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Di d Ch t i ti f E 5 i hibitiDiscovery and Characterization of Eg5 inhibition based ADC

19th RSC-SCI Medicinal Chemistry Symposium Cambridge UK19th RSC-SCI Medicinal Chemistry Symposium Cambridge, UK.September, 2017

Cristina Nieto-Oberhuber, PhD, Novartis Institutes for Biomedical Research Basel SwitzerlandNovartis Institutes for Biomedical Research, Basel, Switzerland

ADC as Cancer TherapeuticsADC schematic: ADC trafficking and release of drug:

Adapted from Solot et al, Nat. Rev. Drug Disc. 2013

Antibody Linker DrugAdapted from Senter et al, Nat. Biotech. 2012

Antibody-Linker-Drug

Modular drug comprised of:• Monoclonal antibody specific to tumor antigen

• Kadcyla™ (Immunogen, Genentech/Roche) is approved for breast cancer (DM1 Maytansine)• Monoclonal antibody specific to tumor antigen

• Chemical linker (cleavable or non-cleavable)

• Potent LMW compound, most commonly

for breast cancer (DM1, Maytansine)

• Adcetris ™ (Seattle Genetics) is approved for NHL/ALCL (Auristatin)

cytotoxic • >/= 30 additional ADCs in the clinic– Sievers, E. L.; Senter, P. D. Annu. Rev. Med.

2013, 64,15.

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ADC as Cancer Therapeutics• IgGs are large hydrophilic molecules (150kDa) that do not diffuse across membranes and are above renal filtration limit (t1/2 ca. 4

weeks in humans)

Antibody and Payload Conjugation

• Serve as a vehicle for selective delivery of the drug to the tumor cell

• The primary routes of clearance of IgG1 are:

– Pinocytosis

– Receptor (target)-mediated endocytosis

• Conjugation Strategies

Adapted from Panowski et. al. 2014. mAbs 6(1):34-45

Challenges of Conventional Conjugation Advantages of DAR controlled conjugates:Challenges of Conventional Conjugation• High DAR species

-Increased toxicity-Rapid clearance leads to poor PK-Reduced stability/high hydrophobicity

• Disruption of native amino acids

Advantages of DAR controlled conjugates:• Improved stability• Improved PK • Reduced off-target toxicity• Increased efficacy• Newer clinical ADCs are focusing on

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s up o o a e a o ac ds• Manufacturing challenges• Mature clinical ADCs are heterogeneous with respect to Drug Antibody Ratio (DAR)

e e c ca Cs a e ocus g ocontrolled DAR ADCs

3

ADC as Cancer Therapeutics

Proteolysis in lysosome MMAE(active cell metabolite)

SGN-35 (AdcetrisTM)Cleavable Linkers

Cleavable linkers are degraded in lysosomes to generate free payload which is the active species

( )

• Pros:

– Rapid and complete release of parent LMW payload as active species

Direct correlation of payload properties to ADC properties (potency permeability hydrophobicity etc)

Spontaneous 1,6 elimination

– Direct correlation of payload properties to ADC properties (potency, permeability, hydrophobicity, etc)

• Cons:

– Potential instability of linker outside tumor cell (i.e. due to extracellular protease activity) leading to unselective delivery of payload and toxicityp y y

Lysosomal enzymes

Proteases(Cathepsins)

-Glucuronidase Thiol redox system

Environmental difference

Clea able linkers can lead to species that ma • Val-Cit• Phe-Lys• Val-Lys• Val-Ala• Gly-Gly-Phe-Gly• Gly-Gly-Gly

Phosphodiesterases pH sensitive

Cleavable linkers can lead to species that may permeate and kill adjacent non-antigen presenting cells.

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Gly-Gly-Gly

4

ADC as Cancer Therapeutics

T SMCC DM1 (K d l TM)

Non-cleavable Linkers

LysosomalProcessing

T-SMCC-DM1 (Kadcyla ):

• Pros:

– (Ideally) Lack of chemical or enzymatic extracellular degradation to prevent release of toxic metabolites

Active Cell Metabolite

– Charged active cell metabolite (aa-linker-payload) possesses intrinsic low permeability with lower potential for off-targeteffects

• Cons:

Properties of charged active metabolite (aa linker payload) which allow to cross the lysosomal membrane and reach– Properties of charged active metabolite (aa-linker-payload) which allow to cross the lysosomal membrane and reachcellular target are unknown

– Cell activity and enzymatic potency of cell metabolite are not predictive of ADC activity

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ADC as Cancer TherapeuticsPayload Selection: Why is Eg5 an Attractive Target

• Eg5 is a motor protein required for centrosome separation

• Inhibition of Eg5 causes monopolar spindle formation, leading tomitotic arrest triggering apoptotic cell death

