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. 31 , 2019 Sheet 13 of 32 US 2019/0328899 Al
HDPD265C-30.16991mg/kg HDP-D265C-30.16993mg/kgHDPre -D265C 30.21152mg/kg HDP D265C-30.21156mg/kg HDP0265C-30.06436mg/kg HDP-D265C-30.12156mg/kgHDP 0265C-3020723mg/kg HDPD265C-30.19696mg/kg vehiclecontrol(PBS,PH7.4) HDPrD2650-30.08806mgkg
FIGURE13 15004 1250 1000 7509 500 ) mm( volume Tumor Patent Application Publication Oct. 31 , 2019 Sheet 14 of 32 US 2019/0328899 A1
Southern startoftreatmentDaysafter SACA Lorena
M
N FIGURE14 ) n( Survival Patent Application Publication Oct. 31 , 2019 Sheet 15 of 32 US 2019/0328899 A1
SY.2.9-***************************is« Dugimi)logconc. NCIH929-cellbinding
) RLUS( fluorescence Msam
MM1SLuccellbinding
FIGURE15 (sonyl aouaosajoni uean Patent Application Publication Oct. 31 ,2 2019 Sheet 16 of 32 US 2019/0328899 A1
FIGURE 16 : ( A ) B16-0040 : J22.9 - ISY -D265C - 30.2115 Aggregation : 0.9 % DAR - 2 Charen D2
Rel,Intensity(%)
D1
nyusun sospension cysymy ** w www.wayne Mass (Da ] Intactmass of unconjugated AB : 146367 From D2 = 148773.5DAR was calculated as - 2
2 30 SDS - gel analyses Western Blot analyses Reducing conditions Non -reducing Reducing conditions Non -reducing conditions conditions 250 250 130 130 - 250 250 - 95 130 130 -
w 55 55 M 95 95 36 36 55 55 — 36 36 ? ? 17 med
1 wwwwww www ??? 2 1 1 2 1 : J22.9 - ISY -D265C - 30.2115 1 : J22.9 - ISY -D265C - 30.2115 2 : J22.9 - ISY -D265C 2 : J22.9 - ISY -D2650 Antibody: anti- amanitin Patent Application Publication Oct. 31 ,2 2019 Sheet 17 of 32 US 2019/0328899 A1
FIGURE 16 : (B ) B16-0049 : J22.9 - ISY - D265C - 30.1699 Aggregation : 1.2 % DAR = 2 Charmann D2 POA 1 0 20nm 22.9-1SY - D266C 30.1699 Sny imat 12uidat
Rel.Intensity(%)
Am
Chile mypapagaweawowyowlosyen kumuhugopohony proponowo qual may pangmagpanawayyy **** Mass (Da ) Intact mass of unconjugated AB : 146366 From D2 148824 DAR was calculated as 2
? 8 23 SDS - gel analyses Western Blot analyses Reducing conditions Non - reducing conditions Reducing conditions Non -reducing conditions
250 250 250 250 130 130 . 130mm 130 95 95 95 55 INT 36 - SUDAN 36 36 36 -
Tweet
17 . 17 . 1 2 1 2 1 2 1 : J22.9 - ISY- D265C - 30.1699 2 2. J22.9 - ISY - D265C 1 : J22.9 - ISY -D265C - 30.1699 Antibody: anti- amanitin 2 : J22.9 - ISY -D2650 Patent Application Publication Oct. 31 ,2 2019 Sheet 18 of 32 US 2019/0328899 A1
FIGURE 17
human cynomolgus mouse PBS plasma plasma plasma pH 7.4 J22.9 - ISY -D265C - 30.2115 Day 0 250mm Human 2 plasma Day 4 13 . Day 10 Cynomolgus Day ? plasma Day 4 Day 10 Day 0 Mouse 36 FE 8 Day 4 plasma Day 10 tay ? 11 PBS Day 4 Day 10 B 1 2 3 5 6 7 8 9 10 11 12 J22.9 -ISY D265C 30.1699 250 Day 0 Human Day 4 130mm plasma Day 10 72 investiti Dayo 11 Cynomolgus Day 4 plasma Day 10 Day 0 Mouse ng plasma Day 4 Dav 10 22 Dayo 23 PBS Day 4 13 14 15 16 17 18 19 20 21 22 23 24 Day 10 Patent Application Publication Oct. 31 ,2 2019 Sheet 19 of 32 US 2019/0328899 A1
FIGURE 18
? J22.9 -ISY -D265C - 30.2115 human plasma cynomolgus plasma mouse plasma PBS pH 7.4
20 120 120 400 100
10-14 10-13 10-12 10-11 1040 105 106 107 10 $ 10-14 10-12 10-12 10-75 100 109 108 107 108 10-4 10-13 10:12 101 1010 104 104 107 104 10-4 10:13 10- 1041 1040 10 10 10 10 Concentration M Concentration M Concentration [ M Concentration M weiteren J22.9 - ISY - D265C - 30.2115 - day J22.9 - SY -D265C - 30.2115 - day 4 J22.9 - ISY -D265C - 30.2115 - day 10
B J22.9 - ISY- D2650-30 . 1699 cynomolgus plasma human plasma 150 150 , mouse plasma PBS PH 7.4 130
30 20
10-14 10-13 10-12 10:44 10-19 104 104 102 104 104 10-18 10-19 10-11 1090 10 10 10 106 10-14 10-13 10-12 1071 10-20 10-9 10- 107 10 € 10-4 10-19 10-12 10-11 10-10 109 10 % 10 % 10 % Concentration (M ) Concentration (M ) Concentration M Concentration ( M )
nevoilo . J22.9 -ISY - D265C - 30.1699 - day 0 er meget J22.9 -ISY - D265C -30.1699 - day 4 J22.9 -ISY - D265C - 30.1699 - day 10 Patent Application Publication Oct. 31 ,2 2019 Sheet 20 of 32 US 2019/0328899 A1
FIGURE 19
36 BCMA 17 -
95 72 55 ... CD138
250 .. POL2A 130 72 p53
36 GAPDH 28 MM.1s NCI-H929 OPM -2 RPMI -8226 U266B1KMS -11 CCRF -CEM MM.19 Luc Patent Application Publication Oct. 31 ,2 2019 Sheet 21 of 32 US 2019/0328899 A1
FIGURE 20
NCI- H929 MM.1Sluc
Cellviability)
Concentration (M ) en J22.9 - ISY -D265C - 30.1699 # $ 22.9 - ISY -D265C - 30.2115 U266B1
CellViability(%) CellViability(%)
www ram Concentration ( M ) Concentration (M ) 0122.9 - ISY -D265C - 30.1699 122.9 - ISY -D265C - 30,2115 OPM - 2 CCRF - CEM 125
CellViability1%) CellViability(%)
0
Concentration ( M ) # J22.9 - ISY - D265C -30.1699 Concentration ( M ) 122.9 - ISY -D265C -30.2115 Patent Application Publication Oct. 31 ,2 2019 Sheet 22 of 32 US 2019/0328899 A1
FIGURE 21
3000 2000 1000
200 PBS ( 10mL /kg ) J22.9 -ISY -D265C - 30.1699 [ 2mg/ kg ) TumorVolume[mm] 150 oblemen J22.9 -ISY -D265C - 30.1699 [4mg /kg ] J22.9 - ISY -D265C - 30.2115 [2mg / kg ) J22.9 - ISY -D265C - 30.2115 [4mg / kg ]
AREA 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 Days After Treatment ( single dose iv ) Patent Application Publication Oct. 31 , 2019 Sheet 23 of 32 US 2019/0328899 Al
J22.9.ISY-D265C30.2115[Img/kg)1xweek J22.91SY-D265C30.2115[0.25mg/kg)1x2weeks J22.91SY-D265C30.2115[1mg/kg]1x2weels J22.94SY-D265C30.2115[0.25mgkg)Txlweek J22.94SY-D265C30.2115[0.5mg/kg]ixtweek J22.9-ISYD265C302115[0.5mg/kg)1x2weeks J22.9.1SY-D265C30.2115[0.5mgkol1x/3weeks J22.91SY-D265C30.2115[imgkg]1x/3weeks J2291SY-D265C30.2115[2mg/kg]1x3weeks J2294SY-D265C30.2115[2mg/kg]2Xweek
PBS[10kg tumorfreeallanimals ##5/9animalsalive WW #6/9animalsalive 100110120
EfficacyofCD269-D265C30.2115inascNCIH929MMXenograft 30405070809060 TreatmentStartDaysAfterof
10200 FIGURE22 3000 2000 1000 ] mm[ Volume Tumor Patent Application Publication Oct. 31 , 2019 Sheet 24 of 32 US 2019/0328899 Al
J22.9-ISYD265C30.1699[2mg/kg] J22.9-ISYD265C30.1699[4mg/kg] J22.9-ISYD265C30.2115[2mg/kg] J22.9-ISYD265C30.2115[4mg/kg]
PBS(10ml/kg] BackgroundLevel
a. 9/10animals:backgroundlevel
11914212835424956637077849198105112 InoculationTumorCellPostDays Treatment(singledoseiv)
FIGURE23(A) 7
0 1011 1010 104 108 107 106 .105 ] s/ p[ Flux Total Patent Application Publication Oct. 31 , 2019 Sheet 25 of 32 US 2019/0328899 Al
Totalfluxdistributionin2and4mg/kggroupsof J22.9-ISYD265C30.2115 0714212835424956637077849198105112119 InoculationTumorCellDaysPost J22.9-ISYD265C30.2115[2mg/kg] J22.9-ISYD265C30.2115[4mg/kg] Treatment(singledoseiv)
10% 108 107 106 .105 ] s/ p [ Flux Total FIGURE23(B) Patent Application Publication Oct. 31 , 2019 Sheet 26 of 32 US 2019/0328899 Al
J22.9-ISYD265C30.2115[0.1mg/kg] J22.9-ISYD265C30.2115[0.3mg/kg] J22.9-ISYD265C30.2115[1mg/kg] J22.9-ISYD265C30.2115[2mg/kg]
PBS[10ml/kg] LevelBackground
98 Hrights 91 *9/10animalsbackground:level 84
35424956637077 EfficacyofATACsinadisseminatingivMM1.S-LucMMXenograft InoculationTumorCellDaysPost
Treatment(singledoseiv) 28 21 14
FIGURE24(A) 07 1011 1010 109 108 107 106. 1057 ] s/ p[ Flux Total Patent Application Publication Oct. 31 , 2019 Sheet 27 of 32 US 2019/0328899 Al
98 91
Totalfluxdistributionin1and2mg/kggroupsof J22.9-ISYD265C30.2115 InoculationCellPostTumorDays 210714283542497756637084 J22.9-ISYD265C30.2115[1mg/kg] J22.9-ISYD265C30.2115[2mg/kg] Treatment(singledoseiv)
100%10 108 107 106. 106 ] s / p [ Flux Total
FIGURE24(B) Patent Application Publication Oct. 31 , 2019 Sheet 28 of 32 US 2019/0328899 Al
* * *
M
8896104112120128 8896104112120128
3mg/kg
Days Days " 081624324048566472808896104112120128 Days
647280162432404856 324048
J22.9-SYD2650-30,2115 1mg/kg 24 ...... 600-0.3mgkg/ . 0.3mg/kg 0.3mg/kg CheWWW**
0 0
O. 0 ALT 52004 AST 5000 3000-1 1000 750 250 LDH 5000 1000 unash Serum UL 52000 08 08 72
Days Days 50 Days 3mg/kg 3mg/kg 40 J22.9-SYD265C30.1699 32 img/kg 24 mg/kg Img/kg ...... 24
0.3mg/kg 0.3mg/kg 0.3mg/kg www.
