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Sutro Biopharma Inc.
Xpress CFTM: A rapid platform for drug development from antibody discovery to manufacturing
August 2014
CONFIDENTIAL 2
Sutro’s Xpress CF™ Platform:
Cell-Free Synthetic Biology
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
Advantages of Sutro’s Xpress
CF™ Platform
• Protein production is separated from biomass, allowing
– Off-the-shelf protein production
– Rapid expression for short make/test cycle
– Expression of toxic proteins
– high titer expression at g/L scales
• Xpress CFTM is an “open” system that can be
manipulated, allowing
– Easy incorporation of non-natural amino acids
– Biochemical optimization of proteins via additives
• An ideal platform for antibody discovery! CONFIDENTIAL 3
Engineered extracts for improved
IgG folding
CONFIDENTIAL 4
IgG
Rapid Execution of Antibody
Discovery Programs
Discover novel antibody fragments using ribosome display and screening
Express antibodies and fragments with Sutro protein synthesis system
Reformat fragments in host of different bispecific and antibody frameworks
Select the best lead candidate based on in vitro and in vivo activity
5
Ribosome Display
CONFIDENTIAL 6
mRNA
Ribosome
Antigen-biotin Antibody
+ Ag No Ag CDR H3
Round 1
Round 2
Round 3
Round 4
Streptavidin
capture
PCR amplify Screen output
Ribosome Display
• Ribosome Display is a selection
technology well-suited for CF
• Built-in bias for variants that
express and fold well in CF
• Selection output translates
directly into CF-based screening
Source of Antibodies and
Integration into the Sutro System
7
Sutro System
Ribosome Display
Phage Display
Cell-Based
Display
B-Cell Based Technologies
Antibody
Engineering
Bispecifics ADCs
Sutro Cell Free
Antibody
Discovery
• Mouse
• Rabbit
• Chicken
• Humanized Mice
• Human B-cells • Bacteria Display
• Yeast Display
• Mammalian
Display Bacteriophage
Display
nnAA Many
Scaffolds
Linker-
warhead
Sutro’s Libraries For Antibody
Discovery
Method Format Framework Diversity Size
Ribosome Display
scFv Optimized Human
Germline Synthetic >1012
Fab Optimized Human
Germline Consensus Synthetic >1012
Phage Display
scFv Optimized Human
Germline Synthetic >109
Fab Naïve Human
Immune Repertoire Natural >109
High Throughput Screening
Capabilities
CONFIDENTIAL 9
Selection (1012)
Primary Screen (1000’s)
Secondary Screen (100’s)
Biacore Affinity
Ligand blocking
ELISA
Cell binding or internalization
Epitope Binning
Screening workflow
10
Colony picking DNA prep Cell-free reaction &
ELISA-based ranking
Flower Plate Expression
Oasis 600
Biacore kinetic analysis ELISA and FACS binding
1000’s
100’s
100’s
Throughput/week
Ligand Blocking
Detailed characterization of
variants
CONFIDENTIAL 11
HT purified protein
Protein
quantitation
ELISA
And Cell
Binding
Kinetic
characterization
Epitope
binning
Cell Killing
(secondary)
Ribosome Display: unique platform for
antibody discovery and optimization
CONFIDENTIAL 12
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
0 500 1000 1500 2000
KD
(n
M)
Recovered protein (ug/mL)
Parent
New lead
Rd1
Rd2
Rd3
+Ag -Ag
Parent
Improved clones
Cell Binding ELISA KD vs protein recovered
Antibody leads can be combined
in multiple bispecific scaffolds
scFv
scFv1-
scFv2
scFv1-Fc-scFv2
scFv1-scFv2- Fc
scFv1-scFc-scFv2
scFv-scFc scFv-Fc
Rapid expression of multiple scaffolds to define product format with
optimal activity and half-life
13
Rapid Screening of Bispecific
Combinations
CONFIDENTIAL 14
HIK
knob Hole x stumpK
A B A B
A A
Bispecific-bridging of 2 targets
with AlphaLISA
CONFIDENTIAL 15
KIH HIK
A B A B
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
CF Engineered
MJ TyrRS +
Sutro Azido nnAA IgG with
DBCO-Based Linker-Warhead
Azido nnAA
Cu Free Click Conjugation Chemistry
DBCO Linker-Peptide Warhead (MMAF)
Data Driven Design:
Production of Many Variants in Hours
Light Chain: 111 Sites Heavy Chain: 133 Sites
SP Number Position TAG site o Mutate Sites in IgG: Choose nnAA sites using
rational 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
18
SKBR3 Binding Assay
Conjugated Variants Compared to Herceptin
Me
an
Flu
ore
sce
nce In
ten
sity
nM
0.01 0.1 1 10 100 10000
500
1000
1500
2000
2500
nM
MF
I
Herceptin
Sutroceptin
E293
K334
R
ela
tive
Ce
ll V
iab
ility
Transform of Data 1
0.0001 0.001 0.01 0.1 10
50
100
150
Rela
tive C
ell
Via
bili
ty
(Cellu
lar A
TP
conte
nt, %
of contr
ol)
ug/ml
T359
K360
N361
Q362
K370
Y373
S375
W381
S383
N384
S136, GY
WT HIC
S136 ERB2 ELISA
CF-Trastuzumab
Pos Cont ADC
DAR=1.6
mg/mL
Cell Killing Assay
Transform of Data 1
0.0001 0.001 0.01 0.1 10
50
100
150
Rela
tive C
ell
Via
bili
ty
(Cellu
lar A
TP
conte
nt, %
of contr
ol)
ug/ml
T359
K360
N361
Q362
K370
Y373
S375
W381
S383
N384
S136, GY
WT HIC
S136 ERB2 ELISA
Conjugation Efficiency (Drug/MAb Ratio)
DAR
0.4
0.7
1.1
DAR
1.1
1.5
1.6
nnAA Incorporation and Expression
Rapid Selection of Optimal Sites for
nnAA Incorporation
0 10 20 30 40 50 60 700
200
400
600
800
1000
1200
1400
1600
Days
Tum
or
Volu
me, m
m3
HC Site 1, DAR 1.84
HC Site 4, DAR 1.97
HC Site 5, DAR 1.97
Vehicle
HerceptinTM,
1x 30mg/kg (t =0), 3x15mg/kg (weekly)
HC Site 1, 15mg/kg
Free Drug, 0.54mg/kg
HC Site 6, DAR 1.96
15mg/kg
Trastuzumab-CF
AB4285 (MMAF)
Unconjugated control
Dose equivalence at DAR of 4.0
Best Single Site ADCs Show Differential
Efficacy (single 15 mg/kg IV dose)
• All treatments are single dose i.v. @ t=0
• Multi-doses of HerceptinTM dosed i.p
• No significant weight loss observed in all treatment groups
All MMAF ADC treated groups vs. vehicle
p < 0.0001**** up to d45
Xpress CF™ High Titers at Multiple Scales
21
Scalable and Efficient
6004002000
800
600
400
200
0
Time, min
[rh
GM
-CS
F],
mg
/L
4L
100L
300 mL
250 uL
a
Zawada et al, (2011) Biotech. & Bioeng.
Rapid Production of
Biotherapeutics
Synthetic
DNA
DAYS 2 4 6
50 µg
protein
HTS plate
format
5 g
protein
5-L
reactor
100 g
protein
100-L
reactor
• Direct linear scale-up from HTS to production scale
• Uses standard bioreactors & downstream equipment
• Minimal, rapid process development
• Gene sequence to drug substance in days
8
Sutro Technology
Speed to the Clinic …