How the chemical design and synthesis of linkers used in antibody drug conjugates drives the success of ADC drug development

Mark Frigerio - Director Chemistry UK, Abzena

9th October, 2018

ELRIG - The European Laboratory Research & Innovation Group

Drug Discovery: Drug Discovery for Small & Large Molecules

2

Antibody Drug Conjugates

• Multiple components and multi-step mechanism of action

• Specific requirements for each step / component. For example, stable in circulation but drug released in tumour cells

Simple concept - complex products

Frigerio and Kyle, (2017), The Chemical Design and Synthesis of Linkers Used in Antibody Drug Conjugates, Curr. Topics in Med. Chem.

• Antibody• Linker• Cytotoxic payload

3

Targeted delivery of drugs using Antibody Drug Conjugates

●●

●

●●

●●

●

●●

●●

●

●

● ●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

● ●

●

●

●

●

●●

●

●●

●

●

●

●●

●●

●●

● ●

●●

●

●

●

●

●●

●●

●

Non-targetedchemotherapy drugs

Antibodies Antibody-drug conjugates (ADCs)

Maximum tolerated dose

Minimum efficacious dose

Maximum tolerated dose

Minimum efficacious dose

Therapeutic window

Adcetris (2011)Kadcyla (2013)MyloTarg (reapproved 2017)Besponsa (2017)>65 ADCs in the clinic

4

Properties of antibodies suitable for attachment of a small molecule

- There are several distinct functionalities present in a native IgG1 antibody that can be exploited for suitable conjugation approaches

1. Fab fragment

2. Fc portion (CH2 and CH3 domains)

3. Heavy chain

4. Light chain

5. Glycosylation site/glycoform

6. Hinge region

5

Chemistry conjugation toolbox for ADC drug development

PayloadConjugating

unitPolymer

Release linker

Targeting protein

Albumin

Native mAb(IgG1/2/4)

DARTs

ABDURIN

AFP

Fab

ScFv

Trauts reagent

Minibody

Engineered cysteines

VH DomainsPEG

Cyclodextrin

Cyclic PEG

Other polymers

ThioBridge™

Maleimide

NHS-ester

CyPEG™

Auristatins

Maytansines

Duocarmycins

Anthracyclines

PBD dimers

DOTA

Desferrioxamine AlexaFluor®

Rhodamine

Biotin

Disulfide

val-cit-PABval-ala-PAB

Dipeptidic

Carbonate

Non-cleavable

RNA

Camptothecins

peptides

Oncology

Imaging & labelling

Fluorescein

Other therapeutics/applications

6

Accessing a suitable toolbox is critical for the design of a successful ADC linker

=

Antibody Spacer Release Drug Attachment

ADC Linker

7

Next generation reagent solutions to ADC linker design are needed to address the limitations in current ADC linkers

Antibody Spacer Release Drug Attachment

ADC Linker

=

Synthon’s trastuzumab / duocarmycin based ADC (SYD985), currently in Phase I trials for the treatment of breast and gastric cancers

8

Approved ADCs: Conjugation and linker chemical approaches

Adcetris® (Seattle Genetics / Takeda-Millenium)

FDA approved Aug 2011 for Hodgkin lymphoma

• 75% ORR in HL patients• 1/3 responders had complete remission

Kadcyla® (Immunogen / Roche-Genentech)

FDA approved Feb 2013 for breast cancer

• 5.8 months longer survival than standard therapy

• Significantly longer PFS

Conjugation to lysine residues

Conjugation to interchain cysteine residues

9

1.0 5.0 10.0 15.0 20.0 24.5

-2.8

0.0

5.0

11.9HIC_current #2700 Kadcyla UV_VIS_3mAU

min

WVL:280 nm

2.3 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0

-3.6

0.0

2.0

4.0

6.0

8.0

10.0HIC_current #2699 Adcetris UV_VIS_3mAU

min

WVL:280 nm

Different conjugation approaches produce ADCs with different drug distributions

DAR 8

DAR 6

DAR 4DAR 2

DAR 0

1 3 5DAR 0

Adcetris® - maleimide conjugation Kadcyla® - lysine conjugation

high DARs

DAR distribution Comparison by Hydrophobic Interaction Chromatography (HIC)

