rapid design and manufacture of single species, site

Rapid Design and Manufacture of Single Species, Site-Specific ADCs using Cell-Free Synthesis with nnAAs

Trevor J. Hallam, PhD Chief Scientific Officer

From Concept to Clinic in 365 days ?

3rd World ADC Summit, San Francisco October 24th, 2012

ADCs – Exciting prospects but . . .

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 Site Specific Cys Conjugation

Heterogeneity translates to poor PK, stability and efficacy

Production of Superior 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

Anatomy of a Disruptive Protein Design and Manufacturing Solution…..

•  Rapid Make-Test –  Fast protein production (from DNA to g’s/L in hours) –  a 1-week cycle time for purification, characterization and testing –  Micro-titre plate evaluation of 100’s of variants in parallel –  Parallel evaluation of optimal production conditions

•  Chemical Diversity –  Incorporation of non-natural amino acids

•  Rapid Scale-up –  Linearly scalable and predictable expression allows for seamless

and rapid progression to specificity testing, efficacy models and GLP tox studies

•  Same system for cGMP manufacture

A Cell-Free Synthetic Biology Platform

Ribosome (Catalyst)

Engineered cell-free extract (E. coli)


Energy

Ribosome (Catalyst)


+ Accessories

(AA, proteins, RNAP, metabolites)


input (DNA)

+

Energy

Ribosome (Catalyst)


+ Accessories



input (DNA)

+

Energy

Ribosome (Catalyst)


output (multi-domain

eukaryotic proteins)

Assembled IgG

+ Accessories


How versatile is cell free (CF) synthesis?

BiTE

(ScFv-Fc)2

ScFv

ScFv-Fc

Fab’2IgG1

•  Examples of Ab Fragments Produced by CF synthesis •  Other examples:

!  Growth factors !  Immunotoxins !  Proteases !  Knottins !  Cellulases !  Hydrogenases

Fab’2Fab

Typical Expression Levels: •  0.25 -1 Gram / Liter (Pre-Optimized) •  6-8 Hour Reaction •  30% Extract

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

Data!driven!design:!!!produc2on!of!many!variants!in!hours!!

o  Mutate"Sites"in"IgG:""Choose!nnAA!sites!using!ra2onal!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!linkerEwarhead!

o  Test:"Assay!conjugated!IgG’s!for!binding!and!cell!killing!

Surface!Scan!

TAG Screen

!

Light!Chain:!!!!111!Sites!Heavy!Chain:!!133!Sites!!!

SP!Number!Posi2on! TAG!site!

Sutro!nnAA!IgG!with!AB3627!Linker+Warhead!

Azido nnAA Cu!Free!Click!Conjuga2on!Chemistry!

DBCO!LinkerEWarhead!

R

R

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

Exp

ress

ion

of P

rope

rly

Fold

ed Ig

G (u

g/m

l)

nnAA Incorporation and Expression

Conjugation Efficiency (Drug/MAb Ratio) DAR: 0.4 0.7 1.1

Target

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

Site-Specific Incorporation of MMAF Yields Potent Cytotoxic ADC

Published Data – Genentech patent

ADC - 3.8 drugs/Ab ADC - 4.1 drugs/Ab ADC – 4.8 drugs/Ab

ug/ml

•  Comparable IC50 to published ADC at equivalent IgG concentration •  More potent at equivalent drug concentration

Cel

l Via

bilit

y

(rel

ativ

e Lu

min

esce

nce)

Cell Viability Assay SK-BR-3 cells at 120 hours

Sutrocep)n+Variants

0.0001 0.001 0.01 0.1 1 100

50

100

150

ug/ml

Rela)ve+Ce

ll+Viability

(Cellular+A

TP+co

ntent,+%+of+C

ontrol)

T135FAB3626+(No+Peg)

S375FAB3626+(No+Peg)S375FAB3627+(++Peg)

A118FAB3626+(No+Peg)

A118FAB3627+(++Peg)

Sutrocep)n++

Hercep)nTM

MMAF+AB3617+Only

MMAF+AB3626+(No+Peg)+Only

MMAF+AB3627+(++Peg)+OnlyH-MC-MMAF [cys] (120 hours) From: US Patent 7,994,135 B2 Aug 9, 2011; Fig 7

µg / ml

%

Cel

l Via

bilit

y

0 5 10 15 20 25 300.01

0.1

1

10

100

Time (days)

Plas

ma

conc

entra

tion

(µg/

mL)

IgG HC S136 AB3627IgG HC AB3627

!!

a5!mg/kg,!BalbEc!mice,!Sutro!!b2mg/kg,!SpragueEDawley!rats!(US7994135B2,!SeaGen!patent!2011)`!

