ** * * ** wwwwwwwwwwwwwwwww * * * 1 * total INTEANAL * reflection microscope * diffusion conditioncondition1 24 objective lens coefficient 1 2 3 4 condition 2 time (cycle ) U . S . Patent Aug . 22, 2017 Sheet 2 of 19 US 9 ,739 , 772 B2 [FIG . 3 ] NT Cetuximab WWWWWWWWWWW 4, 300 wwwwwwwwwwwwwwwwwwwwww . * . TV yuniminiminiminnainiminnasPixel( unit) . .. * * . PM Vedete 400 400 14 mwi i 100 iiiiiiiiiii 93 w 400 * * * * * * * * 400 100 400 100 U . S . Patent Aug . 22, 2017 Sheet 3 of 19 US 9 , 739, 772 B2 [FIG . 4 ] PMT - NT PMT- cetuximab . Y W w * -. 4-46 4.47 * . * jasovedecesorios /.% minimi . eritin .conoscino * * ?:1-i; sensori . * . wwwAwwww * series . wwwS wwwwwwwwww diffusioncoefficient .. verter wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww ( cycle ) 2 4 8 10 2 6 8 WWW 10 .4 EGFR - cetuximab * . -* . .'* . * > .concom ??????? * . diffusioncoefficiento .*5 * e * .???????? . .- * www .- * ?????????????????????????????????????????????????????????????????????? . www wird * o i W cetuximab changeindiffusioncoefficient(%) o PMT EGFR U . S . Patent Aug . 22, 2017 Sheet 4 of 19 US 9 ,739 , 772 B2 (FIG . 6 ) 82 - AR G o heterotrimeric Gi protein berperan seperti ADD ribosylated Gi protein pertussis toxin (DTX ) FZD1 per le per [FIG . 7 ] TX changeindiffusioncoefficient(%) marianna mmmmmmm wwwwwwwwwwww wwwwwww om PMT FZD1 B2- AR U . S . Patent Aug . 22, 2017 Sheet 5 of 19 US 9 ,739 , 772 B2 [FIG . 8 ] diffusioncoefficient(um/s) PMT B2- AR EGFR ( FIG . 9 ] changeindiffusioncoefficient(%) e V o batch1 batch2 batch3m1 batch3 - 2 different batches measured on the same day U . S . Patent Aug . 22, 2017 Sheet 6 of 19 US 9 , 739, 772 B2 [FIG . 10 ) WEK293 cell Hela cel .* * changeindiffusioncoefficient(%) changeindiffusioncoefficient(%) * * i nnan AMT EGFR PMT EGFR C2- cc PMT MEGFR & changeindiffusioncoefficient(%) wwwwwwwwwwwwwwwwwwwwwwwwwwwwwww changeindiffusioncoefficient(%) % wwwwwwwwwwwwwwwww $ @ PMT EGFR HEK293 Hela CHO -K1 cell cell cell (FIG . 11Lind] Cetuximab mAb 199 . 12 mab 528 MAL R U . S . Patent Aug. 22 , 2017 Sheet 7 of 19 US 9 , 739, 772 B2 (FIG . 121 retuxlimAb 199 mAb.12 528 MAD R- 1 NT?????? cetuximab?????? W EGF - Gol - Gele - - olin - often PEGFR ( Y1086 ) EGFR Donau miniintindihittiintindihihiiiiiiiii wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww PErk1/ 2 wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwand ?????????. ?? . ?????????? . ?? . ?? . ?????????? . ?? . ???????????? . ?? . ?????????? . ???????? . ?? . ?????????????? . ???????????????????????? . ?? . ?????????? . ?? . ?? . actin wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww wwwww w wwww herenor * PMT 8 WEGFR * * * * changeindiffusioncoefficient(%) @ o cetuximabapp mAb 199 .12 mAb 528 mAbR- 1 U . S . Patent Aug . 22, 2017 Sheet 8 of 19 US 9 , 739, 772 B2 [ FIG . 141 w fluorescent protein EGFR et ErbB2 trastuzumab Cetuximab ( FIG . 151 1 .01 cetuximab *. trastuzumab actin anibody relativeratio(%) immunoglobulin G antibody fluorescence intensity ( arbitrary unit ) U . S . Patent Aug . 22, 2017 Sheet 9 of 19 US 9 ,739 , 772 B2 [FIG . 