- Hydrophobic interaction chromatography shows drug loading of ADCs

• Lower tolerability

• Accelerated clearance

• High instability

• High hydrophobicity

• Competitive inhibitor• Lower tolerability

• Accelerated clearance

• High instability

• High hydrophobicity

• Competitive inhibitor

10

Seattle Genetics demonstrated why different drug distributions matter

- DAR 2, 4 and 8 purified by HIC from heterogeneous mixture

- Correlation between DAR and PK

- Highly loaded DAR species cleared faster

- Highly loaded DAR species had lower efficacy

Hamblett et al., Clin Cancer Res. 2004. 10, 7063

2

48

24

8

11

Main challenges faced in ADC development

ADC challenge Reasons Efficacy Toxicity

Antigen Antigen heterogeneity (tumour, metastases)Insufficient expression in tumourExpression on healthy cells

↓↓

↑

Payload MOA Resistance of tumour cell to payload MOA ↓

Heterogeneity ofdrug-antibody ratio (DAR)

Naked antibody – competitive inhibitorLow DAR – Insufficient drug deliveredHigh DAR – Fast clearance

↓↓↓ ↑

ADC instability Systemic release of drugDisarming of ADCFragmentation of antibody

↓↓

↑

↑

Suboptimal PK High DAR (fast clearance)Immunogenicity (fast clearance)

↓↓

↑↑

• Homogeneous• Stable• Non-immunogenic

Ideal ADC:

12

Next generation site specific conjugation at accessible disulfides via an addition/elimination reaction mechanism

Disulfide

Reduction

ThioBridge™

Conjugation

Badescu et al. (2014). Bridging disulfides for stable and defined antibody drug conjugates. Bioconjugate Chemistry, 25(6), 1124-1136.

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

-2.0

5.0

10.0

15.0

18.0HIC_current #102 [modified by martin.pabst] PT79-WM002-4 quenched reaction carbon-treated UV_VIS_3mAU

min

WVL:280 nm

DAR 5DAR 3

DAR 4

13

Evaluation of conjugate stability can be demonstrated ex vivo

Method: Alexa Fluor 488 conjugated to trastuzumab using ThioBridge™ or maleimide chemistry and conjugates incubated in rat serum at 37 ⁰C for 48 h; analysed using size exclusion chromatography (SEC)

t = 0 h

t = 48 h

t = 48 h

FragmentsAggregatesAlexa Fluor 488

4 6 8 10 12 14 16 18 20

4 6 8 10 12 14 16 18 20

ThioBridge™ conjugate

Maleimide conjugate

t = 0 h

Cross-conjugation of Alexa Fluor 488 to

albumin

Rel

ativ

eF

luo

resce

nce

mAb AlbuminTime (min)

mAb AlbuminTime (min)

Indicates mAb unstable

vs

Loss of Alexa Fluor 488 and breakdown of mAb seen with maleimide conjugate but not with ThioBridge™ conjugate

14

Enhancing in vivo potency can be achieved through modulation of linker reagent architecture

Five different reagents using the cleavable linker-payload val-cit-PAB-MMAE were prepared

to evaluate the influence of reagent architecture on in vitro and in vivo properties of ADCs

ThioBridge™Format 5, DAR 8

ThioBridge™Format 1, DAR 4

ThioBridge™Format 2, DAR 4

ThioBridge™Format 3, DAR 4

ThioBridge™Format 4, DAR 4

Pabst et al. Modulation of drug-linker design to enhance in vivo potency of homogeneous antibody-drug conjugates J. Control. Release, 2017, 253, 160-164.

