a case study of developing highly hydrophilic...

Innovation in Linker Technologies A Case Study of Developing Highly

Hydrophilic Drug-Linkers

Robert P. Lyon, Ph.D. Sr. Director, Department of Chemistry

2

Overview: areas where novel ADC chemistry offers potential advancement over current technology

• Pharmacokinetics

o High drug loading accelerates clearance, diminishes activity

Limits drug loading to 3-4 per antibody

• Heterogeneity

o Average of 3-4 drugs per antibody generated by partial reduction of

interchain disulfides or lysine conjugation

• Chemical stability

o Maleimide elimination results in loss of drug-linker

3

Generating ADCs through native disulfides

full

reduction

0 2 4 4 6 8

thiols per antibody

8

thiols per antibody

IgG1

Conjugation with maleimido-drug linkers

8

drugs per antibody

W2996 Extraction at 280 nm

min

2.00 4.00 6.00 8.00 10.00 12.00

W2996 Extraction at 280 nm

min

2.00 4.00 6.00 8.00 10.00 12.00

time (min) time (min)

(0)

(2) (4)

(6)

(8)

(8)

Hydrophobic

Interaction

Chromatography

partial

reduction

(excess

TCEP) (limiting

TCEP)

0 2 4 4 6 8

drugs per antibody

4

High drug loading can result in faster clearance

Hamblett,K et al., Clinical Cancer Res. 2004, 10, 7063-7070

0 7 14 21

1

10

100

1000 0 2 4 8

time (days)

c o

n c e n t r

a t i

o n (

m g / m

L )

mc-vc-MMAE loading

1 reduced

disulfide

4 reduced

disulfides

Conjugation with mc-vc-MMAE:

Antibody / ADC Pharmacokinetics

5

High drug loading can result in faster clearance

Hamblett,K et al., Clinical Cancer Res. 2004, 10, 7063-7070

0 7 14 21

1

10

100

1000 0 2 4 8

time (days)

c o

n c e n t r

a t i

o n (

m g / m

L )

mc-vc-MMAE loading

0 1 0 2 0 3 0 4 0 5 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

d a y s p o s t tu m o r im p la n t

tum

or v

olu

me

(m

m3) u n tr e a te d

d o s e : 0 .5 m g /k g

Karpas-299 xenograft

Conjugation with mc-vc-MMAE:

Antibody / ADC Pharmacokinetics

6

Disulfide reduction does not adversely impact antibody pharmacokinetics

capping agent

native IgG1 capped IgG1

1) disulfide reduction

2) thiol capping

0 5 1 0 1 5

1

1 0

1 0 0

tim e (d a y s )

pla

sm

a c

on

ce

ntr

ati

on

(mg

/mL

)

c a p p e d

n a tiv e

2 8 0 n m H I C

W 2 9 9 6 E x t r a c t i o n a t 2 8 0 n m 9 1 5 0 4 7 E ; ; 2 / 1 0 / 2 0 1 2 1 : 1 5 : 5 8 P M P S T

W 2 9 9 6 E x t r a c t i o n a t 2 8 0 n m h 1 F 6 ; ; 3 / 1 3 / 2 0 1 2 4 : 5 2 : 3 8 P M P D T

m in

3 . 0 0 4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 0 1 0 . 0 0

280 nm HIC

W2996 Extraction at 280 nm 915047E; ; 2/10/2012 1:15:58 PM PST

W2996 Extraction at 280 nm h1F6; ; 3/13/2012 4:52:38 PM PDT

min

3.004.005.006.007.008.009.0010.00

retention time (minutes)

native

capped

Hydrophobic Interaction Chromatography Pharmacokinetics

7

Evaluating the role of drug-linker hydrophobicity

• Auristatin drug-linker engineered to eliminate any unnecessary hydrophobic elements and introduce hydrophilic moieties:

‘Auristatin T’

phenylalanine of MMAF

replaced with threonine

valine-citrulline cleavage site

replaced with hydrophilic sequence

self-immolative PAB group

eliminated

8

Apparent hydrophobicity correlates with plasma clearance

• Auristatin ADCs with 8 drugs per antibody evaluated for apparent

hydrophobicity and pharmacokinetics

280 nm HIC

W2996 Extraction at 280 nm h1F6; ; 3/13/2012 7:13:04 PM PDT

W2996 Extraction at 280 nm h1F6-1251; ; 3/13/2012 7:52:57 PM PDT



min

3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

retention time (minutes)

Hydrophobicity

Mouse Pharmacokinetics

(antibody concentration in plasma)

0 2 4 6 8 1 0

0 .1

1

1 0

1 0 0

tim e (d a y s )

[Ab

] ( m

g/m

l)

native h1F6

Auristatin T

mcMMAF

mc-vc-MMAF

ADC ADC:

Lyon, R. P. et al. Nature Biotechnology 33, 733-735 (2015).

• HIC retention time predicts pharmacokinetics reasonably well

9

Cells of mononuclear phagocytic system may mediate the accelerated clearance of hydrophobic ADCs

Kupffer cells hepatic sinusoids

• No observable MPS uptake of unmodified antibody or hydrophilic Auristatin T ADC

• Recognition of hydrophobic ADCs by the MPS may underlie the increased clearance from plasma

perfused rat livers stained with anti-human Fc antibody:

h1F6 h1F6:mc-vc-MMAF8 h1F6:Auristatin T8

(1 hour post-dose)

10

In vivo activity of Auristatin T ADCs increase with drug loading

0 2 0 4 0 6 0 8 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

d a y s p o s t tu m o r im p la n t

Me

an

Tu

mo

r V

olu

me

(m

m3

)

m c -v c -M M A F 4

m c -v c -M M A F 8

U n tr e a te d A u r is ta t in T 4

A u r is ta t in T 8

2 mg/kg 0.5 mg/kg 786-O

(RCC model)

Anti-CD70 ADCs

Hydrophilic drug-linkers can overcome the 4 drug loading limit

• Enable homogeneous conjugates with 8 drugs / antibody

Reverses the situation observed with the hydrophobic mc-vc-MMAF:

4

4 8

8

11

Can PEG can be used to ‘mask’ the hydrophobicity of an MMAE drug-linker?

• Glucuronide cleavable linker less hydrophobic than val-cit

• Prepared with discrete PEG24 in two configurations:

PEG24

‘stretcher’

PEG24

‘side chain’

12

Pharmacokinetics, in vivo activity depend upon PEG configuration

L540cy Hodgkin Lymphoma model

Single dose

cAC10 (anti-CD30) ADCs

8 drugs / antibody

0 2 0 4 0 6 0 8 0 1 0 0

0

5 0 0

1 0 0 0u n tr e a te d

1 m g /k g

1 m g /k g

d a y s p o s t im p la n t

me

an

tu

mo

r v

olu

me

(m

m3)

2 m g /k g

ADC pharmacokinetics

3 mg/kg iv dose

0 7 1 4

0 .1

1

1 0

1 0 0

c A C 1 0 -1

c A C 1 0 -2

c A C 1 0 -3

c A C 1 0

day

tota

l a

nti

bo

dy

(m

g/m

l)

P E G 2 4 s id e c h a in

n o P E G

P E G 2 4 s tr e tc h e r

13

perfused rat livers stained with anti-human Fc antibody:

Appropriate PEG ‘masking’ greatly diminishes hepatic MPS uptake

14

Instability of ADCs generated with maleimide drug-linkers

• Slow elimination via retro-Michael reaction regenerates reactive

drug-linker over several days

• Drug-linker can subsequently react with biological thiols

o e.g. serum albumin

Alley, S.C. et al, Bioconjugate Chemistry 19 (3), 2008, pp 759-765

serum albumin

15

How can drug-linkers conjugated to cysteines be stabilized? • Hydrolysis: a second process in maleimide ADCs:

very slow

X stabilized ADC

16

A potential mechanism to accelerate ring hydrolysis in ADC manufacturing • Intramolecular catalysis:

X

very fast?

stabilized ADC

17

Hydrolysis rates at pH 7.4, 22°C, as a function of amine group spacing

0 6 1 2 1 8 2 4

0

2 0

4 0

6 0

8 0

1 0 0

t im e (h o u rs )

% h

yd

roly

sis

(li

gh

t c

ha

in)

1

t1 /2

(h rs )

0 .4

1 .8

5 .2

1 3 .54

3

2

x

t1/2

(hours)

0.4

1.8

5.2

13.5

x

1

2

3

4

Monitoring hydrolysis by mass spectrometry

vc-MMAE x=1 ‘mDPR’

maleimide

diaminopropionyl

Lyon R.P. et al. Nature Biotechnology 32, 1059-1062 (2014)

18

Stability against drug-linker elimination

H3 L1 H3 L1

H3 H2 H1 H0

L0 L1 H3

H2 L0

L1

retention time (min) retention time (min)

t=0

t=14d Drug loading determined by

reversed-phase HPLC

L1

H3

pH 8.0 buffer, 37 °C, +10 mM NAC

0 1 2 3 4 5 6 7

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

t im e (d a y s )

% d

ru

g r

em

ain

ing

in vitro rat plasma R = val-cit-MMAE

19

Combining stability, homogeneity, and slower clearance

• Inclusion of mDPR into PEGylated MMAE to provide stability

• How long does the PEG chain need to be to overcome

pharmacokinetic liabilities of homogeneous 8-load ADCs?

20

ADC pharmacokinetics profoundly impacted by PEG chain length

• 8 units sufficient to slow clearance to levels

similar to unconjugated antibody

• Incremental pharmacokinetic benefit from

extending PEG chain to 24 units

0 4 8 1 2 1 6 2 0 2 4 2 8

0

1 0

2 0

3 0

4 0

5 0

A D C c le a ra n c e a s a fu n c t io n o f P E G s iz e

P E G le n g th

Cle

ara

nc

e (

mL

·kg

-1·d

ay

-1)

ADC clearance

vs

PEG length

0 5 1 0

0 .1

1

1 0

A D C ra t p h a rm a c o k in e tic s ,

3 m p k , iv d o s e , D A R 8 c o n ju g a te s

T im e (d a y s )

To

tal

Ab

(m

g/m

l)

Ig G -1 0 (n o P E G )

Ig G -9 (a c e ty l)

Ig G -8 (P E G 2 )

Ig G -7 (P E G 4 )

Ig G -6 (P E G 8 )

Ig G -5 (P E G 1 2 )

Ig G -4 (P E G 2 4 )

Ig G n a k e d a n tib o d y

unconjugated antibody

24

12

8

4

2

0 (acetyl)

0 (no lysine spacer)

PEG subunits

0 5 1 0

0 .1

1

1 0



T im e (d a y s )

To

ta

l A

b (

mg

/ml)

Ig G -1 0 (n o P E G )


Ig G -8 (P E G 2 )

Ig G -7 (P E G 4 )

Ig G -6 (P E G 8 )

Ig G -5 (P E G 1 2 )

Ig G -4 (P E G 2 4 )


21

Tolerability inversely correlated with clearance

animals sac’d in PEG 0, 2, and 4

groups due to weight loss

0 5 1 0

-2 5

-2 0

-1 5

-1 0

-5

0

5

1 0

S in g le d o s e m o u s e to le ra b ility

5 0 m p k , ip d o s e , D A R 8 c o n ju g a te s

T im e (d a y s )

% w

eig

ht

ch

an

ge

Ig G -1 0 (n o P E G )


Ig G -8 (P E G 2 )

Ig G -7 (P E G 4 )

Ig G -6 (P E G 8 )

Ig G -5 (P E G 1 2 )

Ig G -4 (P E G 2 4 )

u n tre a te dTolerability in mice, 50 mg/kg

dose tolerated in PEG 8, 12, and 24

groups

0 5 1 0

0 .1

1

1 0



T im e (d a y s )