16 ] PMT 350 3 - EGFRPEST changeindiffusioncoefficient(%) Cetuximab trastuzumab actin antibody (FIG . 17 ] cetuximab Cetuximab Fab2 cetuximab Fab aptamer U . S . Patent Aug . 22, 2017 Sheet 10 of 19 US 9 , 739, 772 B2 [FIG . 18 ] PMT EGFR is * * * * changeindiffusioncoefficient(%) WWWWWWWWWWWWUNNNNNNNNNNN abetöwwwwwwwwwwwwwww when aptamer cetuximab cetuximab Cetuximab [FIG . 19 ] www changeindiffusioncoefficient(%)W Win * * * * * * * w 40 80 120 160 molecular weight (kilodalton ) U . S . Patent Aug . 22, 2017 Sheet 11 of 19 US 9 , 739, 772 B2 [FIG . 20 ] EGFREGFR Dom Icetuximab U . S . Patent Aug . 22, 2017 Sheet 12 of 19 US 9 , 739, 772 B2 [FIG . 21 ] ated cetuximab concentration 1. 0 - - 200. 04. 04 20enthe .06 normalizeddiffusioncoefficient 0 .32 LV wer 0. 64 first measurement 1. 28 second measurement B eatment time point 0 10 20 30 40 time (minute ) U . S . Patent Aug . 22, 2017 Sheet 13 of 19 US 9 , 739, 772 B2 [ FIG . 22] dissociation constant K 0 .62 0 . 19 OM 1 . 0 * wwwwwwwww vojendod693punoq.bewixmajoone qowixnajoojai 01093pun0Q cetuximabunboundEGFR etetter wievi 0 .wwwwwwwwwwwwww 0 0 . 4 0 . 8 wwwww12 ratio of cetuximab - bound EGFA wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww 0 .0 0 . 2 0 . 4 0 .6 0 . 8 1 .0 1. 2 1 . 4 cetuximab (ugimi ) (FIG . 23 ] GFRVIII (mutant ) (EGFRwild type WT ) cetuximab women mAb R - 1 recent U . S . Patent Aug . 22, 2017 Sheet 14 of 19 US 9 , 739, 772 B2 4 ] a cetuximab mAb R - 1 )coefficient(%diffusionchangein 2 o PMT EGFRVIIIwwww [FIG . 25] WA dissociation constant Ko 0 . 36 0 .01 OM 1 . 0 1980 Norway ratioofcetuximab-boundEGFRvIII ratioofCetuximab 18:93punodEGFAVIIIunboundcetuximab 80of . 2 0 . 4 0 .6 0 . 8 1 .0 ratio of cetuximab - BOUND EGFRVII 00 02 04* * * * * * * * * *06 * 08 10* * * * * * * * * * * * * 12* 14 cetuximab (wg /ml ) U . S . Patent Aug . 22, 2017 Sheet 15 of 19 US 9 , 739, 772 B2 (FIG . 26 ] EGER rror 80 -833 - 03 ? } { x } a secondary antibody tertiary antibody (FIG . 27] 5 1. 00 I treatment point www Cetuximab Secondary antibody tertiary normalizeddiffusioncoefficient antibody * * * * ** * * * * * 10 time (minute i ) U . S . Patent Aug . 22, 2017 Sheet 16 of 19 US 9 , 739, 772 B2 [FIG . 28 ) 1. 00 ????????? $ treating point Lappi Cetuximab tertiary antibody y secondary normalizeddiffusioncoefficient antibody wer 0 10 20 time (minute ) [FIG . 29 ) endogenous mese EGFA Fab of EGFA antbody Alexa Fluor 647 14:00 U . S . Patent Aug. 22 , 2017 Sheet 17 of 19 US 9 , 739 ,772 B2 ( FIG . 30 ] fluorescence labeling to mAb done 199 . 12 ( lugim ) www labeling to cetuximab ( Iugem !) fluorescence lebeiing to mab cione 199 . 12 * labeling to cetuximab * cetuximab ( 100ugimi) * cetuximab [100ugimi 10 0 . 81 relativeratio ryyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy relativeratio 0.90 101 102 fluorescence intensity fluorescence intensity (arbitrary unit ) (arbitrary unit ) (FIG . 31] en changeindiffusioncoefficient(%) ene enon !ulingoj?ounww antibody cetuximab U . S . Patent Aug . 22, 2017 Sheet 18 of 19 US 9 , 739, 772 B2 [FIG . 321 immunoglobulin antibody * * Cetuximab (%ijuopuleoouosniDubuaya