15

Different brentuximab ADCs demonstrate similar in vitro potency in CD30+ cells

- ThioBridge™ brentuximab ADCs (DAR 4) were prepared with four different reagents with the same linker-payload (val-cit-PAB-MMAE).

The brentuximab ADCs with MMAE and a cleavable linker had similar

potencies in vitro in Karpas 299 cells as this is driven by drug loading

100 101 102 103 104

0

50

100

MMAE

Adcetris

, average DAR ~4

ThioBridge

Format 1, DAR 4

ThioBridge

Format 2, DAR 4

ThioBridge

Format 3, DAR 4

ThioBridge

Format 4, DAR 4

Conc (pM)

Ce

ll V

iab

ility

[%

]

16

Profiling of brentuximab ADCs with different linker structures demonstrate different efficacy profiles in vivo

ThioBridge™Format 1, DAR 4

ThioBridge™Format 2, DAR 4

ThioBridge™Format 3, DAR 4

Adcetris®Average DAR ~4

ThioBridge™Format 4, DAR 4

The flexibility of linker design allows quick screening of reagent configurations and

selection of optimal ADC via a rapid design → test approach to ADC drug development

- Karpas-299 xenograft model CD30+ at 0.5 and 1 mg/kg (single dose, i.v. administration at day 0)

17

Ease of reagent design allows the rapid production and profiling of ADCs in increasingly complex in vivo evaluations

- By fractionating the dose of brentuximab ADCs (DAR 4) with structurally different linkers can differentiate their in vivo efficacy profiles more clearly

- ADCs were tested in Karpas 299 SCID mouse xenograft model at 0.4 mg/kg (Q4Dx4, i.v. administration)

0 10 20 30 40 500

1000

2000

3000

ThioBridgeTM ADC 2

0.4 mg/kg, Q4Dx4Vehicle

Dosing

Days post treatment

Tum

our

volu

me (

mm

3)

0 10 20 30 40 50 60 70 800

20

40

60

80

100

Kaplan-Meier Plot

VehicleControl ADC

Adcetris

ThioBridge ADC 1

ThioBridge ADC 2

Days

Perc

ent surv

ival

0 10 20 30 40 500

1000

2000

3000

ThioBridgeTM ADC 1Vehicle

0.4 mg/kg, Q4Dx4Dosing

Days post treatment

Tum

our

volu

me (

mm

3)

ThioBridge™Format 1, DAR 4

ThioBridge™Format 2, DAR 4

0 10 20 30 40 500

1000

2000

3000

Adcetris

0.4 mg/kg, Q4Dx4

Vehicle

Dosing

Days post treatment

Tum

our

volu

me (

mm

3)

Adcetris®

0 10 20 30 40 500

1000

2000

3000

Control ADC

0.4 mg/kg, Q4Dx4

Vehicle

Dosing

Days post treatment

Tum

our

volu

me (

mm

3)

Isotype Control ADC

Format 2

Format 1

18

Different linker architectures exhibit species specific serum stabilities: towards development of a suitable translation model

0 100 200 300 400 500 600 700 800 9000

1

2

3

4

Time (h)

Av

era

ge

DA

R

0 100 200 300 400 500 600 700 800 9000.1

1

10

100

1000

ADC 2

Adcetris

ADC 4

ADC 3

ADC 1

Time (h)

mA

b c

on

ce

ntr

ati

on

(

g/m

L) Change in Average DAR

in MiceChange in mAb Concentration

Format 4

Format 1

Format 2

Format 3

Adcetris®

Immobilisation Antigen or anti-Fc Ab on magnetic

beads

EvaluateElute from beads and determine

DAR by HIC

CaptureADC from serum

time

inte

ns

ity

CLEAVAGE

Analytical Method: CD30 affinity capture followed by HIC analysis to determine DAR is a suitable method for in vivo ADC stability evaluation

