INDUS 20190328899A1 IN (19 ) United States ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2019/0328899 A1 Hechler et al. (43 ) Pub . Date : Oct. 31 , 2019 (54 ) AMANITIN ANTIBODY CONJUGATES Publication Classification (51 ) Int. Ci. ( 71) Applicant : Heidelberg Pharma Research GmbH , A61K 47/68 (2006.01 ) Ladenburg (DE ) A61P 35/00 (2006.01 ) CO7K 14/37 (2006.01 ) ( 72 ) Inventors: Torsten Hechler, Lautertal (DE ) ; COOK 16/28 (2006.01 ) Michael Kulke , Ludwigshafen am (52 ) U.S. CI. Rhein (DE ) ; Christian Lutz , Weinheim CPC A61K 47/6831 ( 2017.08 ) ; A61K 47/6867 (DE ) ; Andreas Pahl, Heidelberg (DE ) ; ( 2017.08 ) ; CO7K 16/2878 ( 2013.01 ) ; A61P Christoph Müller , Birkenau (DE ) ; 35/00 ( 2018.01 ) ; CO7K 14/37 (2013.01 ) ; A61K Werner Simon , Hüffelsheim (DE ) ; Anikó Pálfi , Hirschberg a.d. Bergstra (3e 47/6889 ( 2017.08 ) (DE ) (57 ) ABSTRACT (21 ) Appl . No.: The invention relates to a conjugate comprising ( a ) an 16 /470,950 amatoxin comprising ( i) an amino acid 4 with a 6 '- deoxy (22 ) PCT Filed : Dec. 22 , 2017 position ; and ( ii ) an amino acid 8 with an S -deoxy position ; ( b ) a BCMA- binding moiety comprising ( i) the variable ( 86 ) PCT No.: PCT /EP2017 / 084431 domains ofhumanized antibody J22.9 - ISY , and ( ii ) a heavy chain constant region comprising a D265C mutation ; and ( c ) $ 371 ( c ) ( 1 ) , a protease - cleavable linker linking said amatoxin and said ( 2 ) Date : Jun . 18 , 2019 target -binding moiety . The invention furthermore relates to a pharmaceutical composition comprising such conjugate , (30 ) Foreign Application Priority Data particularly for use in the treatment of multiple myeloma. Dec. 23 , 2016 ( EP ) 16206849.8 Specification includes a Sequence Listing . R SCH?1 H?CBTR HN - CH - CO 3CH -CO -A - CH -co -wc 5 ' - co 4 CO 3 ' PRANH CH , CH 2 7 ' HC HO 6 -CH3 CH2 ?? 0 CO - CH -NH OL H A CO - CH RY 8 7 R1 R2 R3 R4 a - amanitin OH OH NH2 OH B - amanitin OH OH OH OH Y - amanitin H OH NH2 OH E - amanitin H OH OH OHOH amanin OH OH OH H amaninamide OH OH NH2 I amanullin H H NH2 OH amanullinic acid H H ?? OH Y -amanin H OH OH H Y - amaninamide I OH NH2 I Patent Application Publication Oct. 31 ,2 2019 Sheet 1 of 32 US 2019/0328899 A1 FIGURE 1 R — 1 CH2 ?.?. ? Y R , 4 ? 5 HN - CH - CO - N - CH - CO - N CHz CO 3 H 4 * 5 6 ' NH 3 " R , CHE ?? 2 HC ?? (? 2 N 6 -N CH , 1 ?? , ?? : CO - CH - N - CO - CH - NH ZI 8 H 7 R , ?Y. B R1 R , R3 R , a - amanitin ?? ?? NH2 ?? B - amanitin ?? ?? ?? ?? y - amanitin ? ?? NH2 ?? E - amanitin ? ?? ?? ?? amanin ?? ?? ?? ?. amaninamide ?? ?? NH2 ? amanullin ? ? NH2 ?? amanullinic acid ? ? ?? ?? y - amanin ? ?? ?? ?. y -amaninamide H ?? NH2 H Patent Application Publication Oct. 31 ,2 2019 Sheet 2 of 32 US 2019/0328899 Al FIGURE 2 PBS, pH 7.4 PBS , pH 7.4 + cysteine heavy chain light chain day 0 day 5 day 0 day 5 Patent Application Publication Oct. 31 , 2019 Sheet 3 of 32 US 2019/0328899 A1 17,0 16,8 CH3 CH2-CH3 16,6 NH HC- CO -NH ym 16,216,4 HO CH2 -NH-CH2 -CO Apment ............ CO NH -NH 15,215,415,615,816,0 Amanitin+Cyst=6dalpha- CH2 - -CH-NH ?.?. O=5 wynamagrantumengumumnyamenganggapngmgatunog DAD-CH1305nm -NH-COCH -CH2OH CO 15,0 -CH 14,8 CH CH2 HN-CH OC-----CH NH2 Amanitin48h+Cyst=alpha- ?3?. CH Minutes DAD-CH1305nm H OH 13111111111 Amanitin+Cyst=24halpha- 12,412,612,813,013,213,413,613,814,014,214,414,6 DAD-CH1305nm mummymwanyamawangumwenyemamynaperymenmampumenemaninyamengenangage alpha-Amanitin+Cyst=0 DAD-CH1305nm 12,012,2 FIGURE3 0 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 -100 MAU Patent Application Publication Oct. 31 , 2019 Sheet 4 of 32 US 2019/0328899 Al CH2-CH3 CH3 16,216,416,616,817,0 NH HCHC CO NH" OH -CO-CH2 -CO-NHCH2 16,0 NH -NH CH2 15,215,415,615,8 -NH-CH H2C -CH beta-Amanitin+Cyst=6d CO DAD-CH1305nm CH2OH CO NH HO CH CH CH CH CH2 OH H?C. HN CH beta-Amanitin+Cyst=48h Minutes DAD-CH1305nm HO beta-Amanitin+Cyst=24h 12,012,212,412,612,813,013,213,413,613,814,014,214,414,614,815,0 DAD-CH1305nm betaAmanitin-+Cyst=0 DAD-CH1305nm FIGURE4 0 1200 1100 1000 900 800 700 600 500 400 300 200 100 -100 MAU Patent Application Publication Oct. 31 , 2019 Sheet 5 of 32 US 2019/0328899 A1 17,0 171TTTTTTTT 16,616,8 ?????????'?????????????? -?????"??"??????????????????? ?????????????????? 14,014,214,414,614,815,015,215,415,615,816,016,216,4 Amanin+Cyst=6d ATTTTTTTTTT DAD-CH1305nm CH2-CHE CH3 ??????????? F Minutes Cys48ht=Amanin+ DAD-CH1305nm -CH----NH---CO-CH2 -NH-CH--CONHCH2 youCH? H2C Cys=24h+tAmanin PITTTTTTTTTTT 12,612,813,013,213,413,613,8 DAD-CH1305nm ??.?? TTTTT CH ennen COOHH2C- CH ??????????'? H?O, Cyst=0Amanin+ DAD-CH1305nm HO ?????????- 12,012,212,4 FIGURE5 0 375 350 325 300 275 250 225 200 175 150 125 100 75 50 25 -25 MAU Patent Application Publication Oct. 31 , 2019 Sheet 6 of 32 US 2019/0328899 A1 16,817,0 HDP30.2105+Cyst=6d 14,014,214,414,614,815,015,215,415,615,816,016,216,416,6 DAD-CH1305nm CH3 CH2-CH3 CO HC- HDP30.2105+Cyst=48h Minutes DAD-CH1305nm CH-NHCO---CH2 -NH-CHCONHCH2 NH H2C CH? HDP30.2105+Cyst=24h 201 DAD-CH1305nm --CH2OH OH CH -CH--NH-CO 12,012,212,412,612,813,013,213,413,613,8 CH CH COOHH2C-- HDP30.2105+Cyst=0 H?C. DAD-CH1305nm De FIGURE6 40 20 260 240 220 200 180 160 140 120 100 80 60 -20 MAU Patent Application Publication Oct. 31 , 2019 Sheet 7 of 32 US 2019/0328899 Al beta-Amanitin SOIZ*0€do Amanitinsentenelinowealpha- ujuewyuitwerking* ??? .**odawcyklus FIGURE7 iSWAMP Patent Application Publication Oct. 31 , 2019 Sheet 8 of 32 US 2019/0328899 Al HDP30.2115 N HN NH HN 'NH H 0 HN HDP30.1699 HN OH HO H HO PHN NH HO abzslysOberlig HO 30.2060HDP NH HN N. HO NH NH OH ?? FIGURE8 HO Patent Application Publication Oct. 31 , 2019 Sheet 9 of 32 US 2019/0328899 Al 567891234 T-D265C30.2115(n.d.)do T-D265C30.2115(n.d.)d4 T-D265C30.2115(n.d.)d10 T-D265C30.1699(2.8)do T-D265C30.1699[2.8)-d4 T-D265C30.1699[2.8)-010 T-D265C30.2060(2.3)do T-D265C30.2060(2-3)-d10 ADCsincubatedinMousePlasma T-D265C30.2060[2.31-04 5 7 2 FIGURE9 250 130 95. 