CFETrastuzumab!Drug!conjugate!pharmacokine2cs!are!in!good!agreement!with!!TrastuzumabE!MMAF!conjugate!literature!

!!!!!!!!!

Sutro!Dataa! SeaGen!Datab!CFETrastuzumab!Drug!

Conjugate!Trastuzumab!MMAF!

Conjugates!!!AUCinf![day/µg/mL]! 248! 299!

Clearance![mL/d/kg]! 8.1! 9!

HalfElife![d]!! 8! 10!

Pharmacokinetics of Cell Free Produced ADCs are Comparable to Cell-Based derived ADCs

CFETrastuzumab!Drug!!Conjugate!

Trastuzumab!MMAF!!Conjugates!

TrastuzumabEvcEPABEMMAFETEG!Total!Ab!TrastuzumabEvcEPABEMMAFETEG!Conjugate!Ab!TrastuzumabEvcEPABEMMAF!Total!Ab!TrastuzumabEvcEPABEMMAF!Conjugated!Ab!

Mean!E/+!SD!ploded!3!mice!per!2me!point!2mg/kg,!Dosed!i.v.!!

0 5 10 15 20 25 30 350

400

800

1200

Days of Treatment

Mea

n Tu

mor

Vol

ume,

mm

3

8.1 mg/Kkg

5.4 mg/kg

Agly Trastuzumab6xHis Drug conjugate 1.6 mg/kg

Vehicle

Trastuzumab

Trastuzumab

(i.p)

CF-Trastuzumab Drug Conjugate (i.v)

Efficacy Study: CF-Trastuzumab DC Dose Response

30 mg/kg 15 mg/kg

Efficacy Study: Agly Trastuzumab DC Dose Response

0 5 10 15 20 25 30 350

250

500

750

1000

1250

Days

Mea

n Tu

mor

Vol

ume,

mm

3

Trastuzumab

SITE 2 ADC

SITE 3 ADC

SITE 1 ADC

Vehicle

Trastuzumab (i.p)

Aglycosylated Trastuzumab Drug Conjugates Sites 1, 2, and 3 (Single Dose 8mg/kg i.v; DAR=1.5)

30 mg/kg 15 mg/kg **** P < 0.0001

****

****

****

****

Site Dependent Impact on Cell Killing Observed!

SMP1018-04

0.00 0.01 0.02 0.030.0070.0

0.5

1.0

1.5

2.0

1.3

SKBR3 Killing IC50 (ug/mL)

DAR

SMP1019-04

0.00 0.01 0.02 0.030.0070.0

0.5

1.0

1.5

2.0

1.2

SKBR3 Cell Killing IC50 (ug/mL)

DAR

SMP1021

0.00 0.01 0.02 0.030.0070.0

0.5

1.0

1.5

2.0

1.3


DAR

SMP1022

0.00 0.01 0.02 0.030.0070.0

0.5

1.0

1.5

2.0

1.3


DAR

SMP1020

0.00 0.01 0.02 0.030.0050.0

0.5

1.0

1.5

2.0

1.3


DAR

SMP1023

0.00 0.01 0.02 0.030.0

0.5

1.0

1.5

2.0


DAR

TAG Scanning: DAR vs. Cell Killing!

Cell!Killing!

DAR!

Efficient Integration of nnAAs and Conjugation Chemistry are key for manufacturing ADCs

•  nnAA Incorporation Efficiency

–  Site Dependence –  RF1 Attenuation –  Multiple nnAA Incorporation

•  Conjugation Efficiency

–  Site Dependence –  Improving Conjugation Kinetics

nnAA Incorporation Efficiency; Site Dependence in HC

A121

nnAA Incorporation Efficiency; Site Dependence in LC

K169

S202 20%

100%

40%

60%

80%

Sup

pres

sion

of A

mbe

r Cod

on

nnAA!Incorpora2on!Efficiency:""

•  "!nnAA!Incorpora2on!Efficiency!•  "!Expression/yield!!

!!

RF1!Adenua2on!!

aa nnAA-tRNA

RF1

Full!length!nnAA!containing!protein!

Truncated!

25

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!OmpTEsuscep2ble!RF1!

Protease!Sensi2ve!RF1!is!inac2vated!during!extract!produc2on!!

Abbrev." Sample"

M! Marker!

P! Pellet!

L! Lysate!

C! Clarified!

1! 1!hour!2me!point!



De.compartmentalized"Extract"Cleaves"RF1"

New!Strains!Have!Similar!Growth!Rates!To!Control!Strain!

0!

5!

10!

15!