U FIGURE25 O 750. ALT 488 52004 100 AST 3000 LDH 5000 3000 3000 2000 1000 Patent Application Publication Oct. 31 , 2019 Sheet 29 of 32 US 2019/0328899 A1
Concentration[M] KMS-11 96h 10-1310-1210-1110-1010-910-810-710-6 TTT
120 110 100 90 80 70 60 50 40 30 20 10 0 ] %[ Viability Cell
Concentration[M] MM.1SLUC 96h 10-1310-1210-1110-10109108107106
FIGURE26(A) 1. 150-1 125 1004 75 50 25 0 -25 -50 ] % [ Viability Cell Patent Application Publication Oct. 31 , 2019 Sheet 30 of 32 US 2019/0328899 Al
106
Concentration[M] MM.19Luc 72h 10-1310-1210-1110-1010910-310-7 -50-Apremte 1501 125 100 754 50 25 O -25 ] % [ Viability Cell
10-6
Concentration[M] Concentration[M] 10-1310-1210-1110-1010910810710-6 MM.19Luc 48h MM.19Luc 120h 10-1310-1210-1110-2010-9108107
-50+TTATTI 0 125" 75 50 25 0 -25 125 75 25 -25 -50-7mperare 1501 100 1504 100 ] % [ Viability Cell ] % [ Viability Cell
Concentration[M] Concentration(M] MM,1SLuc 24h 10-1310-1210-1410-1010-910-8107106 MM.1SLuc 96h 10-1310-1210-1410-1010910810710 -50-pyympopograpprepayment 0 150 125 100 75 50 25 -25 1509 125 100 75 50 25 25 FIGURE26(B) ] % [ Viability Cell ] % [ Viability Cell Patent Application Publication Oct. 31 , 2019 Sheet 31 of 32 US 2019/0328899 Al
10-710-6
Concentration[M] KMS-11 72h 10-1310-1210-111010-910-3
0-Wor 120 110 100 90 80 70 60 50 30 20 10 ] % [ Viability Cell
11111 THAT Concentration[M] TTEET Concentration(M) KMS-11 48h mommy 10-1310-1210-1110-1010-910-10710-6 KMS-11 120h 10-1310-1210-1110-1010-910-810-710 0+TTTTTTTTTTT 120 110 100 90 80 70 60 50 40 30 20 10 1201 110 100 80 70 60 50 40 30 20 10 ] % [ Viability Cell ] % [ Viability Cell
Concentration[M] Concentration[M] KMS-11 KMS-11 10-1310-1210-1110-1010-910-210710-6 24h 10-1310-1210-1110-1010-910-810-710 96h 11.TA
120 110 100 90 80 70 60 50 40 30 20 10 1201 110 100 90 80 70 60 50 40 30 20 10 FIGURE26(C) ] % [ Viability Cell ] % [ Viability Cell Patent Application Publication Oct. 31 , 2019 Sheet 32 of 32 US 2019/0328899 Al
150
Carfilzomib(PR-171)5mg/kg,2xw3w 140 J22.9-ISYD265C30.2115(1.Omg/kg) J22.9-ISYD265C30.2115(4.Omg/kg) J22.9-ISY-McMMAF1.Omg(/kg) J22.9-ISYMcMMAF(4.Omg/kg) 130 PBS(10ml/kg) 120
ws 110 100 7 90 80
70 Daysafterinjection
. 60
50
40
30
20
10
0 1012 1011 .1010 %10 108 107 10 105 FIGURE27 [ s / d ] flux Total US 2019/0328899 Al Oct. 31 , 2019 1
AMANITIN ANTIBODY CONJUGATES develop treatment resistance, leaving a need to develop safe and effective treatments that prolong the duration of remis FIELD OF THE INVENTION sion and improve survival. [0006 ] About 30 % of MM patients will develop renal [0001 ] The invention relates to a conjugate comprising ( a ) insufficiency over the course of disease and 20 % will present an amatoxin comprising (i ) an amino acid 4 with a 6 '- deoxy renal failure , caused by the accumulation and precipitation position ; and ( ii) an amino acid 8 with an S -deoxy position ; of light chains . Renal morbidity is a considerable burden for ( b ) a BCMA -binding moiety comprising (i ) the variable MM patients with incidence of renal replacement therapy for domains of humanized antibody J22.9 - ISY , and ( ii ) a heavy end - stage renal disease due to MM .Novel agents such as the chain constant region comprising a D265C mutation ; and ( c ) proteasome inhibitor bortezomib can successfully restore a protease - cleavable linker linking said amatoxin and said renal function in a considerable proportion of patients and target- binding moiety. The invention furthermore relates to complications due to renal failure can be avoided when a pharmaceutical composition comprising such conjugate, detected and treated early (Dimopoulos et al. , 2008 ). particularly for use in the treatment of multiple myeloma. [0007 ] Even though agents like proteasome inhibitors and / or immunomodulatory drugs have improved outcome for patients , these agents do not cure the disease . Thus , the need BACKGROUND OF THE INVENTION remains for new agents with novel modes of action . The [0002 ] Multiple Myeloma (MM ) is the second most preva knowledge of molecules expressed on the surface of lent hematopoietic malignancy characterized by the prolif myeloma cells resulted in a number of new monoclonal eration of a single clone ofplasma cells derived from B cells antibodies currently in development for new therapies . in the bone marrow . MM ranks as the 15th most common However , most of these molecules are not exclusively type of cancer in the US and is still considered incurable , expressed on MM cells and their potential to treat MM needs with a median survival rate of about 30-60 months . Even to be demonstrated . though advances in the treatment ofMM using conventional [ 0008 ] Numerous drugs and therapies are currently in and novel therapeutics alone or in combination have resulted clinical phase : antibodies like daratumumab targeting CD38 in improved response rate and thus prolonged median sur and elotuzumab targeting SLAMF7 (Signaling Lymphocyte vival of many years , MM is still considered as incurable Activation Molecule Family Member 7 ) , both used alone or disease . MM represents about 0.8-1 % of all cancers world in combination with other drugs ; panabinostat , a histone deacetylase inhibitors (HDAC ) to be used in combination wide and approximately 10 % of all hematological cancers therapy ; chimeric antigen receptor engineered T (CAR - T ) with an estimated global incidence of more than 100.000 cells expressing an anti - BCMA single - chain variable frag new cases a year and a global mortality ofmore than 70.000 ment allowing specific targeting of MM cells . cases yearly (National Cancer Institute SEER database ). [0009 ] MM is characterized by the malignant proliferation [ 0003] The uncontrolled proliferation of plasma cells in of plasma cells , terminally differentiated B -cells which MM results in the abnormal production of monoclonal under normal circumstances are responsible for the mass immunoglobulins ( also known as M -protein ) that can lead to production of immunoglobulins . This progression from renal failure caused by immunoglobulin light chains or plasma cells to malignant myeloma cell is associated with hyperviscosity from excessive amounts of M - proteins . In multiple genetic and oncogenic events including deregula summary , clinically symptomatic MM is characterized by tion of cyclin D1 and c -Myc , and mutations like KRAS , the presence of M - protein in serum and / or urine and end BRAF , FGFR3 ( fibroblast growth factor receptor 3 ) and organ damage, generally involving hypercalcemia , renal TP53 ( Shaffer et al. , 2008 ; Kuehl and Bergsagel , 2012 ; failure , anemia and bone marrow lesions (CRAB features ) Chesi and Bergsagel, 2015 ) . (Bird et al. , 2011 ; Rajkumar et al. 2014 ) . [0010 ] Despite these genetic alterations, the malignant [0004 ] Despite several treatment options , MM is an incur plasma cell remains largely dependent on the bone marrow able disease and almost all patients will eventually develop (BM ) environment. Following binding of the MM cells in resistant or refractory disease . Relapsed MM refers to pro the BM , signalling cascades are activated including acces gressive disease in which at least a partial response was sory growth factors and its ligands secreted by the BM previously achieved following first- line treatment of salvage accessory cells . Components released by these accessory therapy . Refractory MM indicates progressive disease when cells play a critical role in MM to both promote disease and the patient is either unresponsive initially or following escape from immune surveillance . treatment within the last 60 days (Dimipoulos et al. , 2015 ) . [0011 ] Antibody - drug conjugates ( ADCs) aim to take [0005 ] Without any treatment, the median length of sur advantage of the specificity of monoclonal antibodies to vival after diagnosis of a myeloma patient is about three deliver potent cytotoxic drugs selectively to antigen -present years (Kyle and Rajkumar, 2004 ). There are now several ing tumor cells . classes of drugs used in the treatment ofmyeloma : immu [0012 ] The antibody should target a well- characterized nomodulatory drugs ( thalidomide, lenalidomide , pomalido antigen with high and specific expression at the tumor site mide ) , proteasome inhibitors (bortezomib , carfilzomib ) and and low or no expression on normal tissue , thus maximizing stem cell transplantation .Many of the notable drugs in these the efficacy of the ADC while limiting toxicity. classes were approved over the past 10-15 years. Treatments [0013 ] Members of the tumor necrosis factor receptor using these novel drugs and high -dose chemotherapy in ( TNFR ) superfamily and their ligands play a critical role in combination with autologous or allogenic hematopoietic controlling proliferation , differentiation and apoptosis of stem -cell transplant (SCT ) have been shown to improve B - cells . In particular, B -cell activating factor (BAFF ) median survival to five years , which makes this the current ligand - receptor network play a central role in regulating standard therapy . However , many patients suffer relapse or B -cell maturation and differentiation into malignant plasma US 2019/0328899 A1 Oct. 31 , 2019 2 cells . The functionally related BAFF receptors BAFF - R , thatione ) or non - cleavable ( depending on complete degra cyclophilin ligand interactor ( TACI) and B - Cell maturation dation of the antibody after internalization of the ADC ) . antigen (BCMA ) are type III transmembrane proteins lack [0019 ] Finally , with respect to the toxic payload of anti ing a signal- peptide and containing cysteine- rich extracel body - drug conjugates , two main classes of cytotoxic agents lular domains. are currently used in clinical evaluations: drugs that disrupt [0014 ] These receptors promote survival of B -cells at microtubule assembly or compounds that bind to the minor distinct stages of development and thus their expression groove of DNA causing double strand breaks . patterns on B -cells differ depending on the type of B -cells [0020 ] Amatoxins are cyclic peptides composed of 8 and their stage of maturation and activation . BAFF - R amino acids that are found in Amanita phalloides mush expression is not detectable on B - cell precursors but is the rooms (see FIG . 1 ) . Amatoxins specifically inhibit the DNA dominant receptor expressed on mature B - cells . BAFF - R dependent RNA polymerase II of mammalian cells , and mediates the survival of B -cells , sustains the longevity of the thereby also the transcription and protein biosynthesis of the germinal - center reaction and promotes immunoglobulin affected cells . Inhibition of transcription in a cell causes stop class- switch recombination . TACI is expressed on B -cells of growth and proliferation . Though not covalently bound , and activated T -cells and is important in class - switch recom the complex between amanitin and RNA -polymerase II is bination and supports T - cell dependent antibody response . very tight ( K ) = 3 nM ). Dissociation of amanitin from the BCMA, which is almost exclusively expressed on plasma enzyme is a very slow process, thus making recovery of an cells but is absent from naïve and memory B -cells , promotes affected cell unlikely . When the inhibition of transcription B - cell differentiation into plasma cells and thus promotes the lasts too long, the cell will undergo programmed cell death maintenance of humoral immune response . Soluble BCMA (apoptosis ). is elevated in the serum of MM patients and the successful [ 0021] The use of amatoxins as cytotoxic moieties for donor lymphocyte infusion is associated with the formation tumor therapy had already been explored in 1981 by cou of antibodies against BCMA. The level of serum BCMA pling an anti - Thy 1.2 antibody to a - amanitin using a linker found among MM patients associates with clinical status and attached to the indole ring of Trp ( amino acid 4 ; see FIG . 1 ) overall survival. (Belucci et al. , 2005 ; Sanchez et al. , 2012 ) . via diazotation ( Davis & Preston , Science 213 ( 1981) 1385 [ 0015 ] BAFF , APRIL ( a proliferation - inducing ligand ) 1388 ) . Davis & Preston identified the site of attachment as and BCMA perform critical roles in decision -making pro position 7' . Morris & Venton demonstrated as well that cess from B - cell to plasma and MM cell. As B -cells egress substitution at position 7 ' results in a derivative , which from the bone marrow , further maturation into marginal maintains cytotoxic activity (Morris & Venton , Int. J. Pep zone B - cells (MZ B ) is dependent on BAFF. BAFF is also tide Protein Res. 21 ( 1983 ) 419-430 ) . essential for the homeostasis of naïve recirculating B -cells [ 0022 ] Patent application EP 1 859 811 Al (published and MZ B - cells . Downregulation of BAFF - R on plasma Nov. 28, 2007) described conjugates, in which the y C - atom cells is coincident with the upregulation of BCMA , which of amatoxin amino acid 1 of B - amanitin was directly can bind BAFF as well as APRIL . Transcription factor coupled , i.e. without a linker structure , to albumin or to NFKB plays a crucial role in pathways regulation , B - cell monoclonal antibody HEA125 , OKT3 , or PA - 1 . Further proliferation , survival and differentiation . NFKB activity is more, the inhibitory effect of these conjugates on the pro modulated by the inhibitor of KB ( IKB ) proteins . Among liferation of breast cancer cells (MCF - 7 ), Burkitt's lym these, IkBa retains NFKB dimers inactive in the cytoplasm phoma cells (Raji ) and T - lymphoma cells ( Jurkat ) was and , when degraded , releases bound NFKB dimers to trans shown . The use of linkers was suggested, including linkers locate to the nucleus and drive gene expression . Regulation comprising elements such as amide, ester , ether, thioether , of IKB activity through degradation depends on serine disulfide, urea , thiourea , hydrocarbon moieties and the like , phosphorylation by the IKB kinase ( IKK ) (Rickert et al ., but no such constructs were actually shown, and no more 2011 ) . details , such as attachment sites on the amatoxins, were [ 0016 ] Various anti- BCMA antibodies have been gener provided . ated in the past , including murine antibody J22.9 (WO [0023 ] Patent applications WO 2010/115629 and WO 2014/068079 ) and humanized versions thereof (WO 2015 / 2010/115630 (both published Oct. 14 , 2010 ) describe con 166073 ) . jugates, where antibodies, such as anti -EpCAM antibodies [ 0017 ] In order to obtain antibody -drug conjugates , such as humanized antibody huHEA125, are coupled to bifunctional linkers with attachment sites for both the anti amatoxins via ( i ) the y C -atom of amatoxin amino acid 1 , ( ii ) body and the drug are used to join the two components . the 6 ' C - atom of amatoxin amino acid 4 , or ( iii ) via the [0018 ] With respect to conjugation to the antibody, linker C -atom of amatoxin amino acid 3 , in each case either attachment strategies typically rely on cysteine or lysine directly or via a linker between the antibody and the ama residues on the antibody . Ideally , the linker must remain toxins. The suggested linkers comprise elements such as stable in systemic circulation to minimize adverse or toxic amide, ester , ether , thioether, disulfide, urea , thiourea , effects . Upon antigen recognition and binding, the resulting hydrocarbon moieties and the like. Furthermore , the inhibi ADC receptor complex is internalized through receptor tory effects of these conjugates on the proliferation of breast mediated endocytosis . The unconjugated toxin should dem cancer cells (cell line MCF -7 ) , pancreatic carcinoma (cell onstrate high potency to enable efficient killing upon release line Capan - 1) , colon cancer (cell line Colo205 ), and cho from ADC . Release of the toxic payload should only occur langiocarcinoma (cell line OZ ) were shown. inside the target cell and ADC must be stable in plasma. For [0024 ] It is known that amatoxins are relatively non - toxic the release of the toxin from the antibody inside the cell when coupled to large biomolecule carriers , such as anti linkers can be either cleavable (either cleaved by lysosomal body molecules, and that they exert their cytotoxic activity proteases, hydrolysed at low pH in the lysosomal compart only after the biomolecule carrier is cleaved off . In light of ment or released from disulfide bonds by intracellular glu the toxicity of amatoxins , particularly for liver cells , it is of US 2019/0328899 A1 Oct. 31 , 2019 3 outmost importance that amatoxin conjugates for targeted formed from an amatoxin - based antibody conjugate can be tumor therapy remain highly stable after administration in reduced by the addition of cysteine , and in FIGS . 3 and 4 it plasma, and that the release of the amatoxin occurs after is shown that a -amanitin reacts with cysteine . However, the internalization in the target cells . In this context, minor stability of the conjugates comprising a highly toxic ama improvements of the conjugate stability may have drastic toxin is of utmost importance for the envisaged use as a consequences for the therapeutic window and the safety of therapeutic molecule for administration to human beings . the amatoxin conjugates for therapeutic approaches . [0025 ] Patent application WO 2016/142049 describes OBJECT OF THE INVENTION antibody - amanitin conjugates wherein the amanitin payload [0028 ] Thus, there was still a great need for amatoxin is attached to the antibody via specifically engineered cys based antibody - drug conjugates having a high efficacy in teine residues in the antibody heavy chain constant region . target cells , with simultaneously having high stability . The Depending on the nature of the mutated residue , the replace solution to this problem , i.e. the identification of ( a ) an ment of the wild -type amino acid residue may influence the appropriate parental anti -BCMA antibody, ( b ) an appropri activity. ate strategy for attaching the toxic payload via linker to the [0026 ] It has already been reported that amatoxin -based antibody, (c ) an appropriate linker and ( d ) an appropriate antibody conjugates directed at CD269 may offer advan amatoxin variant was neither provided nor suggested by the tages in the treatment of tumor patients ( see Palfi et al .: prior art. “ Preclinical evaluation of anti- CD269 antibody drug conju gates ” , European Journal of Cancer 69 ( 2016 ) , S21 & 28th SUMMARY OF THE INVENTION EORTC -NCI - AACR Symposium on Molecular Targets and [0029 ] The present invention is based on the unexpected Cancer Therapeutics, Munich (Germany ) November observation that a variant form of an amatoxin conjugated to 29 -Dec . 2 , 2016 ; Palfi et al.: “ CD269 - A promising target an anti -BCMA antibody based on antibody J22.9 , wherein for amanitin based ADCs” , Cancer Research 76 (No. 14 the antibody and the amatoxin are linked by a cleavable suppl) ( 2016 ) 2973 & 107th Annual Meeting of the Ameri linker shows an increased stability under stress conditions, can Association for Cancer Research , New Orleans (U.S.A. ) particularly in human plasma, and an improved therapeutic April 16 - Apr. 20 , 2016 ) . However , no details were provided index . about the specific nature of the amatoxin derivative being [0030 ] Thus, in one aspect the present invention relates to used and of the specific linkage between that amatoxin a conjugate according to Formula I comprising ( a ) an derivative and the anti -CD269 antibody. amatoxin comprising (i ) an amino acid 4 with a 6 '- deoxy [0027 ] Thus, significant progress has already been made in position ; and ( ii ) an amino acid 8 with an S -deoxy position ; the development of amatoxin -based conjugates for thera (b ) a BCMA - binding moiety comprising (i ) the variable peutic uses . However, the present inventors have found that domains of the heavy chain according to SEQ ID NO : 1 and constructs based on a- and B - amatoxin were not fully stable the light chain according to SEQ ID NO : 2 of antibody under stress conditions in plasma and resulted in a substan J22.9 -ISY , and ( ii ) a heavy chain constant region comprising tial degree of cross -linked products (see FIG . 2 ). This effect a D265C mutation ; and ( c ) a protease - cleavable linker, was completely unexpected , and the reasons for such insta wherein said BCMA -binding moiety is attached to said bility and cross - linking were unclear. In FIG . 2 it is addi linker via the thiol group of the engineered cysteine residue tionally shown that the amount of cross -linked products at position 265 in the antibody heavy chain . Formula I ?? .