• Unlike other anti-mitotics (taxanes, epothilones & Vinca alkaloids),inhibition of Eg5 does not affect microtubule stability

Normal Mitosis Cdc2/cyclinB(ATP)

g y

• Evidence that Eg5 inhibition only targets dividing cells & thusshould not cause neurotoxicities, unlike microtubule disruptors

• Non ATP competitive allosteric inhibitor

Eg5- P Eg5G2/M

• Non-ATP competitive allosteric inhibitor

10 nMEg5 Inhibitors IC50 vs cell panel

Eg5 Inhibition

-tubulin (green)Chromatin (blue)

Adapted from Mayer et al; Science 1999 286 971-974

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Adapted from Mayer et al; Science, 1999, 286, 971-974

ADC Optimization: Cleavable LinkerIn Vitro Profile

> 50 E 5 l d t t d

Payload 1Eg5 IC50: <0.5 nMKB3.1 GI50: 0.3 nMKB8 5 GI : 0 4 nM

> 50 Eg5 payloads testedwith same linker

Payload 2Eg5 IC50: <0.5 nMKB3.1 GI50: 0.1 nMKB8.5 GI50: 1.2 nM

ADC-1• high oligomerization (> 60%)

ifi ll bi di

KB8.5 GI50: 0.4 nM

ADC-2 • low oligomerization (~ 10%)• specific cell binding and activity

Payload SAR for ADCpotency and aggregationestablished

KB8.5 GI50: 1.2 nM

L1: MC-ValCit-PABC

• unspecific cell binding • specific cell binding and activity• most potent ADC on several cell lines

TBS-ADC-1 MDA-MB-231 DAR: 2.8TBS-ADC-1 MDA-MB-231 Clone 16 DAR: 2.8TBS-ADC-2 MDA-MB-231 Clone 16 DAR: 2.8TBS-ADC-2 MDA-MB-231 DAR: 2.8

bitio

n

• Her2-dependent activity of ADCsinhi

b

MDA-MB-231: Low HER-2 ExpressionMDA-MB-231 Clone 16: High HER-2 Expression

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7

g p

ADC Optimization: Cleavable LinkerIn Vivo Efficacy: Antigen Independent Activity

n = 9/groupNo significant body weight loss in any group1500

SK-OV-3ip)

Payload 2Eg5 IC50: <0.5 nMKB3.1 GI50: 0.1 nMKB8.5 GI50: 1.2 nM

1000

IV Doseolum

e (m

m3

n ±

SEM

ADC-2 • low oligomerization (~ 10%)• specific cell binding and activity

KB8.5 GI50: 1.2 nM

Vehicle0

500IV Dose

Tum

or V

om

ean • specific cell binding and activity

• most potent ADC on several cell lines

1 mg/kg TBS-ADC-2

3 mg/kg TBS-ADC-2

1 mg/kg gH-ADC-2 (isotype)

3 mg/kg gH-ADC-2 (isotype)

Time Post-Implant (Days)

10 20 30 40

3 mg/kg gH ADC 2 (isotype)

Conclusion: The minimal efficacious dose for TBS-ADC-2 is 3 mg/kg; however, we observe significant

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gH-ADC-2 Her2-independent activity in this model.

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MedChem Strategy for Improving ADCs

• 3 distinct linker attachment vectors oriented towards the solvent were identified

Structure Based Design

Stage 1: payload morphing on each region separately using focused set of common linkers

Payload 2 Stage 2: combine positive SAR with best linker and payload options to maximize ADC potency

Key selection criteria for new payload-linker combinations:

• metabolite with strong target inhibition (LOQ ~ 0.5 nM)

• ADC with acceptable level of aggregation (<10%)

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• stable in rodent serum and in lysosome extracts

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MedChem Strategy for Improving ADCs

Scaffold 1 Scaffold 3

Exit Vector Identification

Scaffold 1

Scaffold 3Scaffold 2

Scaffold 2

Payload-2 @ Eg5 site

Linker Toolbox

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ADC Optimization: Non-Cleavable Linker

ADC TBS TBS SK-OV-3ip Anti-c-Kit Anti-c-Kit NCI-H526

A (%) DAR AC / L A (%) DAR AC / L

In Vitro Profile: Antigen Depending Activity

Aggreg. (%) DAR AC50 ng/mL Aggreg. (%) DAR AC50 ng/mL

ADC-3 4.3 5.0 9 5.0 4.2 55

ADC-4 2.6 4.4 17 5.0 4.1 13

ADC-5 2.4 4.6 43 5.0 3.7 55

ADC-6 2.2 4.6 16 4.0 3.5 286

ADC-7 2.5 4.7 12 5.0 3.6 38

TBS-ADC-4 SK-OV-3 DAR: 4.4TBS-ADC-4 MDA-MB-468 DAR: 4.4TBS-ADC-6 SK-OV-3 DAR: 4.6TBS-ADC-6 MDA-MB-468 DAR: 4.6TBS ADC 7 SK OV 3 DAR 4 7