0

1

2

3

4

mouse rat monkey human PBS

Ave

rage

DA

R

Format 4 Format 2

ex vivo Analysis

19

Innate Pharma: Linker chemistry directly correlates to species-specific stability

- Effect of carrying out the deglycosylation as a consequence of linker choice meant retention of FcRn recycling but loss of FcR binding

a

b

c

d

Mouse serum loss of drug was more pronounced with the longer chemical linker

L’Hospice et al, Mol. Pharmaceutics, 2015, 12, 1872

20

Reagent Architecture Plays a Critical Role in Efficacy

ThioBridge™Side-chain PEG, DAR 4

ThioBridge™Short linear PEG, DAR 4

ThioBridge™Long linear PEG, DAR 4

ThioBridge™Bis side-chain, DAR 8

ThioBridge™No PEG, DAR 4

ThioBridge™a-cyclodextrin, (DAR 4)

ThioBridge™b-cyclodextrin, (DAR 4)

ThioBridge™g-cyclodextrin, (DAR 4)

Linear PEG

ThioBridge™Cyclic PEG(13u), DAR 4

ThioBridge™Cyclic PEG(7u), DAR 4

Cyclodextrins Cyclic PEG

21

ADCs produced with cyclodextrins of different sizes showed different potencies in vivo

Extending linker design options: impact of cyclodextrin on in vivo potency

0 10 20 30 40 50 60 70 800

500

1000

1500

2000

Vehicle

0.5 mg/kg

1 mg/kg

Days post tumor induction

Absolu

te tum

our

volu

me (

mm

3)

0 10 20 30 40 50 60 70 800

500

1000

1500

2000

Vehicle

0.5 mg/kg

1 mg/kg

Days post tumor induction

Absolu

te tum

our

volu

me (

mm

3)

0 10 20 30 40 50 60 70 800

500

1000

1500

2000

Vehicle

0.5 mg/kg

1 mg/kg

Days post tumor induction

Absolu

te tum

our

volu

me (

mm

3)

ThioBridge™Cyclodextrin

Format A, DAR 4

ThioBridge™Cyclodextrin

Format B, DAR 4

ThioBridge™Cyclodextrin

Format C, DAR 4

Karpas-299 xenograft model at 0.5 and 1.0 mg/Kg (single dose, i.v. administration at day 14)

0 10 20 30 40 50 60 70 800

500

1000

1500

2000

Vehicle

(0.5 mg/kg)

(1 mg/kg)

Days post tumor induction

Absolu

te tum

our

volu

me (

mm

3)

Adcetris®Average DAR ~4

22

The brentuximab ThioBridge™ Looped PEG ADC showed enhanced

in vivo efficacy in a Karpas299 CD30 +ve cell line over Adcetris®

Extending linker design options: impact of PEG polymer optimisation on in vivo potency

Karpas-299 xenograft model at 0.4 and 0.8 mg/kg (single dose, i.v. administration at day 14)

Adcetris®ThioBridge™ Cyclic PEG(13u) ADC (DAR 4)

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0G 1 : V e h ic le

G 8 : V a ria n t 4 (0 .4 m g /k g )

G 9 : V a ria n t 4 (0 .8 m g /k g )

D a y s p o s t tu m o r in d u c tio n

Ab

so

lute

tu

mo

ur

vo

lum

e (

mm

3)

7 /8 C R

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0 G 1 : V e h ic le

G 1 0 : V a r ia n t 5 (0 .4 m g /k g )

G 1 1 : V a r ia n t 5 (0 .8 m g /k g )

D a y s p o s t tu m o r in d u c tio n

Ab

so

lute

tu

mo

ur

vo

lum

e (

mm

3)

8 /8 C R

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

G 1 2 : A d c e tr is (1 m g /k g )

D a y s p o s t tu m o r in d u c tio n

Ab

so

lute

tu

mo

ur

vo

lum

e (

mm

3)