HO
HN HN
HO - NH
NH -S-( J22.9 -ISY -D265C . ) US 2019/0328899 A1 Oct. 31 , 2019 4
[ 0031 ] In a second aspect, the present invention relates to to reference Thiomab antibody with D265C mutation . Test a pharmaceutical composition comprising the conjugate of itemswere incubated in human plasma at 37 ° C. for 0 an 4 the present invention . days. Cytotoxicity assay were performed on SKBR - 3 cells [0032 ] In a third aspect, the present invention relates to the for 96 h . HDP 30.1699 and HDP 30.2060 based ADCs show conjugate of the present invention , or the pharmaceutical remarkable loss of cytotoxicity after 4 days plasma stressing composition of the present invention , for use in the treatment whereas deoxygenated derivative HDP 30.2115 shows still of cancer in a patient, particularly wherein the cancer is picomolar activity selected from the group consisting of multiple myeloma, [0043 ] FIG . 11 shows cytotoxicity of amatoxin derivatives diffuse large B -cell lymphoma ( DLBCL ) , and chronic lym HDP 30.1699, HDP 30.2060 and HDP 30.2115 conjugated phocytic leukemia (CLL ) , particularly multiple myeloma. to reference Thiomab antibody with D265C mutation . Test items were incubated in mouse plasma at 37 ° C. for 0 an 4 BRIEF DESCRIPTION OF THE DRAWINGS days. Cytotoxicity assay were performed on SKBR - 3 cells [ 0033 ] FIG . 1 shows the structural formulae of different for 96 h . HDP 30.1699 and HDP 30.2060 based ADCs show amatoxins . The numbers in bold type ( 1 to 8 ) designate the remarkable loss of cytotoxicity after plasma stressing standard numbering of the eight amino acids forming the whereas deoxygenated derivative HDP 30.2115 remains amatoxin . The standard designations of the atoms in amino almost unchanged . acids 1 , 3 and 4 are also shown (Greek letters a to y , Greek [ 0044 ] FIG . 12 shows cytotoxicity of amatoxin derivatives letters a to d , and numbers from 1' to 7 ', respectively ) . HDP 30.1699, HDP 30.2060 and HDP 30.2115 conjugated [ 0034 ] FIG . 2 shows the results of a stress testing experi to reference Thiomab antibody with D265C mutation . Test ment in an anti -amanitin Western blot. A trastuzumab items were incubated in PBS at 37 ° C. for 1 an 4 days . amanitin conjugate (Her - 30.0643; lysine conjugation via Cytotoxicity assay were performed on SKBR - 3 cells for 96 6 '- OH ; stable linker ) was incubated for 5 days at 37 ° C. in h . All ADCs show adequate stability to non -enzymatic PBS, PH 7.4 , which led to extensive inter- and intrachain environment. cross - linking ; cross -linking of antibody chains could be [0045 ] FIG . 13 compares the antitumoral activity of dif reduced by addition of free cysteine . ferent amatoxin conjugates based on a tumor- targeting ref [ 0035 ] FIG . 3 shows that a - amanitin shows a strong erence antibody (HDP rep ) -in a xenograft model — single reactivity with cysteine in PBS buffer , pH 7.4 . 1 mg/ mL dose experiment. Depending on linker and toxin structure a -amanitin 10 mg/mL cysteine in PBS , pH 7.4 at 37 ° C. after significant differences in antitumoral activity have been 24 h , 48 h and 6 d RP -HPLC C18 . observed . The deoxy - amanin variant HDP rep 30.2115 (6 [ 0036 ] FIG . 4 shows that ß - amanitin shows a strong deoxy at amino acid 4 and S -deoxy at amino acid 8 ) showed reactivity with cysteine in PBS buffer , pH 7.4 . 1 mg/ mL best antitumoral activity of all amatoxin ADCs, with a B -amanitin 10 mg/mL cysteine in PBS , pH 7.4 at 37 ° C. after significantly better therapeutic index than corresponding 24 h , 48 h and 6 d RP - HPLC C18 . cleavable linker ADC HDP - 30.1699 ( lysine conjugation [ 0037 ] FIG . 5 shows that a 6 '- deoxy variant at amino acid via 6 -OH ; SMO at amino acid 8 ). 4 (“ amanin ” ) shows a reduced reactivity with cysteine. 1 [0046 ] FIG . 14 shows the Kaplan Meier survival analysis mg/ mL amanin 10 mg/mL cysteine in PBS, pH 7.4 at 37 ° C. in a systemic tumor model — single dose experiment with after 24 h , 48 h and 6 d RP -HPLC C18 . reference antibodies . In brief , 2.5x10 tumor cells in 200 uL PBS/ mouse were inoculated intravenously on day 0 . [ 0038 ] FIG . 6 shows that a double deoxy variant HDP Therapy ( single dose , iv) was initiated on day 3 post tumor 30.2105 (6 '- deoxy at amino3 acid 4 and S -deoxy at amino acid 8 ; formula I with R - OR and each R = H ) shows cell inoculation . The deoxy -amanin variant HDP ref- 30.2115 complete absence of reactivity with cysteine . 1 mg/ mL HDP (6 ' -deoxy at amino acid 4 and S -deoxy at amino acid 8 ) 30.2105, 10 mg/ mL cysteine in PBS, pH 7.4 at 37 ° C. after showed superior survival over HDP rer -based conjugates 24 h , 48 h and 6 d RP -HPLC C18 ; * impurity. with a - amanitin derivatives HDP 30.1699 , HDP 30.0880 and [0039 ] FIG . 7 summarizes the results from FIGS . 3 to 6 ; HDP 30.0643 as well as with the corresponding MMAE x axis : reaction time in hours ; y axis : amount of remaining derivative . amatoxin variant in % . [0047 ] FIG . 15 shows that J22.9 - ISY and thiomab J22.9 [0040 ] FIG . 8 shows alpha- amanitin derivative HDP ISY - D265C have comparable binding properties to BCMA 30.1699 with cleavable linker at AA4-6 -OH moiety , alpha positive cells . The results of a flow cytometry - binding amanitin derivative HDP 30.2060 with cleavable linker at assays of antibody variants J22.9 - ISY and J22.9 - ISY -D265C AA1 y -position and double deoxy amatoxin variant HDP revealed comparable binding properties to BCMA -express 30.2115 with cleavable linker at AA1 y -position . ing MM.1S Luc and NCI- H929 cells . [0041 ] FIG . 9 shows Western - Blot analysis of amatoxin [0048 ] FIG . 16 shows the QA results of the production of derivatives HDP 30.1699 , HDP 30.2060 and HDP 30.2115 ( A ) J22.9 - ISY - D265C - 30.2115 , and ( B ) J22.9 - ISY -D265C conjugated to reference Thiomab antibody with D2650 30.1699 . mutation after incubation at 37 ° C. in human plasma, mouse [0049 ] FIG . 17 shows the results of stability experiments : plasma and phosphate buffered saline ( PBS ) for 0 , 4 and 10 J22.9 -ISY - D265C - 30.2115 ( A ) and J22.9 - ISY -D265C -30 . days . Detection was done with a polyclonal anti -amanitin 1699 ( B ) were incubated in human , cynomolgus, or mouse antibody from rabbit and an anti- rabbit antibody conjugated plasma or PBS for control for 0 , 4 , and 10 days at 37 ° C. and to horseradish peroxidase . HDP 30.1699 and HDP 30.2060 analysed in reducing SDS -PAGE and subsequent anti- aman showed considerable cross - links and loss of the amatoxin itin Western Blot. moiety . Double deoxy amanitin variant HDP 30.2115 shows [ 0050 ] FIG . 18 shows the results from experiments analy high stability and significantly reduced cross - links. sing plasma stability and cytotoxic potential . J22.9 - ISY [0042 ] FIG . 10 shows cytotoxicity of amatoxin derivatives D265C - 30.2115 ( A ) and J22.9 - ISY - D265C - 30.1699 ( B ) HDP 30.1699, HDP 30.2060 and HDP 30.2115 conjugated were incubated in PBS for control , or human , cynomolgus or US 2019/0328899 Al Oct. 31 , 2019 5 mouse plasma for 0 , 4 , and 10 days at 37 ° C. and analysed shown for groups treated with 1 mg/ kg (solid line ) and 2 for remaining cellular toxicity potential using BCMA- posi mg/ kg ( dashed line ) of J22.9 -ISY -D265C - 30.2115 . Data tive NCI-H929 cells . indicate that only one animal from each depicted group [ 0051 ] FIG . 19 shows BCMA expression levels on differ shows elevated tumor signals significantly above back ent cell lines used for cytotoxicity assays . Western Blot ground level. analyses using antibodies against BCMA , CD138 , RNA [0057 ] FIG . 25 shows the results from a tolerability study polymerase 2A (POLR2A ) , p53 and GAPDH ( control) . All in cynomolgusmonkeys : Groups of 3 animals were injected cell lines express BCMA, except for CCRF -CEM which was with J22.9 - ISY -D265C - 30.2115 or J22.9 - ISY - D265C - 30 . used a BCMA - negative cell line . Highest BCMA levels are 1699 in escalating doses (0.3 , 1 and 3 mg/ kg ) followed by detected in NCI- H929 cells , lowest in RPMI- 8226 cells . repeated dosing using 3 mg/ kg . ALT: alanine transaminase ; [ 0052 ] FIG . 20 shows results from cellular cytotoxicity AST : aspartate transaminase ; LDH : lactate dehydrogenase . experiments : Viability of BCMA -positive (NCI - H929 , [0058 ] FIG . 26 shows a comparison of the cytotoxic MM.1S , MM.1S Luc, U266B1, OPM - 2 ) and BCMA -nega potential of J22.9- ISY -D265C -30.2115 with the tive ( CCRF - CEM ) cells after incubation for 96 hours with monomethyl auristatin F derivative J22.9 - ISY -MMAF J22.9 - ISY -D265C - 30.1699 and J22.9 - ISY -D265C - 30.2115 on MM.1S Luc ( left panel ) and KMS - 11 cells ( right panel) : was monitored . Cytotoxicity was calculated as half maximal ( A ) comparison at time point 96 h , EC50 of J22.9 - ISY effective concentration ( EC50 ) . D265C -30.2115 : 5.575 10-10 M on MM.1S Luc cells ; 1.716 [0053 ] FIG . 21 shows the antitumor activity in mouse 10-9 Mon KMS . - 11 cells ; EC 50 of J22.9 - ISY -MMAF : 4.812 NCI- H929 subcutaneous xenograft in single dose applica 10-10 M on MM.1S Luc cells ; 6.261 10-10 M on KMS- 11 tion: Female CB -17 Scid mice were subcutaneously inocu cells ; ( B ) time course 24 h to 120 h of cytotoxic potential on lated with 5x106 NCI -H929 cells. Once tumor volume MM.1S Luc cells (J22.9 - ISY -D265C - 30.2115 : ; J22.9 reached 80 mm " , mice ( 8 animals per group ) were treated ISY -MMAF : 1 ); ( C ) time course 24 h to 120 h of cytotoxic intravenously with single doses ( 2 or 4 mg/ kg ) of J22.9 potential on KMS- 11 cells (J22.9 - ISY- D265C - 30.2115 : ISY -D265C -30.2115 or J22.9 - ISY -D265C - 30.1699 , or were J22.9 - ISY -MMAF : treated with PBS as control .. Tumor volumes were moni [0059 ] FIG . 27 shows the results from a study of the tored twice per week . efficacy of J22.9 - ISY - D265C -30.2115 in comparison with [0054 ] FIG . 22 shows the antitumor activity in mouse the monomethyl auristatin F derivative J22.9- ISY- MMAF NCI- H929 subcutaneous xenograft in repeated dose appli and Carfilzomib in a disseminating Xenograft model (MM . cation : Female CB - 17 Scid mice were subcutaneously 1S Luc ). inoculated with 5x10 NCI- H929 cells. Once tumor volume reached 80 mm » , mice ( 9 animals per group ) were treated DETAILED DESCRIPTION OF THE intravenously either once per week (1x /week ) , every two INVENTION weeks (1x /2weeks ) or every 3 weeks (1x /3weeks ) with [ 0060] Before the present invention is described in detail J22.9 - ISY- D265C - 30.2115 ( doses 0.25 mg/kg , 0.5 mg/ kg or below , it is to be understood that this invention is not limited 1 mg/kg ) or were treated with PBS as control. In addition , to the particular methodology, protocols and reagents 2 mg/ kg doses were applied twice per week ( 2x / week ) . described herein as these may vary. It is also to be under Tumor volumes were monitored twice per week . stood that the terminology used herein is for the purpose of [ 0055 ] FIG . 23 shows the antitumor activity in dissemi describing particular embodiments only , and is not intended nating MM.1S Luc xenograft model: ( A ) Female SCID to limit the scope of the present invention which will be beige mice were intravenously injected with 1x107 MM.1S limited only by the appended claims. Unless defined other Luc cells . When reaching a mean flux of 1.5x106-1x107 wise , all technical and scientific terms used herein have the ( 10-14 days after implantation ) 8-10 mice per group were same meanings as commonly understood by one of ordinary treated with single doses ( 2 and 4 mg/ kg ) of J22.9- ISY skill in the art . D265C - 30.1699 , J22.9- ISY -D265C -30.2115 or PBS as con [ 0061] Particularly , the terms used herein are defined as trol. Luciferase activity was monitored by non - invasive described in “ A multilingual glossary of biotechnological bio - imaging twice weekly, 10 minutes after administration terms: (IUPAC Recommendations) ” , Leuenberger, H. G. W , of luciferin ( 10 ul/ g body weight ). ( B ) Individual tumor Nagel, B. and Kolbl, H. eds. ( 1995 ), Helvetica Chimica growth curves for each animal are depicted . Individual Acta , CH -4010 Basel, Switzerland ). Growth curves are shown for groups treated with 2 mg/ kg [0062 ] Throughout this specification and the claims which (solid line ) and 4 mg/ kg (dashed line ) of J22.9 -ISY -D265C follow , unless the context requires otherwise, the word 30.2115 . Data indicate that only one animal from each “ comprise” , and variations such as “ comprises” and “ com depicted group shows elevated tumor signals significantly prising ” , will be understood to imply the inclusion of a above background level . stated integer , composition or step or group of integers or [0056 ] FIG . 24 shows the antitumor activity in a dissemi steps , while any additional integer, composition or step or nating MM.1S Luc xenograft model : dose reduction : ( A ) group of integers , compositions or steps may optionally be Female SCID beige mice were intravenously injected with present as well, including embodiments , where no additional 1x107 MM.1S Luc cells . When reaching a mean flux of integer , composition or step or group of integers, composi 1.5x106-1x10 ? (10-14 days after implantation ) 8-10 mice tions or steps are present. In such latter embodiments , the per group were treated with single doses (0.1 , 0.3 , 1 and 2 term “ comprising ” is used coterminous with “ consisting of " . mg/ kg ) of J22.9 - ISY -D265C - 30.2115 or PBS as control. [0063 ] Several documents are cited throughout the text of Luciferase activity was monitored by non - invasive bio this specification . Each of the documents cited herein ( in imaging twice weekly , 10 minutes after administration of cluding all patents , patent applications, scientific publica luciferin ( 10 ul/ g body weight) . ( B ) Individual tumor growth tions, manufacturer's specifications, instructions, GenBank curves for each animal are depicted . Growth curves are Accession Number sequence submissions etc. ) , whether US 2019/0328899 Al Oct. 31 , 2019 6 supra or infra , is hereby incorporated by reference in its [ 0070 ] In the context of the present invention , the term entirety to the extent possible under the respective patent “ amatoxin ” includes all cyclic peptides composed of 8 law . amino acids as isolated from the genus Amanita and [0064 ] The present invention will now be further described in Wieland , T. and Faulstich H. (1978 ) , which described . In the following passages different aspects of the comprise the specific positions according to (i ) (i.e. where invention are defined in more detail. Each aspect so defined the indole moiety of the amino acid residue tryptophan has may be combined with any other aspect or aspects unless no oxygen - containing substituent at position 6 ', particularly clearly indicated to the contrary . In particular, any feature indicated as being of particular relevance or advantageous where position 6 ' carries a hydrogen atom ) and ( ii ) ( i.e. in may be combined with any other feature or features indi which the thioether sulfoxide moiety of naturally occurring cated as being of particular relevance or advantageous. amatoxins is replaced by a sulfide ), and furthermore [ 0065 ] The present invention is based on a combination of includes all chemical derivatives thereof; further all semi different advantageous elements and features and in particu synthetic analogues thereof; further all synthetic analogues lar on the unexpected observation that a variant form of an thereof built from building blocks according to the master amatoxin conjugated to an anti -BCMA antibody based on structure of the natural compounds (cyclic , 8 amino acids) , antibody J22.9 , wherein the antibody and the amatoxin are further all synthetic or semisynthetic analogues containing linked by a cleavable linker shows an increased stability non -hydroxylated amino acids instead of the hydroxylated under stress conditions , particularly in human plasma, and amino acids, further all synthetic or semisynthetic ana an improved therapeutic index . logues, in each case wherein any such derivative or analogue [0066 ] Thus, in one aspect the present invention relates to carries at least the positions (i ) and (ii ) mentioned above and a conjugate comprising (a ) an amatoxin comprising (i ) an is functionally active by inhibiting mammalian RNA poly amino acid 4 with a 6 '- deoxy position ; and ( ii ) an amino acid merase II. 8 with an S -deoxy position ; (b ) a BCMA -binding moiety [ 0071] Functionally , amatoxins are defined as peptides or comprising the variable domains of the heavy chain accord depsipeptides that inhibit mammalian RNA polymerase II . ing to SEQ ID NO : 1 and the light chain according to SEQ Preferred amatoxins are those with a functional group ( e.g. ID NO : 2 of antibody J22.9 - ISY, and (c ) a protease- cleav a carboxylic group or carboxylic acid derivative such as a able linker , wherein said BCMA - binding moiety is attached carboxamide or hydroxamic acid , an amino group , a to said linker via the thiol group of the cysteine residue at hydroxy group , a thiol or a thiol- capturing group ) that can be position 265 in the antibody heavy chain . reacted with linker molecules or target- binding moieties as [ 0067 ] In a particular embodiment, the BCMA - binding defined above. Amatoxins which are particularly suitable for moiety comprises a heavy chain constant region comprising the conjugates of the present invention are di- deoxy variants a D265C mutation . of a -amanitin , ß -amanitin , y -amanitin , k -amanitin , amanul [0068 ] In a particular embodiment, said protease - cleav lin , or amanullinic acid , or mono - deoxy variants of amanin , able linker is a self - immolative linker . amaninamide , y - amanin , or y - amaninamide as shown in [0069 ] In a particular embodiment, the conjugate is the FIG . 1 as well as salts , chemical derivatives, semisynthetic conjugate according to Formula I. analogues, and synthetic analogues thereof.