Anti-c-Kit-ADC-7 NCI-H526 DAR: 3.6Anti-c-Kit-ADC-5 NCI-H526 DAR: 3.7Anti-c-Kit-ADC-4 NCI-H526 DAR: 4.1Anti-c-Kit-ADC-3 NCI-H526 DAR: 4.2A ti Kit ADC 6 NCI H526 DAR 3 5

ibiti

on

TBS-ADC-7 SK-OV-3 DAR: 4.7

ibiti

on

Anti-c-Kit-ADC-6 NCI-H526 DAR: 3.5

inh

inhi

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SK-OV-3ip: High HER-2 ExpressionMDA-MB-468: Low HER-2 Expression

ADC Optimization: Non-Cleavable Linker

n = 9/groupNo significant body weight loss in any group 1500

n = 5/groupNo significant body weight loss in any group

In Vivo Efficacy: Dose Dependency

1000

1500

olum

e (m

m3 )

±SE

M

NCI-H526

750

1000

me

(mm

3 )SE

M

SK-OV-3ip

IV Dose

500

Tum

or V

om

ean

±

IV Dose

250

500

Tum

or V

olum

mea

n ±

S IV Dose

10 20 300

Time Post-Implant (Days)

5 10 15 20 25 300

Time Post-Implant (Days)

Vehicle

gH-ADC-6 - 9 mg/kg

TBS-ADC-6 - 5 mg/kg

S C /

Vehicle

gH-ADC-6 - 10 mg/kg

Anti-c-Kit-ADC-6 - 5 mg/kg

Anti-c-Kit-ADC-6 - 10 mg/kgTBS-ADC-6 - 10 mg/kg

TBS - 10 mg/kg

Anti-c-Kit-ADC-6 - 10 mg/kg

• TBS-ADC-6 shows improved efficacy compared with antibody alone• No antigen independent activity was observed for ADC using non-cleavable linkers

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No antigen independent activity was observed for ADC using non cleavable linkers• Dose dependent tumor growth inhibition was observed in both models

ADC Optimization: Non-Cleavable LinkerIn Vivo Efficacy Across different ADCs

n= 8/groupNo significant body weight loss in any group

n = 5/groupNo significant body weight loss in any group

SK-OV-3ip

1200

1600

me

(mm

3 )SE

M

1000

1500

me

(mm

3 )SE

M

NCI-H526

400

800

Tum

or V

olum

mea

n ±

S

IV Dose500

Tum

or V

olu

mea

n ±

IV Dose

10 20 30 40 500

Time Post-Implant (Days)

T

5 10 15 20 25 300

Time Post-Implant (Days)

VehicleTBS, 10 mg/kgado-trastuzumab emtansine, 5 mg/kgTBS-ADC-3, 5 mg/kg

Vehicle

Anti-c-Kit-ADC-4Anti-c-Kit-ADC-3

Anti-c-Kit-ADC-5

TBS-ADC-6, 5 mg/kgTBS-ADC-4, 5 mg/kg

• Sub efficacious dosing allowed differentiation of the ADC across the 2 types of tumors

Anti-c-Kit-ADC-6

Anti-c-Kit-ADC-7

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• In-vitro superiority does not translate into superior in-vivo efficacy

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ADC Optimization: Non-Cleavable LinkersPK Profile and Stability Assessment

ADC 6 ADC 6 ADC 4 ADC 4Mean ADC and Total Ab Concentrations in Plasma

Compound ADC-6 ADC

ADC-6 Total Ab

ADC-4 ADC

ADC-4 Total Ab

Cmax (µg/mL) 186 174 152 115

AUCINF DN (hr*kg*µg/mL/mg) 2090 3350 1580 2130

TBS-ADC-4, 1 mg/kg (Total Ab)TBS-ADC-4, 1 mg/kg (ADC)

( g µg g)

t1/2 (hr) 130 148 201 199CL (mL/hr/kg) 0.48 0.3 0.631 0.482

Vss (L/kg) 0.0882 0.0766 0.16 0.142

120Relative DAR

(%)

DAR Evolution per ADC

60

80

100• Integrity of ADC species is conserved throughout the

study since no derived species is observed beside the result of the hydrolysis of the maleimide moiety.

