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 …