Formula I ?? .
HO
HN HN
HO NH
NH
O
-S-( J22.9 - ISY -D265C ) US 2019/0328899 Al Oct. 31 , 2019 7
[0072 ] An amatoxin variant comprising (i ) an amino acid protein of the complement cascade. This interaction is 4 with a 6 '- deoxy position ; and (ii ) an amino acid 8 with an dependent on glycosylation of the antibody at position S - deoxy position was first mentioned in Zhou et al. , Chem Asn297 in the heavy chain constant region . Glycans on IgG1 BioChem 16 (2015 ) 1420-1425 , as one of four diastereom display some heterogeneity but core structure is usually a ers being obtained in a total synthesis approach for amatox fucosylated bi- antennary structure with varying levels of ins . However , the fact that such variants are more stable sialic acid at the antennae. Numerous investigations have under stress conditions in plasma and result in a reduced shown that glycosylation affects binding affinity and loss of degree of cross- linked products (as shown in FIG . 2 ) was not glycosylation completely abrogates binding altogether . publicly known at the effective filing date of the present Absence of glycosylation disrupts the structural integrity of application in December 2016 (see patent application WO the Fc region which is required for optimal binding to the 2017/149077 published on Sep. 8 , 2017 ). Fcy receptor ( for review see Hayes et al. , 2014 ) . However , [ 0073 ] In a particular embodiment, the conjugate of the absence of core fucose of IgG results in improved binding to present invention has a purity greater than 90 % , particularly Fcy receptor and enhanced antibody - dependent cellular greater than 95 % . cytotoxicity (ADCC ) . [0074 ] Monoclonal murine antibody J22.9 has been [0080 ] Binding of the antibody to effector cells or comple obtained using standard hybridoma technology from ment results in ADCC mediated by natural killer cells or C57BL / 6 mice immunized with purified human BCMA complement- dependent cytotoxicity (CDC ) . J22.9 -xi is able extracellular domain (residue 1-54 ) N - terminally fused to to induce strong ADCC and CDC on BCMA- positive glutathione S - transferase (GST ) . Due to instability of the MM.1S cells when mixed with isolated Fc- bearing effector, hybridomas , variable regions of light and heavy chain were peripheral blood mononuclear cells (PBMCs ) from healthy amplified and cloned upstream of the human kappa and donors . IgG1 constant domain , respectively , resulting in chimeric [0081 ] Humanized and chimeric antibody variants bind to J22.9 -xi antibody (Oden et al. , 2015 ) . BCMA -expressing cell lines. No differences in binding [ 0075 ] Antibody J22.9 -xi was humanized based on characteristics were observed for J22.9 - ISY and J22.9 - FSY , sequence alignments and data obtained from crystal struc whereas binding affinity of humanized variant J22.9 - H was ture in order to identify mutations that would potentially much lower. disrupt the binding to BCMA . Briefly , J22.9 -xi Fab fragment [0082 ] In the context of the present invention , the term was generated from full - length antibody by incubation with " purity ” refers to the total amount of conjugates being pepsin and combined with purified 54 amino acid residue present . A purity of greater than 90 % , for example , means BCMA extracellular domain . Isolated complexes were used that in 1 mg of a composition comprising a conjugate of the for crystallization studies and J22.9 - xi BCMA binding present invention , there are more than 90 % , i.e. more than epitopes were analysed in detail (WO2014 / 068079 , 900 pg , of such conjugate . The remaining part, i.e. the WO2015 / 166073 , Oden et al ., 2015 , Marino et al. 2016 ) . impurities may include unreacted starting material and other [0076 ] Based on these analyses , various combinations of reactants , solvents , cleavage products and / or side products . fully humanized gene cassettes were identified and [0083 ] In a particular embodiment, a composition com expressed as full- length antibody . J22.9 -H is the fully prising a conjugate of the present invention comprises more humanized version of J22.9 - xi. J22.9 - ISY and J22.9 - FSY than 100 mg of such conjugate . Thus, trace amount of a are fully humanized versions containing in addition muta conjugate of the present invention that arguably may be tions intended to remove potentially detrimental post - trans present in complex preparations of conjugates of the prior lational modification (PTM ) motifs . art, e.g. from partial reduction of naturally occurring sul [0077 ] J22.9 -xi binds to purified BCMA and also detects foxides, are explicitly excluded . BCMA on human MM cell lines MM.IS , NCI- H929 , [ 0084 ] As used herein , a first compound ( e.g. an antibody ) OPM - 2 and RPMI- 8226 . No binding was detected on is considered to “ specifically bind ” to a second compound BCMA - negative cells . In addition , flow cytometry analyses ( e.g. an antigen , such as a target protein ), if it has a revealed that cells from bone marrow of MM patients were dissociation constant Kj to said second compound of 100 detectable using J22.9 -xi.The affinity of J22.9 -xi to BCMA uM or less , particularly 50 uM or less , particularly 30 uM or is very high with a mean Kd of 54 uM as determined using less , particularly 20 uM or less , particularly 10 uM or less , plasmon resonance (Oden et al. , 2015 ). particularly 5 uM or less, more particularly 1 uM or less , [ 0078 ] BCMA activates nuclear factor KB (NFKB ) path more particularly 900 nM or less , more particularly 800 nM ways and triggers signals important for survival ofMM and or less, more particularly 700 nM or less , more particularly plasma cells through interaction with APRIL and / or BAFF. 600 nM or less, more particularly 500 nM or less , more Affinity of J22.9 to BCMA was shown to be very high and particularly 400 nM or less , more particularly 300 nM or exceeds that of April by 300- and of BAFF by 30.000 - fold less, more particularly 200 nM or less , even more particu (Bossen and Schneider 2006 ) . J22.9 - xi efficiently blocks larly 100 nM or less, even more particularly 90 nM or less, binding of APRIL and BAFF to BCMA . In addition , J22 . even more particularly 80 nM or less , even more particularly 9 -xi interferes with APRIL - induced NFKB activation by 70 nM or less, even more particularly 60 nM or less, even blocking phosphorylation of IKB kinase ( IKK ) and subse more particularly 50 nM or less , even more particularly 40 quent IKBa degradation , leading to reduced DNA - binding nM or less , even more particularly 30 nM or less, even more activity of NFKB . In summary , J22.9- xi interferes with particularly 20 nM or less , and even more particularly 10 nM APRIL - induced NFKB activation in BCMA- positive NCI or less . H929 cells (Oden et al. , 2015 ). [0085 ] The term " antibody or antigen binding fragment [0079 ] Cytotoxic activity of IgG1 antibodies is achieved thereof ”, as used herein , refers to immunoglobulin mol through interaction of the IgG1 with Fcy receptor (FcyR ) on ecules and immunologically active portions of immuno effector cells ( e.g. natural killer cells ) or with the Clq globulin molecules , i.e. molecules that contain an antigen US 2019/0328899 Al Oct. 31, 2019 8 binding site that immunospecifically binds the antigen [ 0090 ] As used herein , “ treat ” , “ treating” or “ treatment ” of BSMA . Thus , the term " antigen -binding fragments thereof" a disease or disorder means accomplishing one or more of refers to a fragment of an antibody comprising at least a the following: ( a ) reducing the severity of the disorder ; ( b ) functional antigen -binding domain . In a particular embodi limiting or preventing development of symptoms character ment, functional antigen - binding domain comprises the vari istic of the disorder (s ) being treated ; ( c ) inhibiting worsen able domains of the heavy chain according to SEQ ID NO : ing of symptoms characteristic of the disorder( s ) being 1 and the light chain according to SEQ ID NO : 2 of antibody treated ; ( d ) limiting or preventing recurrence of the disorder J22.9 - ISY. Also comprised are immunoglobulin - like pro ( s ) in patients that have previously had the disorder ( s ) ; and teins that are selected through techniques including, for ( e ) limiting or preventing recurrence of symptoms in example , phage display to specifically bind to the antigen patients that were previously symptomatic for the disorder BSMA. The immunoglobulin molecules of the invention can (s ). be of any type ( e.g. , IgG , IgE , IgM , IgD , IgA and IgY ), class [0091 ] As used herein , the treatment may comprise admin ( e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass istering a conjugate or a pharmaceutical composition of immunoglobulin molecule . “ Antibodies and antigen according to the present invention to a patient, wherein binding fragments thereof” suitable for use in the present “ administering” includes in vivo administration , as well as invention include, but are not limited to , polyclonal, mono administration directly to tissue ex vivo , such as vein grafts . clonal , monovalent , bispecific , heteroconjugate , multispe cific , human , humanized ( in particular CDR - grafted ) , deim [0092 ] In particular embodiments , a therapeutically effec munized , or chimeric antibodies , single chain antibodies tive amountof the conjugate of the present invention is used . ( e.g. scFv ) , Fab fragments , F ( ab ') 2 fragments , fragments [ 0093 ] A “ therapeutically effective amount” is an amount produced by a Fab expression library , diabodies or tetrabod of a therapeutic agent sufficient to achieve the intended ies (Holliger P. et al ., Proc Natl Acad Sci USA . 90 (1993 ) purpose. The effective amount of a given therapeutic agent 6444-8 ), nanobodies , anti- idiotypic (anti - Id ) antibodies ( in will vary with factors such as the nature of the agent, the cluding , e.g., anti- Id antibodies to antibodies of the inven route of administration , the size and species of the animal to tion ), and epitope- binding fragments of any of the above. receive the therapeutic agent, and the purpose of the admin [ 0086 ] In some embodiments the antigen -binding frag istration . The effective amount in each individual case may ments are human antigen -binding antibody fragments of the be determined empirically by a skilled artisan according to present invention and include , but are not limited to , Fab , established methods in the art . Fab ' and F ( ab ') 2, Fd , single - chain Fvs ( scFv ) , single - chain [ 0094 ] In another aspect the present invention relates to antibodies , disulfide- linked Fvs (dsFv ) and fragments com pharmaceutical composition comprising an amatoxin prising either a VL or VH domain . Antigen -binding antibody according to the present invention , or a conjugate of the fragments , including single -chain antibodies , may comprise present invention of an amatoxin with a target -binding the variable domain ( s ) alone or in combination with the moiety , and further comprising one or more pharmaceuti entirety or a portion of the following : hinge region , CL , cally acceptable diluents , carriers , excipients , fillers , bind CH1, CH2, and CH3 domains. Also included in the inven ers , lubricants , glidants , disintegrants , adsorbents ; and /or tion are antigen -binding fragments also comprising any preservatives. combination of variable domain ( s ) with a hinge region , CL , [0095 ] “ Pharmaceutically acceptable ” means approved by CH1, CH2 , and CH3 domains. In a particular embodiment, a regulatory agency of the Federal or a state government or the antibody or antigen binding fragment thereof comprises listed in the U.S. Pharmacopeia or other generally recog a heavy chain constant region comprising a D265C muta nized pharmacopeia for use in animals , and more particu tion . larly in humans. [0087 ] Antibodies usable in the invention may be from [0096 ] In particular embodiments , the pharmaceutical any animal origin including birds and mammals . Particu composition is used in the form of a systemically adminis larly, the antibodies are from human , rodent (e.g. mouse , rat, tered medicament. This includes parenterals , which com guinea pig , or rabbit) , chicken , pig , sheep , goat , camel , cow , prise among others injectables and infusions . Injectables are horse , donkey, cat , or dog origin . It is particularly preferred formulated either in the form of ampoules or as so called that the antibodies are of human or murine origin . As used ready - for -use injectables , e.g. ready -to -use syringes or herein , “ human antibodies” include antibodies having the single -use syringes and aside from this in puncturable flasks amino acid sequence of a human immunoglobulin and for multiple withdrawal. The administration of injectables include antibodies isolated from human immunoglobulin can be in the form of subcutaneous ( s.c.) , intramuscular libraries or from animals transgenic for one or more human ( i.m.) , intravenous (i.v. ) or intracutaneous ( i.c.) application . immunoglobulin and that do not express endogenous immu In particular, it is possible to produce the respectively noglobulins, as described for example in U.S. Pat . No. suitable injection formulations as a suspension of crystals , 5,939,598 by Kucherlapati & Jakobovits . solutions , nanoparticular or a colloid dispersed systems like, [0088 ] In a second aspect, the present invention relates to e.g. hydrosols . a pharmaceutical composition comprising the conjugate of [0097 ] Injectable formulations can further be produced as the present invention . concentrates , which can be dissolved or dispersed with [ 0089 ] In a third aspect, the present invention relates to the aqueous isotonic diluents . The infusion can also be prepared conjugate of the present invention , or the pharmaceutical in form of isotonic solutions, fatty emulsions, liposomal composition of the present invention , for use in the treatment formulations and micro -emulsions . Similar to injectables, of cancer in a patient , particularly wherein the cancer is infusion formulations can also be prepared in the form of selected from the group consisting of multiple myeloma, concentrates for dilution . Injectable formulations can also be diffuse large B -cell lymphoma (DLBCL ), and chronic lym applied in the form of permanent infusions both in in -patient phocytic leukemia (CLL ), particularly multiple myeloma. and ambulant therapy, e.g. by way of mini- pumps . US 2019/0328899 A1 Oct. 31 , 2019 9
