Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism...
Transcript of Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism...
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Producing Homogeneous ADCs with Combination Warheads
Trevor J. Hallam, PhD Chief Scientific Officer
October 15, 2013
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ADCs – State of the Art. . .
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
# drugs / Ab # drugs / Ab # drugs / Ab
Random Lysine Conjugation Cysteine Conjugation: S-S bonds Site Specific Conjugation
Heterogeneity translates to sub-optimal PK, stability and efficacy
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Success is a matter of Design
Efficacy Resistance Safety
Antigen Choice of Epitope Tumor Heterogeneity (inter and intra Healthy tissue Density Antigen mutation or shedding expression Internalization rate Therapy-induced changes
Alterations in apoptotic pathways
Antibody / Tumor Distribution/Disposition Stability Scaffold Fc functionality and PK Non-specific Fc binding
ADCC and CDC activity
Conjugation Specificity Stability
Linkers Cleavable or non-cleavable Immunogenicity Stability Physicochemical props
Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered metabolism Immunogenicity
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Next Generation ADC’s? Homogeneous and Multi-functional….
Multiple Warhead-Linker Combinations • Warheads with distinct mechanisms of
action that are synergistic – lower payloads improves TI
• Eliminate effects of tumor heterogeneity due to variation in cell proliferation
• Overcome resistance due to toxin transport or metabolism
Single or Multiple Antigen Recognition • Broaden target population, range of
indications • Enhance internalization • Eliminate effects of heterogeneous
antigen expression • Overcome resistance due to treatment
induced changes in antigen expression, shedding
• Leverage avidity effects to improve TI
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Sutro: A Cell-Free Synthetic Biology Platform
input (DNA)
+
Energy
Ribosome (Catalyst)
Engineered cell-free extract (E. coli)
output (multi-domain
eukaryotic proteins)
Assembled IgG
+ Accessories*
(AA, proteins, RNAP, metabolites)
*To be incorporated into strain for commercial production
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High Titers at Any Scale
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Rapid Execution of Antibody Discovery Programs
Discover novel an-body fragments using ribosome display and cell free screening
Express an-bodies and fragments with cell free protein synthesis
Reformat an-bodies/ fragments in a whole host of different frameworks
Choose the “Best Lead” based on in vitro and in vivo ac-vity
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Production of Homogeneous ADCs Using Non-Natural AA Incorporation
The Non-Natural Amino Acid Advantage 1. Controlled stability: nnAA chemical space provides
alternatives to cysteine or lysine for creating stable MAb~drug junction
2. Homogeneity: site-specific conjugation using orthogonal chemistries regulates number and location of drugs attached to Mab
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Translation of nnAA-Containing Proteins Enables Site-Specific Conjugation input (DNA)
nnAA
Ribosome (Catalyst)
cell-‐free extract (E. coli)
NNN
mRNA NNN UAA
Stop
tRNA
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Data Driven Design: Production of Many Variants in Hours
Light Chain: 111 Sites Heavy Chain: 133 Sites
SP Number Posi-on TAG site
o Mutate Sites in IgG: Choose nnAA sites using ra-onal design, or just make all of them!