20!

25!

30!

35!

40!

45!

50!

0! 5! 10! 15! 20! 25!

OD 5

95"

Time"(hrs)"

Growth"Profiles"SBJY001"

SBHS015"

SBHS016"

SBHS017"

0!

0.1!

0.2!

0.3!

0.4!

0.5!

0.6!

0.7!

0.8!

SBJY001! SBHS015! SBHS016! SBHS017!

µ avg"(hr.1)"

Total"Average"Growth"Rates"

High"Growth"Rate"is"Historically"Predicave"of"Good"Extract"Performance"

RF1- Strain Engineered Extract Boosts nnAA-Protein Production

Strain:!!RF1!Adenua2ng!Modifica2on!

WT nnA

A

WT

nnA

A

Control!Extract! Engineered!Extract!

Suppression of amber codon in HC

A121 0"

100"

200"

300"

400"

500"

WT" V422" S415" Q418" P343" G341" K320" F404" Q342" T299" Y373" N297" F405" S136"

IgG"soluble"yield"(m

g/L)" RF1+"extract"

RF1N"extract"

WT 1 2 3 4 5 6 7 8 9 10 11 12 C

Incorporation of p-Acetyl Phe into IgG at three selected sites

HC"A121"

LC"K16

9"

LC"S20

2"

HC"A121"

LC"K16

9"

LC"S20

2"

RF1+" RF1."

nnAA

"IgG"yield"

HC"A121"

LC"K16

9"LC"S20

2"HC

"A121"

LC"K16

9"LC"S20

2"

HC"A121"

LC"K16

9"LC"S20

2"HC

"A121"

LC"K16

9"LC"S20

2"

RF1+" RF1."

RF1+" RF1."

nnAA"IgG"

HC"

LC"

truncated"Su

ppre

ssio

n

0"1" 2" 3" 4" 5" 6"

Yield&

0%#

20%#

40%#

60%#

80%#

100%#

1# 2# 3# 4# 5# 6#

Supp

ression*

•  Incorpora2on!of!mul2ple!nnAAs!in!each!! !!IgG!LC!and!HC!

•  Enables!Mul2ple!(4,6,8,10+)!site.specific"combinaaons"of"warheads/IgG"

•  Intractable!using!cellEbased!systems!or!wildEtype!extract!due!to!significant!accrued!losses!in!yield!

•  Combina2ons!of!sites!screened!for:!•  nnAA!Incorpora2on!efficiency/expression!•  Conjuga2on!Efficiency!•  Stability!•  PK/Potency!in!vivo!

!

warheads

RF1!Deple2on!through!Strain!Engineering:!Extract!Can!Produce!IgG!w/!Mul2ple!nnAA’s!

4 nnAA IgG Yields Using Combinations of 2 LC and 2 HC Sites

#  nnAA: nn-AA1 #  Synthetase 3

#  nnAA: pN3nnAA #  Synthetase 1

0!

100!

200!

300!

400!

500!

600!

IgG"yield"(m

g/L)"

TAG"site"(LC/HC)"

4!nnAA!combos! 4!nnAA!combos!

WT!PC

R!

WT!PC

R!

WT!

plasmid!

WT!

plasmid!

Several!4!nnAA!combos!(2LC+2HC)!express!similarly!to!WT!

PCR Based Expression PCR Based Expression

Conjuga2on!Efficiency!

Azido nnAA Cu!Free!Click!Conjuga2on!Chemistry!

DBCO!LinkerEWarhead!

R

R

Position affects Kinetics of Conjugation

2.50 3.00 4.00 5.00 6.00 7.00 8.00 9.00 9.50-50

200

400

700 CR 2012-07-26 R NG HTS #7 [modified by labuser, 2 peaks manually assigned] UV_VIS_1mAU

min

unco

njug

ated

,35.

57

2

singl

y,51

.20

doub

ly,13

.16

WVL:214 nmHIC

mA

U

Retention Time (Min)

Variant" t1/2(Doubly)"Hrs"Posi2on!1! 6.2!

Posi2on!2! 39.7(est)!

Posi2on!3! ND!

Position 3

Position 2

Position 1

Conjugation Efficiency

Conjugation. Oxime ligation between pAcPhe on the antibody and alkoxy- amine-functionalized auristatin was carried out in 100 mM acetate buffer pH 4.5 with 100 µM (5 mg/mL) Fab and 3 mM AF (30-fold excess) for 1–2 d at 37 °C. IgG was conjugated at 66.7 µM (10 mg/mL) and 1.3 mM AF (20-fold excess) for 4 d at 37 °C.