[0098 ] It is possible to add to parenteral drug formula agents can be added as further adjuvants. For the production tions , for example , albumin , plasma, expander, surface of lyophilisates scaffolding agents like mannite , dextran , active substances, organic diluents , pH - influencing sub saccharose , human albumin , lactose , PVP or varieties of stances, complexing substances or polymeric substances, in gelatine can be used . particular as substances to influence the adsorption of the target -binding moiety toxin conjugates of the invention to proteins or polymers or they can also be added with the aim EXAMPLES to reduce the adsorption of the target- binding moiety toxin conjugates of the invention to materials like injection instru [0102 ] In the following, the invention is explained in more ments or packaging -materials , for example , plastic or glass . detail by non - limiting examples : [0099 ] The amatoxins of the present invention comprising a target- binding moiety can be bound to microcarriers or Example 1 nanoparticles in parenterals like, for example , to finely dispersed particles based on poly (meth )acrylates , polylac tates, polyglycolates , polyamino acids or polyether ure Synthesis of Amatoxin -Linker HDP 30.2115 thanes . Parenteral formulations can also be modified as depot preparations, e.g. based on the " multiple unit prin [ 0103] Linkage of antibody to toxin can occur either to ciple ” , if the target - binding moiety toxin conjugates of the cysteine or lysine residues , a specific tag or non - natural invention are introduced in finely dispersed , dispersed and amino acids of the antibody . In order to obtain homogenous suspended form , respectively , or as a suspension of crystals ADC products with specific DAR of 2 , site -specific conju in the medicament or based on the “ single unit principle ” if gation to cysteine in combination with genetic engineered the target- binding moiety toxin conjugate of the invention is Thiomabs is preferred . Key attributes of the linker include enclosed in a formulation , e.g. in a tablet or a rod which is the requirement to be stable in plasma in order to prevent the subsequently implanted . These implants or depot medica uncontrolled release of the toxic payload into the circulation , ments in single unit and multiple unit formulations often and on the other hand the toxin needs to be released within consist of so called biodegradable polymers like e.g. poly the cell after internalization of the ADC upon target binding . esters of lactic acid and glycolic acid , polyether urethanes , Release of the toxin inside the target cell can occur either via polyamino acids, poly (methacrylates or polysaccharides. cleavable or non -cleavable linkers. Cleavable linkers can be [0100 ] Adjuvants and carriers added during the production cleaved from the payload via a variety of mechanism of the pharmaceutical compositions of the present invention including acidic degradation as consequence of lower pH formulated as parenterals are particularly aqua sterilisata inside the cell compared to circulation and protease cleavage ( sterilized water ), pH value influencing substances like, e.g. by the protease cathepsin B or thiol- disulfide exchange organic or inorganic acids or bases as well as salts thereof, attributed to the more reductive intracellular environment. buffering substances for adjusting pH values , substances for Non -cleavable linkers require complete lysosomal pro isotonization like e.g. sodium chloride, sodium hydrogen teolytic degradation generating a payload with a charged carbonate , glucose and fructose, tensides and surfactants , lysine or cysteine residue from the antibody. respectively , and emulsifiers like , e.g. partial esters of fatty acids of polyoxyethylene sorbitans (for example , Tween® ) [0104 ] The main advantage of non - cleavable compared to or, e.g. fatty acid esters of polyoxyethylenes ( for example , cleavable linkers is their increased plasma stability . While it Cremophor® ) , fatty oils like , e.g. peanut oil , soybean oil or is apparent that activities of ADCs containing non -cleavable castor oil , synthetic esters of fatty acids like , e.g. ethyl linkers are less predictable , cleavable linkers raise the con oleate , isopropyl myristate and neutral oil ( for example , cerns of non - specific cytotoxicity . Miglyol® ) as well as polymeric adjuvants like , e.g. gelatine , dextran , polyvinylpyrrolidone , additives which increase the [0105 ] However, in previous experiments , particularly in solubility of organic solvents like , e.g. propylene glycol, vitro experiments , with numerous different linkers , ADCs ethanol, N , N - dimethylacetamide, propylene glycol or com containing non -cleavable linkers have been shown to be less plex forming substances like, e.g. citrate and urea , preser toxic than ADCs with a cleavable linker , and the majority of vatives like, e.g. benzoic acid hydroxypropyl ester and amanitin -based ADCs based on cleavable linkers were more methyl ester, benzyl alcohol, antioxidants like e.g. sodium effective compared to non - cleavable linker constructs . Thus , sulfite and stabilizers like e.g. EDTA . a cathepsin B - cleavable linker has been chosen for the drug [0101 ] When formulating the pharmaceutical composi substance . tions of the present invention as suspensions in a preferred [0106 ] The A - amanineamide- linker - toxin HDP 30.2115 is embodiment thickening agents to prevent the setting of the synthesized by a multistep approach using solid phase target -binding moiety toxin conjugates of the invention or, peptide synthesis whereas the bicyclic octapeptide is ini tensides and polyelectrolytes to assure the resuspendability tially assembled in a linear fashion . Starting point is a of sediments and /or complex forming agents like, for example , EDTA are added . It is also possible to achieve hydroxyproline resin immobilisation , followed by C - termi complexes of the active ingredient with various polymers . nal coupling of dihydroxyisoleucin (HDP 30.0477 ) . The Examples of such polymers are polyethylene glycol, poly remaining six amino acids are coupled by Fmoc strategy . styrene , carboxymethyl cellulose, Pluronics or polyethyl The first cyclisation (right hand ring ) occurs upon acidic ene glycol sorbit fatty acid ester. The target -binding moiety cleavage from resin following the ‘ Savige - Fontana ’mecha toxin conjugates of the invention can also be incorporated in nism . To make this happen , Tryptophan is incorporated in its liquid formulations in the form of inclusion compounds e.g. oxidized form , ‘HPI (HDP 30.0079 ), as final amino acid . with cyclodextrins. In particular embodiments dispersing The second ring is formed by macrolactamisation using US 2019/0328899 A1 Oct. 31 , 2019 10 moderate to high dilution . The linker compound (HDP Step 1 : 4 -Hydroxy -pyrrolidine - 1,2 -dicarboxylic acid 30.2109 ) is synthesized in six linear steps and is finally 2 -allyl ester 1- (9H - fluoren - 9 - ylmethyl) ester (HDP introduced under standard coupling conditions, after depro 30.0013 ) tection . [0110 ] OH 1. Synthesis of Synthetic Dideoxy Precursor Molecule K
[0107 ] The synthesis of the dideoxy precursor molecule K 1. Cs2CO3 is described in WO 2014/009025 in Example 5.5 . 85 % MeOH RT 2. AllBr K DMF RT
Com . Source 353.38 C20H19NO5 NH HN
NH N
HO
HDP 30.0013 393.44 C23H23NOS [ 0108 ] Compound K may be deprotected by treatment [0111 ] FmocHypOH ( 10.0 g , 28.3 mmol) was suspended in 100 ml 80 % MeOH and Cs2CO3 (4.6 g , 14.1 mmol ) was with 7 N methanolic NHz solution ( 3.0 ml) and stirring added . The suspension was stirred at 50 ° C. for 30 minutes overnight. until complete dissolution . The reaction mixture was con centrated to dryness and resolved in 100 ml DMF. Allyl bromide ( 1.6 mi, 3.6 g , 29.7 mmol) was added dropwise and 2. Synthesis of Synthetic Dideoxy Precursor HDP 30.2105 the reaction was stirred over night at RT. DMF was distilled off and the residue dissolved in tert -butylmethyl ether. [ 0109 ] An alternative dideoxy precursor molecules com Precipitates were filtered and the clear solution was prising a COOH group instead of the carboxamide group poundabsorbed was on purifiedCelite prior on 220column g Silicagel chromatography with an . nThe -hexane com/ at amino acid 1 can be synthesized (HDP 30.1895 ) and ethyl acetate gradient. deprotected to result in HDP 30.2105 . [0112 ] Yield : 11.5 g , 100 % Step 2 : Resin Loading (HDP 30.0400 ) HO [0113 ]
HO NH HN THP - Resin PPTS 80 ° C. NH
HO HDP 30.0013 HDP 30.2105 393.44 C23H22NOS US 2019/0328899 Al Oct. 31, 2019 11
-continued [0114 ] HDP 30.0013 (5.0 g , 14.1 mmol) , pyridinium 4 -toluenesulfonate ( 1.33 g , 5.3 mmol) were added to a suspension of 1,3 -dihydro - 2H -pyran - 2- yl- methoxymethyl resin (5.0 g, 1.0 mmol/ g THP -resin ) in 40 ml dichloroethane . PS The reaction was stirred at 80 ° C. overnight. After cooling the resin was filtered and extensively washed with dichlo 0."!!! roethane , dimethylformamide , acetonitrile , dichloromethane and tert -butylmethylether . [0115 ] Loading was 0.62 mmol/ g ( determined by UV spectroscopy of the fluorene methyl group after deprotec tion ) . HDP 30.0040 Step 3 : Solid Phase Precursor Synthesis (HDP 393.44 30.1894 ) C23H23NOS [0116 ]
PS 10
HDP 30.0400 393,44 C23H23N05
PS ZT 0.1111
LZ
HN 7
OH HDP 30.1894 1447,69 C74H94N ,0195 US 2019/0328899 Al Oct. 31, 2019 12
Resin Pre - Treatment: minutes. The resin was washed with N -methyl - 2 - pyrroli [0117 ] HDP 30.0400 (0.5 g , 0.31 mmol) was treated with done (Note : No deprotection after coupling of the final N ,N -dimethylbarbituric acid (483 mg, 3.1 mmol) and amino acid ). Pd (PPh3 )4 (69 mg, 0.06 mmol) . The resin was shaken over [0121 ] All other amino acids were coupled following the night at RT. Thereafter the resin was extensively washed above protocol, weightings are shown below : with dichloromethane , N -methyl - 2 -pyrrolidone , acetoni trile , dichloromethane and tert -butylmethyl ether and dried [0122 ] 0.63 mmol, 498 mg Fmoc Asp (OAII ) OH under reduced pressure . [ 0123 ] 0.63 mmol, 738 mg Fmoc Cys ( Tri )OH Coupling Procedure : [0124 ] 0.63 mmol, 375 mg Fmoc GlyOH [0118 ] All reactants and reagents were dissolved in dichlo [0125 ] 0.63 mmol, 445 mg FmocIleOH romethane / N - methyl- 2 - pyrrolidone containing 1 % Triton [0126 ] 0.63 mmol, 375 mg Fmoc GlyOH X100 (Solvent A ) . [0127 ] 0.38 mmol, 242 mg N - Boc -HPIOH (HDP 30.0079 ) [0119 ] HDP 30.0477 (257 mg, 0.38 mmol ) was dissolved in 3.0 ml Solvent A and treated with 3.0 ml of a 0.2 N [0128 ] 4,5 -Diacetoxy - 2 - amino - 3 -methyl -pentanoic acid solution PyBOP (333 mg, 0.63 mmol, 2.0 eq ) , 3.0 ml of a 0.2 tert- butyl ester , hydrochloride (HDP 30.0477) was synthe N solution HOBt ( 130 mg, 0.63 mmol, 2.0 eq ) and 439 pl sized as described in WO 2014/009025 . DIEA (4.0 eq ) . The reaction was heated to 50 ° C. for 8 [0129 ] N - Boc -HPIOH (HDP 30.0079 ) was prepared minutes by microwave irradiation (20 W , CEM microwave according to Zanotti , Giancarlo ; Birr Christian ; Wieland reactor ) and was washed with N -methyl - 2 -pyrrolidone after Theodor; International Journal of Peptide & Protein coupling . Research 18 (1981 ) 162-8 . Deprotection : Step 4 : HDP 30.1895 [0120 ] Deprotection was performed by addition of 6.0 ml 20 % piperidine in N -methyl - 2 - pyrrolidone at 50 ° C. for 8 [0130 ]
O NI
PS N NH
Steps
OH HDP 30.1894 1447 , 69 C 74H94N , O10S US 2019/0328899 Al Oct. 31 , 2019 13
-continued
HN N NH HN
NH ??"