0

20

40

0 200 400 600 800Time (Hours)

• The DAR of 2 ADC’s is decreasing over time. At 28 days, the DAR is about 50% from the initial value for TBS-ADC-6 and TBS-ADC-4.

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0 200 400 600 800

Each plot corresponds to an average of the data derived from the 3 rats

ADC Optimization: Non-Cleavable LinkersPK/PD Assessment

Tumor PK

Putative Catabolite

10 mg/kg TBS ADC 6 dose (N 3 per

pHH3 IHC PD

10 mg/kg TBS-ADC-6 dose (N = 3 per group)

Vehicle 6 hr 24 hr 48 hr 72 hr 96 hr 168 hr gH-ADC-6

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Anti-HER2-ADC-624 hr

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ADC Optimization: Non-Cleavable LinkersIn-Vivo Efficacy: Antigen Depend Activity

n= 8/groupNo significant body weight loss in any group

SKOV3ip Xenograft (Her2+ ckit-)600

)

NCI-H526 Xenograft (Her2- cKit+)2500

3 )

g y g y g p

400

olum

e (m

m3 )

n ±

SEM

IV Dose

1000

1500

2000

Volu

me

(mm

3

an ±

SEM

0

200

Tum

or V

om

ean

0

500

1000

Tum

or V

mea IV Dose

Vehicle

gH-ADC-6 - 10 mg/kg

Time Post-Implant (Days)10 11 12 13 14 15 16

0

Time Post-Implant (Days)0 5 10

0

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gH ADC 6 10 mg/kg

Anti-c-Kit-ADC-6 - 6.5 mg/kg

TBS-ADC-6 - 10 mg/kg

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Conclusions

• Identification and validation of a novel MoA (Eg5, motor protein inhibiton) as an ADC ( g p )target

• ADCs with Val-Cit cleavable linker display high aggregation and target-independent activity

• Structure Based Drug Design identified new linker attachment points which retained payload potency and displayed optimized properties

• ADCs optimization was achieved through combined linker-payload (L-P) SAR p g p y ( )

• Several non-cleavable L-P combinations using Eg5 inhibitors which exhibited in-vitroantibody dependent activity were identified

• In vivo efficacy was demonstrated with multiple ADC candidates targeting selectively• In-vivo efficacy was demonstrated with multiple ADC candidates targeting selectively HER-2+ cell lines or c-KIT+ cell lines

• A cross-over efficacy study using L-P ADC-6 conjugated to a HER-2 and a c-Kit antibody supports ADC selectivity by demonstrating antigen dependent tumor regression in micesupports ADC selectivity by demonstrating antigen dependent tumor regression in mice

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Acknowledgement

Grazia Piizzi Alexei Karpov

Mauro ZuriniPiotr Martyniuk

Patrick CheneYannick Mesrouze

Bernhard GeierstangerWeijia Oup

Marc Lafrance Robert GrotzfeldMarion Lacaud-BaumlinLionel DoumampouomStephanie Lagasse

Piotr MartyniukPatrick SchindlerJean-Marc SchlaeppiThierry BessonMichele CoulotEdwige Fongue

Yannick MesrouzeC. Uli BialuchaTinya AbramsUrsula Jeffry Sanela BilicWolfgang Hackl

Weijia OuBill Mallet Kristine VenstromPeiyin WangPayman AmiriMina AikawaStephanie Lagasse

Darryl JonesMelanie VelayEmilie JolyEnrique Blanco

Edwige FongueTorsten KuipeBrendan KerinsMikias WoldegiorgisEric FangMi h l Kiff

Wolfgang Hackl Christie FantonJochen EisfeldPaul Kwon David RewolinskiBjoern Gruenenfelder

Mina AikawaMike DoyleLi ZhangDylan DanielEdward LorenzanaYoko Oei

Nikolaos DrososEtienne RichardFaouria Boinali-DervisagicPavel FedoseevFrancesca Perruccio

Michael KiffeBernard FallerStephan GrueningerBrian GrandaNancy Lewicki

Bjoern GruenenfelderSandy Huynh Bill SellersEmma LeesMike DillonBill Sellers

o o OeSuzy ClarkMark Knapp Robert EllingPatrick RudewiczYing-Bo Chen

Paul BarsantiTetsuo Uno Rainer KneuerAnne BaslerMajid Ghoddusi

Melissa RamonesAmin KamelKaren WangVladimir CapkaDaniel Wall

Bill SellersScott LesleyThomas PietzonkaAndreas MarzinzikCasrten SpankaJudith Abraham

gKeshi WangHarry Sterling Gavin DollingerKathy MillerAlessandro PalumboMajid Ghoddusi

Zhen Wang Kan ZhuJudith AbrahamSteve Bender

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