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Producing Homogeneous ADCs with Combination Warheads Trevor J. Hallam, PhD Chief Scientific Officer October 15, 2013

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Page 1: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

1

Producing Homogeneous ADCs with Combination Warheads

Trevor J. Hallam, PhD Chief Scientific Officer

October 15, 2013

Page 2: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

Page 3: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

Page 4: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

Page 5: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

Page 6: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

6

High Titers at Any Scale

Page 7: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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  

Page 8: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

Page 9: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

Page 10: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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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11

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

Page 12: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

12

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

Page 13: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

13 13

N

C

RF1

Page 14: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

Page 15: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

15

Conjugation Efficiency

Azido nnAA Cu  Free  Click  Conjuga-on  Chemistry  

DBCO  Linker-­‐Warhead  

R

R

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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  

Page 17: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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.    

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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  

 

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19

Learnings from ADC Specific Site Conjugation Variants

Page 20: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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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21

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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22

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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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      

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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    

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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

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26

Site Transfer across Scaffolds: IgG1 vs. scFv-Fc

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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

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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  

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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

Page 30: Producing Homogeneous ADCs with Combination Warheads · 2018. 9. 12. · Warheads Potency Mechanism becomes redundant Bystander effect Payload MDR/Pgp status Systemic release Altered

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

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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