HO HDP 30.1895 1012 , 11 C46H61N90158 phase chromatography and finally quenched with 100 ul Elimination[0131 ] The from resin Resin was andshaken B -Ring with Formation10 ml trifluoroacetic water . The mixture was concentrated by reduced pressure acid /dichloromethane 50:50 (v /v ) plus 10 % methanol for 30 and re -dissolved in 1-2 ml methanol. Precipitation of the min and finally eluted into a 50 ml flask . The resin was product was performed by dropwise addition to 20 ml washed twice with methanol ( 10 ml each ) . The combined diethyl ether. The precipitate was washed twice with diethyl ether and dried under reduced pressure. The next step was eluates2-4 ml weremethanol concentrated. The methanolic in vacuum solutionand re -was suspended dropped in performed without further purification . twice into 50 ml cold diethyl ether for peptide precipitation , After centrifugation the precipitate was washed with diethyl [0135 ] Mass spectrometry: [ M + Na] " , 1034.6 etherprecipitate ( 2 times was ) and solubilized dried under in approx reduced. 4-5 pressure ml methanol. The white (0.5 Ester Deprotection: ml per 100 mg) and purified by preparative reverse phase column chromatography . Approximately 100 mg crude pre romethane[0136 ] To, diethylbarbituricthe crude cyclisation acid (22.3 product mg, 118.52.5 mlumol dichlo) and cipitate were purified per run . Fractions were analyzed by Pd (PPhz ). ( 27 mg, 23.7 umol) were added . The reaction was mass spectrometry , combined and methanol distilled off stirred at RT overnight. The reaction can be monitored by under reduced pressure. The aqueous phase was freeze RP - HPLC . After complete conversion , the mixture was dried . added dropwise to 20 ml cooled diethyl ether and the [ 0132 ] Yield : 24.4 mg, 23.7 pmol precipitate washed twice with diethyl ether . After drying at (0133 Mass spectrometry : [ M + H ] * , 1030.5 reduced pressure the precipitate was dissolved in methanol A - Ring Formation matography( 1.0 ml) and. purified by preparative reversed phase chro [0137 ] Yield : 15.0 mg [in0134 25 ml ) dimethylformamideThe above freeze dried and intermediatetreated with diphenylphoswas dissolved [0138 ] Mass spectrometry : [ M + H ] " , 972,3 ; [ M + Na] * , phorylazide (63 ul, 1185 umol, 5 eg ) and diisopropylethyl 994.5 amine ( 201 ul, 1185 pmol, 5 eq ) . The reaction was stirred Step 5 : HDP 30.2105 overnight ( 20 hours ). Conversion was monitored by reverse [0139 ]
N
NH HN 7N NH3 in MeOH RT NH HO
HO HDP 30.1895 1012 , 11 C46H61N ,015S US 2019/0328899 Al Oct. 31 , 2019 14
-continued OH N
HO NH HN
NH N NH
HO HDP 30.2105 887,97 C39H53N ,O13S [0140 ] HDP 30.1895 ( 15.0 mg, 15.3 pmol) was dissolved in 7 N methanolic NH , solution (3.0 ml) and stirred over -continued night. Conversion was checked by mass spectrometry . After complete conversion the reaction was concentrated in vacuum , suspended in 80 % tert -butanol and lyophilized . fmoc Product was purified by preparative HPLC . [0141 ] Yield : 12.1 mg ( 0142 ] Mass spectrometry : [ M + H ] " , 888,0 ; [ M + Na ] * , 910.2 3. Synthesis of Synthetic Dideoxy Precursor HDP 30.2115 Fmoc - Val- OSu (HDP 30.1343 ) [ 0143 ] 436 , 47 ?? . C24H24N206
HO [0146 ] This compound is prepared according to R. A. HN Firestone et al, U.S. Pat. No. 6,214,345 . Fmoc - Val- OH (20.24 g ; 59.64 mmol) and N -hydroxysuccinimide (6.86 HN g = 1.0 eq . ) in tetrahydrofuran ( 200 ml) at 0 ° C. were treated with N , N - dicyclohexylcarbodiimide ( 12.30 g ; 1.0 eq. ) . The HO.11 N ZI mixture was stirred at RT under argon atmosphere for 6 h NH and then the solid dicy bhexyl urea (DCU ) by duct was filtered off and washed with THF and the solvent was removed by rotavap . The residue was dissolved in 300 ml NH dichloromethane , cooled in an ice bath for 1 h and filtered again to remove additional DCU . The dichloromethane was evaporated and the solid foam ( 26.51 g ) was used in the next step without further purification . Step 2 : Fmoc - Val- Ala - OH (HDP 30.1414 ) [0144 ] dideoxy precursor molecule comprising a thiol reactive group with cleavable linker can be synthesized from [0147 ] example 2 product in 7 steps as follows: Step 1 : Fmoc - Val- OSu (HDP 30.1343 ) [ 0145 ] NH2 HO DSC fmoc DIPEA HO . fmoc THF L - Alanine 89,09 C3H -NO2 L - Valine Fmoc- Val- OSu (HDP 30.1343 ) 339 , 39 436 , 47 C20H21N04 C24H24N206 US 2019/0328899 Al Oct. 31, 2019 15
-continued -continued boc
fmoc fmoc HO N
Fmoc - Val- Ala -OH (HDP 30.1414 ) HDP 30.1713 410 , 47 614 , 75 C23H26N205 C35H42N406
[0148 ] Step 2 product is prepared in analogy to P. W. [0150 ] Step 2 product HDP 30.1414 ( 1.76 g ; 4.28 mmol ) Howard et al . US 2011/0256157 . A solution of L -alanine and 4 -[ ( N -Boc ) aminomethyl ] aniline ( 1.00 g ; 1.05 eq. ) were (5.58 g ; 1.05 eq .) and sodium hydrogen carbonate (5.51 g ; dissolved in 26 ml abs . tetrahydrofuran . 2 -Ethoxy - N 1.1 eq . ) in 150 ml water was prepared and added to a ( ethoxycarbonyl) -1,2 -dihydroquinoline ( EEDQ 1.11 g ; 1.05 solution of HDP 30.1343 ( 26.51 g ; max . 59.6 mmol) in 225 eq. ) was added and the mixture was stirred at RT, protected ml tetrahydrofuran . The mixture was stirred for 50 h at RT . from light. With ongoing reaction a gelatinous matter is After consumption of starting material the solution was formed from the initially clear solution . After 40 h the partitioned between 240 ml of 0.2 M citric acid and 200 ml reaction mixture was diluted with 25 ml of tert -butylmethyl of ethyl acetate . The aqueous layer was separated and ether (MTBE ) and stirred for 1 h . Subsequently the precipi extracted with ethyl acetate ( 3x200 ml) . The combined tation is filtered off with suction , washed with MTBE and organic layers were washed with water and brine (300 ml dried in vacuo to 2.30 g (85 % yield ) of a white solid . each ) dried (MgSO4 ) and the solvent was evaporated to [0151 ] " H NMR (500 MHz, DMSO -d6 ) d 9.87 (s , 1H ), approx . 200 ml. Pure product precipitated at this time and 8.11 ( d , J = 7.1 Hz, 1H ) , 7.88 ( d , J = 7.5 Hz, 2H ) , 7.74 ( q , was filtered off. The mother liquor was evaporated to J = 8.4 , 7.9 Hz, 2H ) , 7.51 ( d , J = 8.2 Hz, 2H ) , 7.45-7.23 ( m , dryness and the residue was stirred 1 h with 100 mlMTBE 7H ), 7.17 (d , J = 8.3 Hz , 2H ), 4.44 (p , J = 7.0 Hz, 1H ), to result additional crystalline material . The two crops of 4.36-4.17 ( m , 3H ) , 3.96-3.89 ( m , 1H ) , 2.01 (hept , J = 6.9 Hz , product were combined to 18.01 g ( 74 % ) white powder . 1H ) , 1.39 ( s , 9H ) , 1.31 ( d , J = 7.1 Hz , 3H ) , 0.90 ( d , J = 6.8 Hz, (m.p .: 203-207 ° C.) 3H ), 0.87 ( d , J = 6.8 Hz, 3H ) . [0152 ] 13C NMR (126 MHz, DMSO - d6 ) 8 170.84 , 170.76 , 156.04 , 155.63, 143.77 , 143.69, 140.60 , 137.41, 134.99 , MS [M + Na ] + found: 410.94 ; calc .: 411.19 ( C23H27N205 ) 127.50 , 127.26 , 126.93 , 125.22 , 119.95 , 118.97 , 77.60 , ( ESI+ ) [M + Na] * found : 433.14 ; calc.: 433.17 (C23H2 - N205) 65.62, 59.95 , 48.86 , 46.62 , 42.93 , 30.28 , 28.16 , 19.06 , [2M + H ] * found : 842.70 ; calc .: 843.36 ( C46H52N Na010 ) 18.10 , 18.03 . Step 4 : H - Val - Ala - PAB - NHBoc (HDP 30.1747 ) Step 3 : Fmoc - Val- Ala -PAB -NHBoc (HDP 30.1713) [ 0153 ] [ 0149 ] boca . HNED2 fmoc DMF boc
+ HDP 30.1713 614 , 75 C35H42N406 boc NH2 N-( tert . - Butoxycarbonyl) 4 -aminobenzylamine NH2 222 , 29 C12H18N202 HDP 30.1747 392, 50 EEDO C20H32N404 fmoc HO THF IZ [0154 ] Step 3 compound HDP 30.1713 ( 1.230 g , 2.00 mmol) was placed in a 100 ml flask and dissolved in 40 ml HDP 30.11414 dimethylformamide (DMF ) . Diethyl amine ( 7.5 ml) was 410 , 47 added and the mixture was stirred at RT. The reaction was C23H26N205 monitored by TLC ( chloroform /methanol / HOAC 90 : 8 : 2 ) . After consumption of starting material ( 30 min ) the volatiles US 2019/0328899 A1 Oct. 31, 2019 16 were evaporated and the residue was co - evaporated with 40 [0157 ] ' H NMR ( 500 MHz, DMSO -do ) 8 9.75 ( s, 1H ) , ml fresh DMF to remove traces of diethyl amine. The crude 8.09 ( d , J = 7.1 Hz, 1H ) , 7.98 ( d , J = 8.4 Hz, 1H ) , 7.52 ( d , J = 8.6 product was used without further purification for the next Hz , 2H ) , 7.29-7.23 ( m , 1H ) , 7.16 ( d , J = 8.5 Hz , 2H ) , 6.98 ( s , step . 2H ) , 4.39 ( p , J = 7.1 Hz, 1H ) , 4.13 (dd , J = 8.4 , 6.7 Hz, 1H ) , 4.06 ( d , J = 6.1 Hz, 2H ) , 3.67-3.56 ( m , 2H ) , 2.49-2.41 ( m , 2H ), 1.96 ( h , J = 6.8 Hz , 1H ) , 1.39 ( s , 9H ), 1.30 ( d , J = 7.1 Hz , MS [MH ] " found : 393.26 ; calc .: 393.25 ( C20H33N404) 3H ), 0.86 ( d , J =6.8 Hz, 3H ), 0.82 ( d, J= 6.8 Hz, 3H ) . 13C ( ESI+ ) [M + Na] * found : 415.35 ; calc .: 415.23 ( C20H32N NaO4) NMR ( 126 MHz, DMSO -do ) d 170.80 , 170.63 , 170.60 , 169.72 , 155.65 , 137.45 , 134.94 , 134.44 , 127.26 , 118.95 , [2M + H ] * found : 785.37 ; calc .: 785.49 ( C40H65N208) 77.62 , 57.71 , 48.92 , 42.95 , 33.96 , 33.64 , 30.17 , 28.17 , 19.02 , 18.06 , 17.82 . Step 5 : BMP - Val - Ala -PAB - NHBoc (HDP 30.2108 ) Step 6 : BMP -Val - Ala - PAB - NH2 (HDP 30.2109 ) [ 0155 ] 10158 ] boc BMPS boca M = 266 , 21 NH2 DIPEA TFA DMF HDP 30.1747 392, 50 HDP 30.2108 543 , 63 C20H32N404 C27H37N507 H2N
boc Qytt HDP 30.2109 443 , 51 C22H29N505 TFA salt : 557,53 sienHDP 30.2108 543, 63 C27H37N507 [0159 ] Step 5 product HDP 30.2108 (400 mg, 736 pmol) was dissolved in 4,000 ul trifluoroacetic acid and stirred for [0156 ] Crude step 4 product HDP 30.1747 (max 2.00 2 min . Subsequently the volatiles were evaporated at RT and mmol) was dissolved in 40 ml DMF, 3-( maleimido ) propi the remainders were co - evaporated twice with 4,000 ul onic acid N -hydroxysuccinimide ester (BMPS 532 mg; 1.0 toluene . The residue was dissolved in 5,000 ul 1,4 -dioxane / eq. ) and N - ethyldiisopropylamine (510 ul, 1.5 eq .) were water 4 : 1 , solidified in liquid nitrogen and freeze -dried : 410 added and the mixture was stirred 3 h at RT After consump mg (quant . ) colorless powder. tion of starting material HDP 30.1747 ( TLC : chloroform / methanol/ HOAC 90 :8 :2 ) the volatiles were evaporated and MS [ M + Na] + found : 415.35 ; calc .: 466.21 ( C22H29NaOs ) the residue is stirred with 50 mlMTBE until a fine suspen ( ESI+ ) [ 2M + H ] * found : 887.13 ; calc.: 887.44 (C44H59N10010 ) sion was formed ( 1 h ). The precipitate was filtered off with suction , washed with MTBE and dried . The crude product ( 1.10 g ) was dissolved in 20 ml dichloromethane /methanol [0160 ] ' H NMR ( 500 MHz, DMSO -do ) d 9.89 ( s , 1H ) , 1: 1 , kieselgur (15 g ) was added and the solvents were 8.13 ( d , J= 6.9 Hz, 1H ), 7.99 (d , J= 8.2 Hz, 1H ), 7.66-7.60 (m , stripped off. The solid material was placed on top of an 80 2H ) , 7.41-7.34 ( m , 2H ) , 6.98 ( s , 2H ) , 4.39 (p , J = 7.1 Hz, 1H ) , g silica gel column and eluted with a linear gradient of 4.11 (dd , J = 8.2 , 6.6 Hz , 1H ) , 3.97 ( 9 , J = 5.6 Hz , 2H ) , 0-10 % methanol in dichloromethane . Product fractions were 3.69-3.58 ( m , 2H ), 2.49-2.40 (m , 2H ) , 1.96 (h , J= 6.8 Hz, combined and evaporated to 793 mg ( 73 % over two steps ) 1H ) , 1.32 ( d , J = 7.1 Hz, 3H ) , 0.86 ( d , J = 6.8 Hz , 3H ) , 0.83 ( d , amorphous solid . J = 6.7 Hz, 3H ) . [0161 ] 13C NMR (126 MHz , DMSO - do ) d 171.24 , 170.78 , MS [ M + Na ] found : 566.24 ; calc .: 566.26 (C27H37Na07 ) 170.72 , 169.85 , 158.12 (9 , J = 33.2 Hz , TFA ), 158.25, 157.99 , ( ESIT ) 157.73 , 139.19 , 134.53 , 129.45 , 128.52 , 119.02 , 116.57 ( q , J = 296.7 Hz, TFA ), 57.78 , 49.08 , 41.90 , 34.00 , 33.68 , 30.21 , 19.07 , 18.16 , 17.76 . US 2019/0328899 Al Oct. 31 , 2019 17
Step 6 : HDP 30.2115 [0162 ]
?? ,
HO SIL HN N HN HDP 30.2109 TBTU DIEA HO.lt DMF NH RT
OH HDP 30.2105 887 , 97 C39H53N9013S ?? .