Synthesis of site-specific antibody-drug conjugatesusing unnatural amino acidsJun Y. Axupa,b, Krishna M. Bajjuric, Melissa Ritlandd, Benjamin M. Hutchinsa,b, Chan Hyuk Kima,b,Stephanie A. Kazanea,b, Rajkumar Haldera,b, Jane S. Forsythd, Antonio F. Santidriand, Karin Stafind, Yingchun Lue,Hon Trane, Aaron J. Sellere, Sandra L. Biroce, Aga Szydlike, Jason K. Pinkstaffe, Feng Tiane, Subhash C. Sinhac,Brunhilde Felding-Habermannd, Vaughn V. Smiderc,1, and Peter G. Schultza,b,1

aDepartment of Chemistry, cDepartment of Molecular Biology, dDepartment of Molecular and Experimental Medicine, and bThe Skaggs Institute forChemical Biology, The Scripps Research Institute, La Jolla, CA 92037; and eAmbrx, Inc., La Jolla, CA 92037

Contributed by Peter G. Schultz, July 20, 2012 (sent for review January 27, 2012)

Antibody-drug conjugates (ADCs) allow selective targeting ofcytotoxic drugs to cancer cells presenting tumor-associated surfacemarkers, thereby minimizing systemic toxicity. Traditionally, thedrug is conjugated nonselectively to cysteine or lysine residues inthe antibody. However, these strategies often lead to heteroge-neous products, whichmake optimization of the biological, physical,and pharmacological properties of an ADC challenging. Here wedemonstrate the use of genetically encoded unnatural amino acidswith orthogonal chemical reactivity to synthesize homogeneousADCs with precise control of conjugation site and stoichiometry.p-Acetylphenylalanine was site-specifically incorporated into ananti-Her2 antibody Fab fragment and full-length IgG in Escherichiacoli and mammalian cells, respectively. The mutant protein wasselectively and efficiently conjugated to an auristatin derivativethrough a stable oxime linkage. The resulting conjugates demon-strated excellent pharmacokinetics, potent in vitro cytotoxic activ-ity against Her2+ cancer cells, and complete tumor regression inrodent xenograft treatment models. The synthesis and character-ization of homogeneous ADCs with medicinal chemistry-like controlover macromolecular structure should facilitate the optimization ofADCs for a host of therapeutic uses.

breast cancer | protein conjugation

Amajor challenge in the development of new drugs is theability to selectively modulate a specific target in the tissue

of interest while minimizing undesirable systemic side effects.In the past decade, antibody-drug conjugates (ADC) have shownconsiderable promise as anticancer agents by preferentially target-ing cytotoxic drugs to cells presenting tumor-associated antigens(1–4). It is hypothesized that the ADCs are endocytosed afterbinding cell-surface antigens and the antibody molecule is de-graded in the lysosome, releasing the cytotoxic drug into thecytosol (5, 6). Antibody-drug conjugates have had significantachievements as well as notable failures in the clinic. The firstFood and Drug Administration-approved ADC, gemtuzumabozogamicin (Mylotarg; Wyeth/Pfizer), was a conjugate of theDNA cleaving agent calicheamicin to an anti-CD33 antibody forthe treatment of acute myelogenous leukemia (7). Despite earlyencouraging clinical activity and fast-track approval, Mylotarg wasremoved from the market because of toxicity and lack of efficacyin larger trials (8). More recently, however, brentuximab vedotin(Adcetris/SGN-35; Seattle Genetics), an anti-CD30 auristatinconjugate, became the first drug approved for Hodgkin’slymphoma since 1977 (9, 10), and there are many more ADCsin development (11–15). Although the approval of Adcetrisshows the potential of ADCs to impact major unmet needs inoncology, the withdrawal of Mylotarg shows that further work isnecessary to optimize this new class of drugs.In contrast to small molecules, in which chemically defined

structures are synthesized in an attempt to improve drug proper-ties, the most widely used technologies for constructing antibody-drug conjugates rely on nonspecific electrophilic modification of

cysteine or lysine residues. There is limited stoichiometric controlbecause of the large number of disulfide bonds and lysine residuesin antibody molecules, which leads to a typical distribution of zeroto eight toxins per antibody (16, 17). In addition, coupling occursat many different sites leading to a heterogeneous mixture ofADCs, the components of which likely have distinct affinities,stabilities, pharmacokinetics, efficacies, and safety profiles.Genentech reported a site-specific ADC (Thiomab) that wassynthesized by introducing an additional cysteine into anti-bodies (18, 19). Thiomab showed similar efficacy to randomlylabeled ADCs despite having fewer (only two) drugs per an-tibody, but had an improved therapeutic index and betterpharmacokinetics in rodents. These results support the notionthat precise chemical control over the site and stoichiometryof conjugates can lead to improved therapeutics. However,this methodology has not been generally adopted, likely in partbecause of challenges in scalability, which involves disulfidebond reduction and reoxidization steps, and the instability ofmaleimide-thiol conjugates (20).Here we report the synthesis of chemically defined ADCs

