producing homogeneous adcs with combination warheads · 2018. 9. 12. · warheads potency mechanism...
TRANSCRIPT
1
Producing Homogeneous ADCs with Combination Warheads
Trevor J. Hallam, PhD Chief Scientific Officer
October 15, 2013
2
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
3
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
4
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
5
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
6
High Titers at Any Scale
7
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
8
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
9
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
10
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
13 13
N
C
RF1
14 14
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
15
Conjugation Efficiency
Azido nnAA Cu Free Click Conjuga-on Chemistry
DBCO Linker-‐Warhead
R
R
16
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
17
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.
18
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
19
Learnings from ADC Specific Site Conjugation Variants
20
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
23
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
24
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
25
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
26
Site Transfer across Scaffolds: IgG1 vs. scFv-Fc
27
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
28
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
29
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
30
• 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
31
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
32
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
33
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.
34
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
35
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
36
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
37
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
38
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
39
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
40
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