HO
HN HN
HO NH
NH
N N N H
HDP 30.2115 ???? 1313 , 47 C61H80N14017S
[0163 ] HDP 30.2105 (15.0 mg, 16.5 umol) were treated Example 2 with 429 ul of a 0.1 M solution of HDP 30.2109 (25.2 umol , 1.5 eq ), 492 ul of 0.1 M TBTU ( 25.2 umol, 1.5 eq ) and 492 ul of 0.2 M DIEA (49.1 umol, 3.0 eq ) at RT. The reaction Properties of Constructs Based on HDP 30.2105 was monitored by RP - HPLC . After completion the reaction was quenched with 100 ul H20 stirred for 15 minutes and [0166 ] Various experiments were performed to compare injected onto a preparative RP -HPLC . the properties of constructs based on di- deoxy amanitin [0164 ] Yield : 12.2 mg , 56 % derivative HDP 30.2105 with those of other amanitin vari [0165 ] Mass spectrometry : 1313.2 [ M + H ] * , 1335.5 ants . The results of these experiments are shown in FIGS. 2 [ M + Na ] * to 14 . US 2019/0328899 Al Oct. 31, 2019 18
Example 3 TABLE 1 EC50 [ M ] Generation and Expression of Thiomab Antibody Compound on THP - 1 J22.9 - ISY - D265C T - D265C - 30.0643 ( stable linker; lysine coupling ) 1.9 x 10-8 T - D265C - 30.0880 ( stable linker ; cysteine coupling ) 4.0 x 10-8 (0167 ] Conjugation of the antibody with linker and toxin T -D265C - 30.1699 ( cleavable linker; cysteine coupling ) 2.0 x 10-8 can occur to lysine or cysteine residues , resulting in highly T - A118C - 30.0643 ( stable linker; lysine coupling ) 2.8 x 10-10 variable drug -antibody ratio (DAR ). Since potency and T - A118C - 30.0880 ( stable linker ; cysteine coupling ) 7.0 x 10-10 toxicity is strongly influenced by DAR , homogeneity and T- A118C - 30.1699 ( cleavable linker; cysteine coupling ) 4.4 x 10-10 comparability of the ADC with predictable DAR is favour Her- 30.0880 (wild - type ) 4.6 x 10-10 able . Antibodies with engineered reactive cysteine residues Her- 30.0643 (wild -type ) 1.2 x 10-10 ( thiomabs) allow for site - specific conjugation and therefore , amino acid aspartic acid at position 265 has been exchanged [0171 ] Results from characterization of the chimeric and to cysteine (D265C ) . Thus, the resulting antibody variant sequence -optimized variants of J22.9 revealed improved contains two introduced cysteines at each chain of the Fc binding properties of the sequence optimized variants J22. region , which serves as coupling site for the toxin - linker 9 - ISY and J22.9- FSY to BCMA compared to J22.9 - H , with compound and allows production of ADCs with DAR = 2. no obvious difference for ISY- or FSY - variant. Since use of [0168 ] Sequence modifications in the Fc- region of the plasmids expressing J22.9 - ISY antibody variant resulted in antibody can have dramatic influence in linking antibody improved titers when transiently expressed in cell culture, mediated immune responses with cellular effector functions , since residues in the Fc - region are responsible for interac variant J22.9- ISY was chosen for conjugation and further tion with IgG Fcy receptor (FcyR ) . Interactions of IgG with evaluation . the FcyR play crucial role in cellular effector functions [0172 ] The nucleic acid coding sequence for the heavy including release of inflammatory mediators , endocytosis of chain of monoclonal antibody J22.9 -ISY -D265C was immune complexes , antibody - dependent cellular cytotoxic obtained from the heavy chain sequence of humanized ity ( ADCC ) and complement- dependent cytotoxicity antibody J22.9 - ISY (see WO 2014/068079 and WO 2015 / (CDC ) . Preserving these effector functions in an ADC might 166073 ) as described in WO 2016/142049 . In addition to the contribute to the anti- tumor activity of the ADC . However , exchange D265C , J22.9 -ISY - D265C additionally contains a this assumption might prove to be incorrect as these effector functions are generally weak and , importantly , might com mutation R214K (see SEQ ID NO : 1 ) . pete with ADC internalization , reduce or abolish their spe [0173 ] In order to produce amanitin -conjugate J22.9 - ISY cific toxin - related activity and increase off- target toxicity D265C - 30.2115 , antibody J22.9 - ISY - D265C was transiently due to ADC uptake in antigen -negative cells via Fc - receptor. expressed in Expi293FTM cells co - transfected with plasmids Several ADCs currently in pre -clinical and clinical devel for the heavy and light chain . In an additional approach , opment are based on IgG2 and IgG4 which are very inef antibody J22.9 - ISY -D265C was produced in CHO cells ficient in these effector functions ( Trail , 2013 ; Peters and stably transfected with plasmids expressing heavy and light Brown , 2015 ). chain . The antibody was purified from the cell culture [0169 ] Detailed mapping of binding sites on human IgG1 supernatant using protein A chromatography followed by gel to FcyRI, FcyRII , FcyRIII and to neonatal Fc receptor filtration . Antibody produced transiently from Expi293FTM ( FcRn ) revealed a crucial role of aspartic acid at position or from stable CHO cells are comparable conjugation to 265. Replacement of aspartic acid in IgG2 and IgG1 com payload , in binding to BCMA and cytotoxicity on BCMA pletely abolished interaction with FcyR (Baudino et al. , expressing cells . 2008 ; Shields et al. , 2001) . [0174 ] In order to test if amino acid exchange in Thiomab [0170 ] The efficiency of antibody - induced effector func J22.9 -ISY -D265C did not interfere with binding to BCMA, tions in Trastuzumab ( Trademark Herceptin® ) and corre binding of J22.9 - ISY and J22.9 - ISY -D265C to BCMA sponding thiomab antibodies with amino acid exchanges at expressing cells was compared . The binding property of the position 265 (D265C ) or 118 ( T118C ) for control were engineered thiomab J22.9 - ISY -D265C to BCMA - positive tested on a macrophage cell line THP - 1 . This monocytic cell NCI- H929 and MM.1S - Luc cells was identical to J22.9 - ISY line expresses multiple FcyRs and allows for testing anti antibody ( see FIG . 15 ) . body -mediated phagocytosis ( Ackerman et al. 2011) . Cyto toxicity on THP- 1 cells was reduced by at least two orders Example 4 of magnitude with thiomab D265C compared to thiomab A118C , containing a cysteine mutation at position A118 not reducing the effector function , and non -engineered antibody. Synthesis of Conjugate J22.9 -ISY - D265C - 30.2115 This effect of reduced cytotoxicity with thiomab engineered at amino acid 265 is not due to conjugation of the toxin to [0175 ] Conjugation of HDP 30.2115 to 10 mg J22.9 - ISY the engineered cysteine , since , in contrast to T- D265C - 30 . D265C 1699 containing a cleavable linker conjugated to the engi [0176 ] 10 mg Thiomab J22.9- ISY -D265C in PBS buffer neered cysteine , in the case of T - D265C - 30.0643 a stable will be used for conjugation to HDP 30.2115 . linker is conjugated to lysine residues of the antibody. Thus , antibody - dependent effector functions were surprisingly [0177 ] Adjust antibody solution to 1 mM EDTA : highly reduced due to D265C exchange in thiomabs (see [0178 ] 2 ml antibody solution ( 10.0 mg) +20 ul 100 mm Table 1) . EDTA , pH 8.0 US 2019/0328899 A1 Oct. 31, 2019 19
[0179 ] Amount antibody : 10 mg = 6.8x10-8 mol [0198 ] Adjust protein concentration to 5.0 mg/ ml ( 3.4x [0180 ] Uncapping of cysteines by reaction of antibody 10-5 M ) and bring to sterile conditions by filtration . Store at with 40 eq . TCEP : 4 ° C. [0181 ] 2 ml antibody solution (6.8x10-8 mol) +54.5 ul 50 Example 5 mM TCEP solution ( 2.72x10- mol) Characterization of Drug Substance [0182 ] Incubate for 3 h at 37 ° C. on a shaker. J22.9 - ISY -D265C - 30.2115 [0183 ] Two consecutive dialyses at 4 ° C. in 2.0 11xPBS , [0199 ] 1. Production and Release Testing 1 mM EDTA , pH 7.4 in a Slide - A - Lyzer Dialysis Cassette [0200 ] In order to characterize an ADC variant based on 20'000 MWCO , first dialysis ca. 4 h , second dialysis HDP 30.2115 , it was compared to a variant based on HDP overnight 30.1699 (see Table 1) .
OH
HO H HN
HN `8 * 1110 HO. 111 NH IZ
HN NH NH2
O
HDP 30.1699
[0184 ] Concentrate to ca. 4.0 ml using Amicon Ultra [0201 ] HDP 30.1699 contains amanitin from natural Centrifugal Filters 50'000 MWCO . source in contrast to fully synthetic amanitin derivative HDP [0185 ] Oxidation by reaction of antibody with 20 eq . 30.2115 . Due to the chemical synthesis , there are the fol dehydroascorbic acid ( dhAA ) : lowing differences between the two compounds : HDP [0186 ] ca. 2 ml antibody solution ( 6.8x10-8 mol) +27.2 ul 30.2115 contains a thioether bridge and a core tryptophan moiety , whereas HDP 30.1699 contains a sulfoxide bridge fresh 50 mM dhAA solution ( 1.36x10- mol) and a 6 -hydroxytryptophan moiety . The absence of 6 -hy [0187 ] Incubate for 3 h at RT on a shaker . droxytryptophan in HDP 30.2115 requires linkage of the [0188 ] Conjugation with amanitin using 4 eq . HDP antibody to the aspartic acid in contrast to HDP 30.1699 30.2115 and quenching with 25 eq . N -acetyl - L -cysteine : were 6 -hydroxytryptophan is used for the linkage . In both [0189 ] Solubilize 0.7 mg HDP 30.2115 in 70 ul compounds a cathepsin B cleavable linker was used . DMSO - 10 ug/ ul [0202 ] Antibody J22.9 - ISY -D265C was conjugated to [0190 ] ca. 2 ml antibody solution (= 9.5 mg; 6.54x10-8 compounds HDP 30.1699 and HDP 30.2115 using mol) +34.4 ul HDP 30.2115 (= 344 ug ; 2.62x10-7 mol) . maleimido - chemistry as described above and tested for [0191 ] Incubate 1 h at RT. aggregates, DAR and product quality in analytical SEC [0192 ] Quench by addition of 16.4 ul 100 mM N - acetyl HPLC , Mass spectrometry , SDS- Page and Western Blot. L - cysteine ( 1.64x10- mol) . [0203 ] Test results from B16-0040 and B16-0049 are [ 0193] Incubate 15 min at RT (or overnight at 4 ° C.) . presented exemplarily in FIG . 16 . [0194 ] Purify reaction mix with 1x PD - 10 columns equili [0204 ] Stability of J22.9 - ISY- D265C - 30.2115 and J22.9 brated with 1x PBS, pH 7.4 . Identify protein - containing ISY -D265C - 30.1699 was tested following incubation at 37 ° fractions with Bradford reagent on parafilm and bring C. for 0 , 4 , and 10 days in PBS , human , cynomolgus or protein -containing fractions together . mouse plasma. In contrast to J22.9 - ISY - D265C - 30.1699 , [ 0195 ] Dialysis of antibody solution at 4 ° C. overnight in Western Blot analyses surprisingly revealed a particularly 2.0 I PBS , pH 7.4 and Slide- A - Lyzer Dialysis Cassettes high stability throughout the time course of the experiment 20'000 MWCO . for J22.9 - ISY- D265C -30.2115 in PBS or plasma (see FIG . [0196 ] Determination of protein concentration by UV 17 ) . Thus the ADC - containing the synthetic amanitin -de spectra (absorption at 280 nm ). rivative showed superior stability compared to the ADC with [0197 ] Determination of DAR by LC - ESI -MS - analysis . amanitin from natural source . US 2019/0328899 A1 Oct. 31 , 2019 20
[0205 ] Plasma stability of both ADC variants was con [0206 ] Cytotoxicity potentials of both ADCs were tested firmed in a cell- based cytotoxicity assay on BCMA- positive on five BCMA - positive MM cell lines (NCI - H929 , MM.IS NCI- H929 cells ( see Table 2 ) after incubation for 0 , 4 and 10 Luc, MM.IS , U266B1, and OPM - 2 ) and one BCMA -nega days in PBS , human , cynomolgus or mouse plasma . Incu tivecytotoxic control activity cell line in the(CCRF picomolar -CEM )range. Both with ADCs slightly show supehigh bation in PBS or plasma over up to 10 days has almost no rior toxicity of J22.9 - ISY -D265C - 30.1699 on some cell influence on the cytotoxic potential of J22.9- ISY -D265C lines . Cytotoxicity of J22.9 - ADCs is strongly dependent on 30.2115. In contrast, the cytotoxicity and thus the stability of BCMA -expression levels on cell surface with no toxicity J22.9 - ISY -D265C - 30.1699 was clearly reduced after incu observed in non -BCMA expressing control cells . In addí bation in human , cynomolgus or mouse plasma, as already cellstion , withcorrelate both amanitinwith poor - ADCs BCMA low -EC50 expression values (on see OPM Table -2 observed in Western Blot analyses ( see FIG . 18 ) . 3 / FIG . 19 and Table 4 / FIG . 20 ) . TABLE 2
human cynomolgus mouse PBS EC50 plasma plasma plasma pH 7.4 J22.9 - ISY day 0 2.8 x 10 -10 2.5 x 10-10 1.2 x 10 -10 2.4 x 10-10 D265C day 4 2.7 x 10-10 2.5 x 10-10 1.8 x 10-10 2.4 x 10-10 30.2115 day 10 1.0 x 10-9 2.9 x 10-10 6.0 x 10-10 4.1 x 10-10 J22.9 - ISY day 0 1.8 x 10-10 2.0 x 10-10 8.6 x 10-11 1.6 x 10-1 D265C day 4 3.7 x 10-7 2.1 x 10-9 2.0 x 10-9 3.6 x 10-10 30.1699 day 10 6.0 x 10-10
TABLE 2 CD269 CD138 protein protein J22.9 - ISY (Western (Western binding Cell line blot) blot ) ( FACS ) MM.1S Multiple myeloma cell ( B ++ +++ ++ lymphoblast) established from the bone marrow of a 55 - year old man with plasma cell leukemia at relapse after chemotherapy MM.1S MM.1S cells expressing ++ +++ ++ Luc luciferase NCI Multiple myeloma cell ( B +++ ++ +++ H929 lymphoblast ) cell established from a 62 -year - old white woman with myeloma at relapse U266B1 Multiple myeloma cell (B ++ +++ + lymphoblast ) cell derived from peripheral blood of a 53 - year old patient with an IgE myeloma OPM - 2 Multiple myeloma cell ( B + +++ + lymphoblast ) cell established from the peripheral blood of a 56 - year -old woman with multiple myeloma ( IgG lambda ) in leukemic phase (relapse , terminal) KMS - 11 Multiple myeloma cell ( B + +/ + lymphoblast) cell established from patient with multiple myeloma RPMI Multiple myeloma cell (B +/ + + 8226 lymphoblast ) cell established from the peripheral blood of a 61- year- old man with multiple myeloma CCRF T lymphoblastoid line - ??? established from the peripheral blood of a 3 - year -old Caucasian girl with acute lymphoblastic leukemia (ALL ) at relapse ( control cell line ) US 2019/0328899 A1 Oct. 31 , 2019 21
TABLE 4 EC50 [ M ]: NCI- H929 MM.1S Luc MM.1S U266B1 OPM - 2 J22.9 - ISY 7.0 x 10-11 6.5 x 10-11 1.7 x 10-10 7.3 x 10-11 7.9 x 10-9 D265C 30.1699 J22.9 - ISY 9.7 x 10-11 2.3 x 10-10 2.9 x 10-10 1.4 x 10-10 1.4 x 10-7 D265C 30.2115