in which the site and stoichiometry of conjugation are con-trolled using genetically encoded unnatural amino acids withorthogonal chemical reactivity relative to the canonical 20 aminoacids. This approach produces homogeneous molecular entities,rather than mixtures, and allows for medicinal chemistry-like optimization of physical properties, efficacy, pharmaco-kinetics, and safety profiles. An orthogonal amber suppressortRNA/aminoacyl-tRNA synthetase (aaRS) pair is used to site-specifically incorporate p-acetylphenylalanine (pAcPhe) in responseto an amber nonsense codon into antibodies recombinantlyexpressed in either bacterial or mammalian cells (21–24). Theketo group of pAcPhe can be selectively coupled to an alkoxy-amine derivatized drug of interest to form a stable oxime bondin excellent yield (25–27). To illustrate this concept, a C-terminalalkoxy-amine auristatin derivative was conjugated to trastu-zumab (Herceptin, anti-Her2) mutants containing pAcPhe atone of several positions in the antibody constant regions. Theresulting ADCs showed excellent efficacy and pharmacokineticsin an in vivo rodent xenograft model against mammary fat padtumors induced by Her2-transduced MDA-MB-435 humanbreast cancer cells.

Author contributions: J.Y.A., B.M.H., V.V.S., and P.G.S. designed research; J.Y.A., K.M.B.,M.R., B.M.H., J.S.F., A.F.S., S.L.B., and A.S. performed research; J.Y.A., K.M.B., B.M.H.,C.H.K., S.A.K., R.H., J.S.F., K.S., Y.L., H.T., A.J.S., and F.T. contributed new reagents/analytictools; J.Y.A., K.M.B., M.R., B.M.H., C.H.K., S.A.K., R.H., A.F.S., S.L.B., A.S., J.K.P., F.T., S.C.S.,B.F.-H., V.V.S., and P.G.S. analyzed data; and J.Y.A., V.V.S., and P.G.S. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1211023109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1211023109 PNAS Early Edition | 1 of 6

BIOCH

EMISTR

Y

Materials"and"Methods"Secaon"Oxime!Liga2on!

Novel!nnAAs!with!Boosted!Conjuga2on!Kine2cs!

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!AzE!nnAA!#1!AzEnnAA!#2!AzEnnAA!#3!

AzidoEnnAA!

DBCO!

New!Chemistry!offers!Improved!Kine2cs,!Flexibility!

R

R

Top Sites: Nearly Complete Conjugation

5 x10

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1 1.1 1.2

Counts vs. Deconvoluted Mass (amu) 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500 152000

150933.50

149553.94 148173.08 147023.90 152132.08 149967.26

HC(Site"1)"DAR:"1.80"

5 x10

0 0.2 0.4 0.6 0.8

1 1.2 1.4

Counts vs. Deconvoluted Mass (amu) 148000 148500 149000 149500 150000 150500 151000 151500 152000 152500

150878.41

149522.99 148668.60 148180.50 150264.69 147708.73


5 x10

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

Counts vs. Deconvoluted Mass (amu) 145000 146000 147000 148000 149000 150000 151000 152000 153000

150793.38

149410.37 151896.68 148367.28 146826.54 145697.07


5 x10

0 0.2 0.4 0.6 0.8

1 1.2 1.4

145000 145500 146000 146500 147000 147500 148000 148500 149000 149500 150000

149057.78

149372.07 147629.94 146532.75 148437.61 145220.64

LC(Site"4)"DAR:"1.95"

0 1

2

Some sites are completely conjugated in under 4 hours!

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]!5E20uM!5x!M!excess!!!Linker/warhead!Room!temp.!!

Cell.Free"Manufacturing:"GMP"Facility"a"Criacal"Element"

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

Sutro’s technology enables Best-in-Class ADCs….

•  Data-driven assessment of many potential ADC variants ensures optimized positioning for suppression, expression, conjugation efficiency, payload, cell killing, stability and pharmacokinetic properties

•  Flexible and rapid scaling means pharmacodynamic and exploratory toxicology assessment studies can be front-loaded into the discovery phase

•  Material for GLP Tox studies and clinical studies can be delivered in days/weeks from selection of Clinical Development Candidate

•  365 days?

rapid design and manufacture of single species, site

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