1 /8 C R

L e ge nd

ThioBridge™ Cyclic PEG(7u) ADC (DAR 4)

23

Conjugation Site and Linker Architecture Can Impact FcgRIIIa binding and influence the therapeutic window

Ligand KD (M) RMAX (RU) Chi² (RU²)

Naked Antibody 1.54E-06 35.7 1.24

ThioBridge™ Format 2 6.10E-06 28.3 0.00648

Kadcyla 9.61E-07 37 0.886

ThioBridge™Format 2

KadcylaNaked Antibody

Trastuzumab ADCs show different potencies in

triggering the FcgRIIIa pathway.

Potential safety implications

ThioBridge™Format 2

Analytical Method: SPR

Analytical Method: Cell Reporter Assay

Kadcyla

Naked Antibody

Opsonized cancer target cells are co-incubated with engineeredJurkat effector cells expressing the FcgRIIIa receptor. Signalinitiated in engineered Jurkats is detected by luminescence.Reporter cells supplied by Promega.

24

ADC drug development process flow and support across scientific groups is driven by linker design and synthesis

ADC Production In vitro Potency2D CellTitre Glo®Limited exposure3-D CellTitre Glo®By-stander effectStress test

Cellular PDInternalisationCell cycle arrestSOC resistant cellsADCC/ADCP/CDCCell binding

DAR purityIntact mass analysisAggregationEndotoxin testing

In vivo efficacy

Mouse xenograftSC/orthotopic/PDX models

In vivo PKMouse PKRat PKNHP PK

SafetyFree drug quantificationFcg receptor bindingImmunogenicityRat MTD*NHP tolerability*

FormulationHeat stress aggregationTurbidity & opalescenceThermal shiftLyophilisationAccelerated stability

0 100 200 300 400 500 600 700 800 9000.1

1

10

100

1000

ADC 2

Adcetris

ADC 4

ADC 3

ADC 1

Time (h)

mA

b c

on

ce

ntr

ati

on

(

g/m

L)

Conc (pM)

Ce

ll V

iab

ility

[%

]

100 101 102 103 104

0

50

100

MMAE

Adcetris

ThioBridge™ ADC 1

ThioBridge™ ADC 2

ThioBridge™ ADC 3

ThioBridge™ ADC 4

Apoptosis assay (Caspase-3 and -7 activity) - 48hrs incubationAnti-PSMA Conjugates included in mouse xenograft study 140294

Compound Concentration (nM)

Ap

op

tosis

(Fo

ld o

ve

r U

ntr

eate

d c

ells)

0.001 0.01 0.1 1 100

1

2

3

4

5

6

7

8 PT074-BM004

PT074-BM005

PT073-MP007

PT74-TK002

PT073-MP005

PT073-MP006

PT074-BM006

PT074-TK007

PT55-ED006-3

PT55-TK004-2

PT55-TK003-1

PT55-ED006-1

PT55-ED006-2

Bioimaging

Tritium labellingChelator PET imaging

Reagent ProductionToxin synthesisRoute optimisationPurity by HPLCStructure by MS & NMR

Ex vivo Stability

Serum m/r/m/h stabilityCathepsin B digestionLysosomal digestion

Pilot productionToxin synthesisToxin-linkers & ADCDoE process optimisationLonger term stability

GMP ManufactureToxin 100’s g scaleToxin-linker kg scaleADC 100’s g scale

The process flow demonstrates the required capabilities for supporting each step of ADC development

25

Summary of linker chemistry

- Linker compatibility with the protein including optimal attachment points

- Match the linker with the payload, and the target environment

- Reintroduction of known and robust chemistries updated for use in novel linkers

- The archive of chemical transformations remains a source of inspiration for new and innovative approaches to the ADC and linker bioconjugate linker chemistries

- Medicinal chemistry approaches are now driving the ADC field

Thank you