Example 6 mg/ kg . Already single dose application of 0.3 mg/kg resulted in complete tumor eradication ( see FIG . 24 ) . In - Vivo Pharmacological Activity in Multiple Myeloma Xenograft Model Example 7 1. Subcutaneous Xenograft Model Non -Human Primate (NHP ) Tolerability Study [ 0212 ] J22.9 - ISY - D265C - 30.2115 and J22.9 - ISY - D265C Single Dosing 30.1699 were assessed for a dose -escalating tolerability [ 0207 ] Antitumor activity of J22.9 - ISY - D265C - 30.2115 study in cynomolgus monkeys . Groups of 3 animals were and J22.9 - ISY -D265C - 30.1699 was tested in a subcutaneous injected with J22.9 - ISY -D265C - 30.1699 at days 1 (0.3 NCI- H929 (see Table 3 for details of cell line ) mouse mg/ kg ) , 22 ( 1 mg/ kg ) , and 44 ( 3 mg/ kg ), or with J22.9 - ISY xenograft model . D265C - 30.2115 at days 1 (0.3 mg/ kg ) , 21 ( 1 mg/ kg ) , and 42 , [0208 ] Single treatment with 2 mg/kg doses resulted in 64 , 84,106 ( 3 mg/ kg each time point ). Animals were moni initial response followed by regrowth of tumor in 7 of 8 mice tored over time for biochemical and haematological blood for J22.9 - ISY - D265C -30.1699 and 6 of 8 mice for J22.9 parameters ( see FIG . 25 ), urinalysis , body weight, food ISY -D265C - 30.2115 . At 4 mg/ kg doses, complete tumor consumption, clinical signs and mortality . In addition , blood remission was reached in 8 of 8 mice for J22.9 - ISY -D265C samples were collected for pharmacokinetic studies. At the 30.1699 and 7 of 8 mice for J22.9 - ISY -D265C - 30.2115 and end of the experiments tissue samples are used for histo mice stayed tumor free for more than 100 days (the time pathological examinations. course of the experiment) . At doses of 2 mg/ kg J22.9 - ISY [0213 ] Up to 3 mg/ kg doses of both compounds were well D265C - 30.2115 showed slightly superior anti -tumor effi tolerated with no signs of kidney damage by serum param cacy compared to J22.9 -ISY -D265C - 30.1699 . No statisti eters and unaffected body weight and food consumption . cally significant body weight reduction was observed , Doses of 3 mg/ kg resulted in increased but transient levels except for J22.9 - ISY -D265C - 30.1699 at 4 mg/ kg doses ( see of liver enzymes ( ALT, AST ) and the unspecific inflamma FIG . 21) . tory marker LDH . Repeated dosing of 3 mg/ kg J22.9 - ISY D265C - 30.2115 did not result in further increase of liver Repeated Dosing enzymes or LDH . [0209 ] The efficacy of drug substance J22.9 - ISY- D265C Example 7 30.2115 was further evaluated in a repeated dose setting in the subcutaneous NCI- H929 xenograft model . Compared to Cytotoxic Potential of J22.9 - ISY- D265C - 30.2115 in the single dose application described above , doses were Comparison to Monomethyl Auristatin F Derivative reduced to 1,0.5 and 0.25 mg/ kg and applied either once per [0214 ] Cytotoxicity potentials of J22.9 - ISY -D265C -30 . week ( 1x / week ) , every two weeks ( 1x / 2 weeks ) or every 2115 was compared to the interchain conjugate J22.9 - ISY three weeks ( 1x / 3 weeks ). Repeatedly applied doses of 1 MMAF on two BCMA -positive MM cell lines (MM.1S Luc , mg/ kg already resulted in tumor regression , and with and KMS- 11) . Both ADCs show high cytotoxic activity in repeated 2 mg/ kg dosing complete tumor remission could be the picomolar range after 96 h with slightly superior toxicity observed (see FIG . 22 ). of J22.9 -ISY -MMAF (see FIG . 26A ) . The efficacy of both compounds was compared in a timeline experiment on the 2. Intravenous Xenograft Model two cell lines. Cytotoxicity was measured after 24 , 46 , 72 , Single Dosing 96 , and 120 hours (see FIGS. 26B + 26C ). J22.9 - ISY -D265C 30.2115 unfolded the full cytotoxicity after 96 and 120 [0210 ] The antitumor activity of J22.9 - ISY -D265C -30 . hours , whereas the MMAF conjugate showed already after 1699 and J22.9 - ISY -D265C - 30.2115 was further tested in a 48 and 72 hours a full blown cytotoxic potential. This disseminating intravenous MM.1S Luc xenograft model at indicates that the amanitin derivative needs more time to doses of 2 and 4 mg/ kg . Both compounds showed very unfold its cytotoxic potential and to drive the cells into effective and comparable antitumor activity with complete apoptosis due to its mode of action than MMAF . tumor remission for more than 100 days (see FIG . 23A ). Only 1 of 10 mice for both compounds showed slight Example 8 regrowth of tumor after initial response for over 90 days ( see FIG . 23B ). Efficacy of J22.9 -ISY -D265C - 30.2115 in a [0211 ] In the disseminating MM.1S Luc xenograft model Disseminating Xenograft Model doses of drug substance J22.9 - ISY -D265C - 30.2115 were [0215 ] The antitumor activity of J22.9 - ISY- D265C -30 . further reduced to single dose application of 1 , 0.3 and 0.1 2115 was compared to the interchain conjugate J22.9 - ISY US 2019/0328899 Al Oct. 31 , 2019 22
MMAF in a disseminating intravenous MM.1S Luc xeno [0234 ] Rickert R C et al . Signaling by the tumor necrosis graft model at doses of 1 and 4 mg/ kg . The MMAF factor receptor superfamily in B - cell biology and disease. conjugate showed a very fast initial response after five days Immunol Rev. 2011 November ; 244 ( 1 ) : 115-33 at both doses and a regrowth of the tumor after 20 and 50 days, respectively . J22.9 - ISY - D265C -30.2115 in contrast [0235 ] Sanchez et al.Serum B -cell maturation antigen is showed a slower initial response after 10 days at both doses , elevated in multiple myeloma and correlates with disease but a longer period until regrowth of the tumor after 50 and status and survival. Br J Haematol. 2012 September ; 100 days , respectively (see FIG . 27 ) . Thus, the amatoxin 158 (6 ): 727-38 conjugate showed a better efficacy in terms of tumor free [0236 ] Shaffer A L et al. IRF4 addiction in multiple survival despite its slower onset of action (see FIGS . 26 ( A ) myeloma. Nature . 2008 Jul. 10 ; 454 ( 7201) :226-31 to (C )) . [ 0237 ] Shields R L et al . High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma REFERENCES RII , Fc gamma RIII , and FcRn and design of IgG1 [0216 ] Ackerman M E et al. A robust , high - throughput variants with improved binding to the Fc gamma R. J Biol assay to determine the phagocytic activity of clinical Chem . 2001 Mar. 2 ; 276 ( 9 ) :6591-604 antibody samples. J Immunol Methods. 2011 Mar. 7 ; [0238 ] Trail P A. Antibody drug conjugates as cancer 366 ( 1-2 ): 8-19 . therapeutics. Antibodies 2013 , 2 ( 1 ), 113-129 . [0217 ] Baudino L et al . Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and [0239 ] Wieland T, Faulstich H. Amatoxins , phallotoxins, IgG2b Fc -associated effector functions. J Immunol. 2008 phallolysin , and antamanide: the biologically active com Nov. 1 ; 181( 9 ) :6664-9 . ponents of poisonous Amanita mushrooms. CRC Crit Rev [ 0218 ] Belucci R et al. Graft -versus -tumor response in Biochem . 1978 December; 5 ( 3 ) : 185-260 patients with multiple myeloma is associated with anti body response to BCMA , a plasma- cell membrane recep [0240 ] Zhao L , May J P , Blanc A , Dr. Dietrich D J , tor. Blood . 2005 May 15 ; 105 ( 10 ) : 3945-5 Loonchanta A , Matinkhoo K , Pryyma A , and Perrin D M. [ 0219 ] Bird J M et al. Guidelines for the diagnosis and Synthesis of a Cytotoxic Amanitin for Biorthogonal Con management of multiple myeloma 2011. Br J Haematol. jugation . ChemBioChem 16 ( 2015) 1420-1425 2011 July ; 154 (1 ): 32-75 [0220 ] Bossen C , Schneider P. BAFF , APRIL and their SEQUENCES receptors: structure , function and signaling . Semin Immu nol. 2006 October ; 18 ( 5 ) : 263-75 [0221 ] Chesi M , Bergsagel P L. Advances in the patho [0241 ] genesis of multiple myeloma. Int J Lab Hematol. 2015 May ; 37 Suppl 1 : 108-14 Humanized J22.9 heavy chain J22.9 - ISY - D265C [0222 ] Davis MB , Preston J F. A Conjugate of a -amanitin ( SEQ ID NO : 1 ) : and Monoclonal Immunoglobulin G to Thy 1.2 Antigen is EVOLVESGGG LVQPGGSLRL SCAASGFTFS RYWISWVRQA Selectively Toxic to T Lymphoma Cells . Science 213 (1981 ) 1385-1388 PGKGLVWVGE INPSSSTINY APSLKDKFTI SRDNAKNTLY [ 0223 ] Dimopoulos M Aet al. Current treatment landscape for relapsed and /or refractory multiple myeloma. Nat Rev LOMNSLRAED TAVYYCASLY YDYGDAYDYW GOGTLVTVSS Clin Oncol . 2015 January; 12 ( 1 ) :42-54 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS [0224 ] Dimopoulos M A et al. Pathogenesis and treatment of renal failure in multiple myeloma. Leukemia . 2008 WNSGALTSGV HTFPAVLOSS GLYSLSSVVT VPSSSLGTQT August ; 22 ( 8 ) : 1485-93 [ 0225 ] Hayes J M et al. Glycosylation and Fc receptors . YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG Curr Top Microbiol Immunol. 2014 ; 382 : 165-99 [ 0226 ] Kuehl W M , Bergsagel P L. Molecular pathogen PSVFLFPPKP KDTLMISRTP EVTCVVVCVS HEDPEVKFNW esis ofmultiple myeloma and its premalignant precursor. J Clin Invest. 2012 October , 122 ( 10 ) :3456-63 YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK [ 0227 ] Leukemia . 2009 Jan ;23 ( 1 ): 3-9 EYKCKVSNKA LPAPIEKTIS KAKGQPREPO VYTLPPSREE [ 0228 ] Kyle RA, Rajkumar S V. Multiple myeloma. N Engl J Med . 2004 Oct. 28 ; 351 (18 ) : 1860-73 . MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV [0229 ] Marino S F, Olal D , Daumke O. A complex water network contributes to high -affinity binding in an anti LDSDGSFFLY SKL TVDKSRW QQGNVFSCSV MHEALHNHYT body -antigen interface . Data Brief. 2015 Dec. 19 ; 6 :394-7 QKSLSLSPGK [ 0230 ] Morris P W , Venton D L. Regiospecific amine substitution into a - amanitin with retention of inhibitory Humanized J22.9 light chain ( SEQ ID NO : 2 ) : properties against eukaryotic class II RNA polymerase . EIVMTQSPAT LSVSPGERAT LSCKASQSVE SNVAWYQQKP Int. J. Peptide Protein Res. 21 ( 1983 ) 419-430 [0231 ] Oden et al. Potent anti - tumor response by targeting GQAPRALIYS ASLRFSGIPA RFSGSGSGTE FTLTISSLOS B cell maturation antigen (BCMA ) in a mouse model of EDFAVYYCQQ YNNYPLTFGA GTKLELKRTV AAPSVFIFPP multiple myeloma. Mol Oncol. 2015 August ; 9 ( 7 ) : 1348-5 [ 0232 ] Peters C , Brown S. Antibody -drug conjugates as SDEOLKSGTA SVCLLNNFY PREAKVQWKV DNALQSGNSO novel anti- cancer chemotherapeutics . Biosci Rep . 2015 Jun . 12 ; 35 (4 ). ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG [ 0233 ] Rajkumar S V et al . International Myeloma Work LSSPVTKSFN RGEC ing Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol. 2014 November ; 15 ( 12 ): e538-4 US 2019/0328899 Al Oct. 31, 2019 23
SEQUENCE LISTING
< 160 > NUMBER OF SEQ ID NOS : 2 < 210 > SEQ ID NO 1 < 211 > LENGTH : 450 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Humanized J22.9 heavy chain J22.9 - ISY - D265C < 400 > SEQUENCE : 1 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Ser Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp Gly Gin 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Cys Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr US 2019/0328899 A1 Oct. 31 , 2019 24
- continued
340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
< 210 > SEQ ID NO 2 < 211 > LENGTH : 214 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Humanized J22.9 light chain < 400 > SEQUENCE : 2 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gin Ser Val Glu Ser Asn 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Leu Arg Phe Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 US 2019/0328899 Al Oct. 31 , 2019 25
1. A conjugate according to Formula I comprising ( a ) an consisting of multiple myeloma, diffuse large B -cell lym amatoxin ; (b ) a BCMA -binding moiety comprising (i ) the phoma (DLBCL ) , and chronic lymphocytic leukemia variable domains of the heavy chain according to SEQ ID ( CLL ) . NO : 1 and the light chain according to SEQ ID NO : 2 of 11. A method of treating cancer in a patient, the method antibody J22.9 -ISY , and (ii ) a heavy chain constant region comprising administering to the patient the conjugate of comprising a D265C mutation ; and ( c ) a protease - cleavable claim 3 , wherein the cancer is selected from the group linker , wherein said BCMA- binding moiety is attached to consisting of multiple myeloma, diffuse large B - cell lym said linker via the thiol group of the cysteine residue at phoma (DLBCL ) , and chronic lymphocytic leukemia position 265 in the antibody heavy chain , (CLL ) . wherein said amatoxin is characterized by comprising (i ) 12. A method of treating cancer in a patient, the method an amino acid 4 with a 6' - deoxy position ; and (ii ) an comprising administering to the patient the conjugate of amino acid 8 with an S -deoxyposition . claim 6 , wherein the cancer is selected from the group
Formula I ?? ,
HO 0
HN HN HOUS111 NH NH N
NH
N ?. H -S-( J22.9 - ISY - D265C )
2. The conjugate of claim 1 , wherein said BCMA -binding consisting of multiple myeloma, diffuse large B - cell lym moiety comprises the heavy chain according to SEQ ID NO : phoma (DLBCL ), and chronic lymphocytic leukemia 1 and the light chain according to SEQ ID NO : 2 . (CLL ) . 3. The conjugate of claim 1 , wherein said BCMA- binding 13. A method of treating cancer in a patient, the method moiety is an IgGl. comprising administering to the patient the pharmaceutical 4. A pharmaceutical composition comprising the conju composition of claim 4 , wherein the cancer is selected from gate of claim 1. the group consisting of multiple myeloma , diffuse large 5. A method of treating cancer in a patient, the method B -cell lymphoma (DLBCL ), and chronic lymphocytic leu comprising administering to the patient the conjugate of kemia (CLL ). claim 1, wherein the cancer is selected from the group 14. A method of treating cancer in a patient, the method consisting of multiple myeloma, diffuse large B -cell lym comprising administering to the patient the pharmaceutical phoma (DLBCL ), and chronic lymphocytic leukemia composition of claim 7 , wherein the cancer is selected from ( CLL ) . the group consisting of multiple myeloma, diffuse large 6. The conjugate of claim 2 ,wherein said BCMA -binding B - cell lymphoma (DLBCL ) , and chronic lymphocytic leu moiety is an IgGl. kemia (CLL ). 7. A pharmaceutical composition comprising the conju 15. A method of treating cancer in a patient, the method gate of claim 2 . comprising administering to the patient the pharmaceutical 8. A pharmaceutical composition comprising the conju composition of claim 8 , wherein the cancer is selected from gate of claim 3 . the group consisting of multiple myeloma, diffuse large 9. A pharmaceutical composition comprising the conju B - cell lymphoma (DLBCL ) , and chronic lymphocytic leu gate of claim 6 . kemia (CLL ). 10. A method of treating cancer in a patient, the method 16. A method of treating cancer in a patient, the method comprising administering to the patient the conjugate of comprising administering to the patient the pharmaceutical claim 2 , wherein the cancer is selected from the group composition of claim 9 , wherein the cancer is selected from US 2019/0328899 A1 Oct. 31 , 2019 26 . the group consisting of multiple myeloma, diffuse large B -cell lymphoma (DLBCL ) , and chronic lymphocytic leu kemia (CLL ).