o Produce nnAA IgG: Incorporate nnAA at 100’s of chosen sites
o Conjugate: Conjugate nnAA with appropriate chemistry
o Purify: Separate conjugated IgG away from unincorporated linker-‐warhead
o Test: Assay conjugated IgG’s for binding and cell killing
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0.1 1 10 100 10000
1000
2000
3000
nM
Rela%ve'MFI R61
S63S65R66T69D70T72T74S76S77WT
0.01 0.1 1 10 100 10000
500
1000
1500
2000
2500
nM
MFI
HerceptinSutroceptinE293K334
Rapid Selection of Optimal Sites for Expression, Conjugation, Binding and Killing
SKBR3 Binding Assay Conjugated Variants Compared to Herceptin
Mea
n Fl
uore
scen
ce In
tens
ity
CF-Trastuzumab
Pos Cont ADC DAR=1.6
ug/mL
nM
Cell Killing Assay
Rel
ativ
e C
ell
Viab
ility
nnAA Incorporation Efficiency
nnAA Incorporation and Expression
Conjugation Efficiency (Drug/MAb Ratio) DAR: 0.4 0.7 1.1
Transform of Data 1
0.0001 0.001 0.01 0.1 10
50
100
150
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
ug/ml
T359K360N361Q362K370Y373S375W381S383
N384S136, GYWT HIC
S136 ERB2 ELISA
Transform of Data 1
0.0001 0.001 0.01 0.1 10
50
100
150
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
ug/ml
T359K360N361Q362K370Y373S375W381S383
N384S136, GYWT HIC
S136 ERB2 ELISA
DAR: 1.1 1.5 1.6
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Translation of nnAA-Containing Proteins Enables Site-Specific Conjugation input (DNA)
nnAA
Ribosome (Catalyst)
cell-‐free extract (E. coli)
NNN
mRNA NNN UAA
Stop
tRNA
RF1
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13 13
N
C
RF1
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RF1 M74R RF1 E76K
RF1 E84K RF1 A85R RF1 E87R
RF2 D312
RF2 D104
RF2 L103
RF2 E94
RF2 D92
RF2 G320
RF1 T293R
RF2 M310 RF1 N296K
N
C
Design of OmpT-‐suscep-ble RF1
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Conjugation Efficiency
Azido nnAA Cu Free Click Conjuga-on Chemistry
DBCO Linker-‐Warhead
R
R
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Novel Azido nnAAs with Boosted Conjuga-on Kine-cs
DBCO z
[Azido], mM0 0.5 1 1.5 2 2.5 3
k obs
, sec
-1
0
0.002
0.004
0.006
pAzF
pMeAzF
AB 3562
7x
z Az-‐Me-‐Phe (pAMF) Az-‐nnAA #2 Az-‐Phe
Azido-‐Phe
DBCO
New Chemistry offers Improved Kine-cs, Flexibility
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Novel nnAAs with Boosted Conjugation Kinetics; Best Sites Completely Conjugated in Under 4 hrs
0 5 10 15 200.0
0.5
1.0
1.5
2.0
DA
R
Time (hr)
Site 1 ADCSite 2 ADCSite 3 ADCSite 4 ADCSite 5 ADC
[MAb] 5-‐20uM 5x M excess Linker/warhead Room temp.
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warheads
Multiple warhead payload; single species ADC
• Incorpora-on of mul-ple nnAAs in each IgG LC and HC
• Intractable using cell-‐based systems or wild-‐
type extract due to significant accrued losses in yield
• Enables Mul-ple payloads (4,6,8,10+) of specifically posi-oned warheads/IgG
• Combina-ons of sites screened for:
• nnAA Incorpora-on efficiency/expression • Conjuga-on Efficiency • Stability • PK/Potency in vivo
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Learnings from ADC Specific Site Conjugation Variants
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SKBR3 Cell Killing 1K cells/well5-day Treatment
0.0001 0.01 1 100 100000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
Site 2 DM1 ADC
Site 10 DM1 ADC
Site 1 DM1 ADC
Site 5 DM1 ADC
Site 6 DM1 ADC
Site 4 DM1 ADC
DM1 only
SKBR3 Cell Killing 1K cells/well5-day Treatment