To

tal

Ab

(m

g/m

l)

Ig G -1 0 (n o P E G )


Ig G -8 (P E G 2 )

Ig G -7 (P E G 4 )

Ig G -6 (P E G 8 )

Ig G -5 (P E G 1 2 )

Ig G -4 (P E G 2 4 )



24

12

8

4

2

0 (acetyl)


PEG subunits

0 5 1 0

0 .1

1

1 0



T im e (d a y s )

To

ta

l A

b (

mg

/ml)

Ig G -1 0 (n o P E G )


Ig G -8 (P E G 2 )

Ig G -7 (P E G 4 )

Ig G -6 (P E G 8 )

Ig G -5 (P E G 1 2 )

Ig G -4 (P E G 2 4 )


22

Antitumor activity parallels ADC PK

0

4

4

5

2

0

untreated

24

12

8

4

0

PEG

subunits

anti-CD19 ADCs at

3 mg/kg single dose

RL model of DLBCL

mostly cures with

PEG 8, 12, and 24

no cures in absence of PEG

0 5 1 0

0 .1

1

1 0



T im e (d a y s )

To

tal

Ab

(m

g/m

l)

Ig G -1 0 (n o P E G )


Ig G -8 (P E G 2 )

Ig G -7 (P E G 4 )

Ig G -6 (P E G 8 )

Ig G -5 (P E G 1 2 )

Ig G -4 (P E G 2 4 )



24

12

8

4

2

0 (acetyl)


PEG subunits

0 5 1 0

0 .1

1

1 0



T im e (d a y s )

To

ta

l A

b (

mg

/ml)

Ig G -1 0 (n o P E G )


Ig G -8 (P E G 2 )

Ig G -7 (P E G 4 )

Ig G -6 (P E G 8 )

Ig G -5 (P E G 1 2 )

Ig G -4 (P E G 2 4 )


cures

(5 mice/group)

0 2 0 4 0 6 0 8 0

0

3 0 0

6 0 0

9 0 0

1 2 0 0


me

an

tu

mo

r v

olu

me

(mm

3)

0 2 0 4 0 6 0 8 0

0

3 0 0

6 0 0

9 0 0

1 2 0 0


me

an

tu

mo

r v

olu

me

(mm

3)

23

Summary of PEG length findings

• PEG chain of 8-24 units resulted in ADCs with pharmacokinetics similar to


o These ADCs exhibited greater tolerability in a rodent model

o Greater exposure of these ADCs also improved activity in xenografts

• Appropriately configured PEG chain can improve the apparent therapeutic

index on both sides of the activity / tolerability ratio

24

Overview: areas where novel ADC chemistry offers potential advancement over current technology

• Pharmacokinetics

o High drug loading accelerates clearance, diminishes activity

Limits drug loading to 3-4 per antibody

o Hydrophilic and polymer-masked drug-linkers overcome PK limitations

Lyon, R. P. et al. Nature Biotechnology 33, 733-735 (2015).

• Heterogeneity

o Average of 3-4 drugs per antibody generated by partial reduction of interchain disulfides or lysine

conjugation

o Drug-linkers designed for hydrophilicity allow for uniform 8-load ADCs

• Chemical stability

o Maleimide elimination results in loss of drug-linker

o Self-stabilizing maleimide through rapid ring hydrolysis

Lyon R.P. et al. Nature Biotechnology 32, 1059-1062 (2014)

• These advances can be combined and applied to new drug classes

25

Acknowledgements

Synthetic Chemistry

Tim Bovee

Svetlana Doronina

Patrick Burke

Joe Hamilton

Scott Jeffrey

Protein Chemistry

Chris Leiske

Bioanalytical Chemistry

Jocelyn Setter

Josh Hunter

Animal Models

Marti Anderson

Research Leadership

Peter Senter

Dennis Benjamin

Jonathan Drachman

a case study of developing highly hydrophilic...

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