55 36. 28 17. 123456789 123456789 ADCsincubatedinHumanPlasma ADCsincubatedinPBS SIA 250 130 95 7255 36. 17 250 130 95 55 36 17 Patent Application Publication Oct. 31 , 2019 Sheet 10 of 32 US 2019/0328899 A1 T-D265C30.2115CLd4 4.049e-011 Concentration(M) 10-1410-1310-1210-1110-1010-910-710-8 T-D265C30.2115CLinHumanplasma T-D265C30.2115OLdo 0+TTTTTTTTTTTTTTTTTTTTTTTTATTTT 2.013e-011 EC50 120 110 100 90 80 70 60 50 40 30 20 10 ] % [ Viability 107 10-8 T-0265C30.206004 2.715e-010 Concentration[M] T-D266C30,206004 T-D2650-30,2060-00 T-D265C30.206000 T-D265C30.2060inHumanplasma 10-1410-1310-1210-1110:10109 3.856e-011 EC50 1207 110 100 90 80 70 60 50 40 30 20 10 0 afterincubationinHumanplasmaCytotoxicityonSKBR-3cells ] % [ Viability T-D2650-30.169904 3.022e-010 Concentration[M] 10710-1410-1310-1210:1110-1010-910-8 T-D265C30.1699-04 T-D265C30.1699inHumanplasma T-D2650-30.1699do 2.3589-011 FIGURE10 0+TTTTTTTTTTTTT EC50 120 110 100 90 80 70 60 50 40 30 20 10 ] % [ Viability Patent Application Publication Oct. 31 , 2019 Sheet 11 of 32 US 2019/0328899 Al 10-7 T-D2650-30.2115CL04 3.091e-011 Concentration[M] T-D2650-30.2115CL04 T-D265C30.2115CLinMouseplasma 10-1410-1310-1210-1110-1010-910-8 T-D2650-30.2115CLdo 2.130e-011 C50 120my 110 100 90 80 70 60 50 40 30 20 10 0TTTTTTTTTTTTTTTTTTTTT ] % [ Viability 10-7 10-8 TITLE T-0265C30.2060-04 1.285e-010 10-9 Concentration[M] T-D266C30.200004 T-D265C30.2060do T-D265C30.2060inMouseplasma 2.954e-011 10-1410-1310-1210-1110-10 EC50 110 904 .80 70 60 50 40 304 20 10 0TTTTTTTTTTT 1207 100 plasmaSKBR-3cellsafterincubationinMouseCytotoxicityon ] % [ Viability T-D265C30.169904 4.8660-010 Concentration[M] T-D265C30.1699.d4 10-810-1410-1310-1210-1110-1010-910-7 T-D265C30.1699do T-D265C30.1699inMouseplasma 1.887e-011 FIGURE11 TTTTTTTTTTOtto EC50 120 110 1007 90 80 70 60 50 40 30 204 10 ] % [ Viability Patent Application Publication Oct. 31 , 2019 Sheet 12 of 32 US 2019/0328899 Al 10-7 10-8 T-D265C30.2115CLd4 4.7292-011 Concentration[M] T-D265C30.2115CLdo T-D265C30.2115CLd4 T-D265C30.2115CLinPBS "11HET T-0265C30.2115CLdo 10-1410-1310-1210-1110-1010-9 3.080e-011 120 110 100 90 80 70 60 50 40 30 20 10 0 ] % [ Viability 10-7 10-8 -04TD2650-302060 9.937e-011 Concentration[M] T-D2650-30.206004 T-D265C30.2060inPBS T-D265C30.2060do 10-1410-1310-1210-1110-1010-9 5.665e-011 1207 110 100 90 80 70 60 404 30 20 10 PBSinafterincubationcellsSKBRCytotoxicity-3on ] % [ Viability ?????????????????????????????????????????????? 10-8 T-0265C30.169904 5.980e-011 Concentration(M) T-D265C30.169904 T-D265C30.1699inPBS T-D2650-30.1699do 10-1410-1310-1210-1110-2010-9 2.344e-011 FIGURE12 0 120 110 100 90 80 70 60 50 40 30 20 10 ] % [ Viability Patent Application Publication Oct.