0.0001 0.01 1 100 100000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
Site 2 Gemcitabine ADC
Site 10 Gemcitabine ADC
Site 1 Gemcitabine ADC
Site 5 Gemcitabine ADC
Site 6 Gemcitabine ADC
Site 4 Gemcitabine ADC
SC108 Gemcitabine only
SKBR3 Cell Killing 1K cells/well5-day Treatment
0.0001 0.01 1 100 100000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f co
ntro
l)
Site 2 MMAE ADC
Site 10 MMAE ADC
Site 1 MMAE ADC
Site 5 MMAE ADC
Site 6 MMAE ADC
Site 4 MMAE ADC
AB4546 MMAE only
Site of Conjugation and Killing Activity With Different Cytotoxin Warheads
SKBR3 Cell Killing 1K cells/well5-day Treatment
0.0001 0.01 1 100 100000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
Site 2 MMAF ADC
Site 10 MMAF ADC
Site 1 MMAF ADC
Site 5 MMAF ADC
Site 6 MMAF ADC
Site 4 MMAF ADC
AB4285 MMAF only
MMAF
GEMCITABINE DM1
MMAE
Trastuzumab-‐CF site specific ADC cell killing ac-vity on SKBR3 cells
Free drug
LC Site 2 LC SIte 10
HC SIte 1
HC Site 5HC Site 6
HC Site 4
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Cell Killing 1K cells/wellTreatment of IgG2 HC Site5 ADC
0.0001 0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
KPL-4, 1k, d3
SKBR3, 1k, d5
JIMT-1, 1k, d6
SKOV3., 1k, d6
Cell Killing 1K cells/wellTreatment of IgG2 HC Site 1 ADC
0.0001 0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
KPL-4,1k, d3
SKBR3, 1k, d5
JIMT-1, 1k, d6
SKOV3, 1k, d6
Trastuzumab-CF IgG1 and IgG2 Isotype Drug Conjugates Are Comparable
Cell Killing 1K cells/wellTreatment of IgG1 HC Site 5 ADC
0.0001 0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
KPL-4,1k, d3
SKBR3, 1k, d5
JIMT-1, 1k, d6
SKOV3, 1k, d6
Trastuzumab resistant lines
Trastuzumab sensi-ve lines
IgG1 IgG2
HC Site 5 ADC
Cell Killing 1K cells/wellTreatment of IgG1 HC Site 1ADC
0.0001 0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
nM
Rel
ativ
e C
ell V
iabi
lity
(Cel
lula
r ATP
con
tent
, % o
f con
trol)
KPL-4,1k, d3
SKBR3, 1k, d5
JIMT-1, 1k, d6
SKOV3, 1k, d6
HC Site 1 ADC Trastuzumab-‐CF Single Site MMAF ADC
HC-‐Site DAR KPL-‐4 1k, d3
SKBR3 1k, d5
IC50, nM
IgG2 Site 1 1.5 0.11 0.053
Site 5 1.9 0.071 0.035
IgG1 Site 1 1.5 0.064 0.04
Site 5 1.9 0.076 0.04
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0 10 20 30 40 500
500
1000
1500
2000
Days
Tum
or V
olum
e, m
m3
15mg/kg
10mg/kg
5mg/kg
15mg/kg
15mg/kg
Vehicle
Brentuximab = Adcetris, anti-CD30Trastuzumab = Herceptin, anti Her2CF = Cell free, Agly protein
Brentuximab-CF HC-Site 5 MMAF Conjugate, DAR 1.97
Trastuzumab-CF HC-Site 5Unconjugated
Trastuzumab-CF HC-Site 5 MMAF Conjugate, DAR 1.97
• n =10 each treatment group • All treatments are single dose, i.v. @ t=0 • No significant weight loss observed in any treatment groups
Dose-‐dependent efficacy of a single conjuga-on-‐site homogeneous ADC
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0 10 20 30 40 50 60 700
400
800
1200
1600
Days
Tum
or V
olum
e, m
m3
IgG HC site 1, DAR 1.840
IgG HC site 4, DAR 1.973
IgG HC site 5, DAR 1.970
Vehicle
Trastuzumab,
1x 30mg/kg (t =0), 3x15mg/kg (weekly)
IgG HC site 1
Free Drug, 0.54mg/kg
IgG HC site 6, DAR 1.959
15mg/kg Trastuzumab-CF
MMAF Conjugates
15mg/kgTrastuzumab-CF
Dose equivalence at DAR of 4.0
• n =10 each treatment group • All treatments (except Trastuzumab) are single intra-venous dose @ t=0 • Trastuzumab multiple dose group dosed i.p • No significant weight loss observed in any treatment group
Efficacy is Conjuga-on-‐Site Dependent
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HC-S136 LC-S7SKBR3 3000Cells/well Day 5 treatment
0.001 0.01 0.1 1 100
25
50
75
100
125
nM
% V
iabi
lity
Trastuzumab-CF- LC-Site 2
Trastuzumab-CF- HC-Site 1
Trastuzumab-CF- HC Site 1/LC-Site 2
0.001 0.01 0.1 1 100
25
50
75
100
125
nM
% V
iabi
lity
Trastuzumab-CF HC-Site 11 /HC-Site 5
Trastuzumab-CF HC-Site 11
Trastuzumab-CF HC-Site 5
0.001 0.01 0.1 1 100
25
50
75
100
125
nM
% V
iabi
lity
Trastuzumab-CF- HC Site 5/LC-Site 10
Trastuzumab-CF- LC Site 10
Trastuzumab-CF- HC Site 5
Characterizing Multiple Site Specific MMAF ADC
Trastuzumab-‐CF Single Site vs. MulW Site MMAF ADC
IC50, nM DAR
HC -‐ Site 5 0.23 1.8
LC -‐ Site 10 0.13 1.5
HC -‐ Site 5 -‐ LC Site 10 0.06 4.0
HC -‐ Site 5 0.23 1.8
HC -‐ Site 11 0.08 1.4
HC -‐ Site 5 -‐ HC Site 11 0.03 3.8
HC-‐Site 1 0.06 1.9
LC-‐Site 2 0.07 1.5
HC Site 1 -‐ LC Site 2 0.03 3.9
Mul--‐Site ADCs are more potent than single site ADCs
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Multiple Site ADC is more Efficacious than Single Site ADC at Equivalent Dose
• KPL-‐4 Orthotopic breast cancer model • ADC and Control, Single dose, i.v. • Trastuzumab-‐CHO, mul-ple dose, i.p.
0 10 20 30 40 50 60 700
200
400
600
800
1000
1200
1400
1600
Days
Tum
or V
olum
e, m
m3
Trastuzumab-HC Site 1 MMAF ADC
Trastuzumab-CF HC Site 1, unconjugated
Vehicle
Free Drug, 0.54mg/kg
Trastuzumab-CHO1x30mg/kg (t =0), 3x15mg/kg (weekly)
Trastuzumab-HC Site 1 - LC Site 2 MMAF ADC
n =1015mg/kg
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Site Transfer across Scaffolds: IgG1 vs. scFv-Fc
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Site Specific ADC: IgG1 and scFv-Fc Comparison In vitro Cell Binding and Killing
Site 4 MMAF ADC Scaffold DAR
Cell Killing IC50, nM
Cell Binding Kd, nM
IgG1 HC-CF 2.0 0.034 2.3
ScFv-Fc-CF 2.0 0.06 5.0
Trastuzumab-CHO NA NA 2.0
Binding to SKBR3 CellsDetection : Alexa647 labeled polyclonal
anti-human IgG (H+L)
0.1 1 10 100 10000
25
50
75
100
125
nM
% B
indi
ng
Trastuzumab-CHO
ScFv-Fc-CF Site 4 MMAFIgG1-CF HC Site 4 MMAF
Cell Binding Cell Killing SKBR3 1000cells/well Day 5 Treatment
0.001 0.01 0.1 1 100
50
100
150
nM%
Via
bilit
y
Trastuzumab-CHO
IgG1-CF HC Site 4 MMAF
ScFv-Fc-CF SIte 4 MMAF
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Site Specific ADC: IgG1 and scFv-Fc Comparison PK and conjugate stability in vivo
Site 4 MMAF ADC Scaffold
Terminal t1/2 (day)
Cl (mL/day/kg)
IgG1 HC-‐CF 11.2 7.6
ScFv-‐Fc-‐CF 11.9 6.2
DAR Analysis by Pull down/LCMS
30min d3 d7 d14 d210.0
0.5
1.0
1.5
2.0
DA
RDays
IgG1-CF HC Site 4 MMAF
ScFv-Fc-CF Site 4 MMAF
0 5 10 15 20 25 300.1
1
10
100
Time (days)
Pla
sma
IgG
(ug/
ml)
IgG1 HC-CF Site 4 MMAF
scFv-Fc-CF Site 4 MMAF
PK Profile In vivo Stability of ADC
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scFv-Fc Drug Conjugate Scaffolds are very effective
• n =10 each treatment group • All treatments are single dose, i.v. @ t=0 • No significant weight loss observed in any treatment groups
5 15 25 35 45 550
300
600
900
1200
1500
1800
Time (days)
Tum
or V
olum
e, m
m3 Vehicle
IgG HC Site 5
IgG
scFv-Fc (vH-vL) Site 5
scFv-Fc (vL-vH) Site 5
15mgkgTrastuzumab-CF MMAF ConjugatedDAR 1.97
15mg/kgTrastuzumab-CF
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• Incorpora-on of two different nnAAs in mul-ple copy number in IgG LC and HC
• Enables Mul-ple payloads (4,6,8,10+) of two
different specifically posi-oned warheads/IgG
• Combina-ons of sites screened for:
• nnAA Incorpora-on efficiency/expression • Conjuga-on Efficiency • Stability • PK/Potency in vivo
warheads
Combination Warheads; single species ADC
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Toward a dual nnAA incorporation system
The goal: A system to incorporate 2 distinct nnAA’s with mutually orthogonal chemistries (azide and tetrazine) into a single protein in a single CF reaction
The components*: 2 synthetases 2 nnAAs (azide and tetrazine) 2 tRNAs (can decode TAG, TAA, TGA, or 4-base codon) *must be orthogonal to each other and to E.coli
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Combination Warheads: Development of Dual Payload ADCs
input (DNA)
nnAA1
Ribosome (Catalyst)
cell-‐free extract (E. coli)
mRNA
NNN
NNN UAA
otRNA1 NNN
NNN UAA
otRNA2
nnAA2
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Discovery of novel synthetases at Sutro
• 2000 synthetase variants from a rationally-designed library are expressed in individual CF reactions.
• Synthetase from aaRS expression reaction is added to GFP (K49TAG) reporter cell free reactions +/- nnAA.
• Desired synthetases will mediate amber suppression and GFP fluorescence in the presence but not absence of nnAA.
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Rationally-designed MjYRS library
Tyr32
Ile159
Asp158
Leu65 Gln109
Phe108 Library Positions
Tyr32 = Ala, Val, Leu, Thr Leu65 = Ala, Leu, Ile, Val Phe108 = Phe, Tyr, Trp Gln109 = Met, Leu, Ile, Gln Asp158 = Ala, Gly Ile159 = Ala, Gly, Val, Ser
4x4x3x4x2x4 = 1536 unique variants
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Testing ~2000 clones against 3 different nnAA’s
No nnAA nnAA #1 nnAA #2 nnAA #3
CF1: 100ul 96W aaRS Expression
CF2: 20ul 384W GFP TAG Assay
Each aaRS variant can be tested under 4 conditions
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Identification of a high fidelity, proprietary pAMFRS
• pAMFRS variant B3 produces potent ADCs with DAR of 1.9
• Fidelity of pAMF incorporation >99.8% by peptide LC-MS/MS
• Novel amino acid sequence
Trastuzumab-‐CF HC Site 1 MMAF conjugates
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Tetrazine nnAA’s to enable a second conjugation chemistry
• Tetrazine moiety enables retro Diels-Alder conjugation • 30x faster than copper-free click, complete conjugation in minutes • Compatible with and orthogonal to azide-click chemistry • Pyridyl-derivative nnAAs are more soluble than phenylalanine
derivatives
nnAA2
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LC synthesized with nnAA1
HC synthesized with nnAA2 in presence
of LC-nnAA1
LC-nnAA1/HC-nnAA2 conjugated to
drug1 and drug2
nnAA1
Drug2
Drug1
nnAA2
nnAA1
Dual Warhead ADC Concept
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Drug1 on HC
Drug1 DAR
Drug2 on LC Drug2 DAR
Total DAR
MMAF 1.8 SN-38 1.4 3.2
MMAF 1.8 PBD dimer 1.5 3.4
MMAF 1.9 Gemcitabine 1.6 3.6
PBD dimer 1.9 MMAF 1.7 3.7
Gemcitabine 2.0 MMAF 1.6 3.5
DAR Analysis of Combination Warheads
DAR=3.7
PBD Dimer/ MMAF Dual Warhead ADC
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Homogeneous, Multispecific Antibody Drug Combination Conjugates
– Multiple warheads
• to target synergistic mechanisms to improve safety • to simultaneously target potential resistance pathways
– Multiple epitope targeting • to increase internalization rate of an antigen
– Multiple antigen targeting • to increase apparent affinity to less densely expressed
antigens • to give better specificity over normal healthy tissues • to address antigen expression heterogeneity of tumor