BT8009: A bicyclic peptide toxin conjugate targeting Nectin-4 (PVRL4) displays efficacy in preclinical tumour models

Mike RigbyAACR Annual Meeting 2019 Atlanta 4479

Bicycles®: a new therapeutic modality

• Bicycles® are bicyclic peptides :

• Highly constrained: sub-nM binding affinities

• Large surface area and large binding footprint, so can disrupt protein-protein interactions

• New Chemical Entity classification and fully synthetic molecules

• Easily conjugated to deliver additional functionality; e.g. chelated radionucleotides, fluorescent dyes, affinity tags, PK extenders and cytotoxic agents

• Mw of 1.5-2kDa compared with 150kDa for an antibody

2 Bicycle AACR 2019 #4479

Scaffold

Loop 1 Loop 2

AA

AA AA

AA

AAScaffold

Loop 1 Loop 2

AA

Bicycle AACR 2019 #4479

Bicycles®️: phage display to derive hits

Linear peptide

Bicycle DNA Sequence

Gene III

Phage particle

Bicycle

Chemical modification with scaffold

3Amplify

2Select

1Cyclise

POC in 6 wk, Optimised

lead in 9mnth

• Enormous diversity (upto 1017)• Sequence, loop size, symmetry, scaffolds

• Evolution driven• Informed selection• Low synthetic burden

Phage panning

3

Bicycle AACR 2019 #4479

Nectin-4: Biological rationale

• Nectin-4; cell adhesion molecule

• Widely expressed during development

• restricted expression in maturity – epithelial cells e.g. skin, airways, esophagus/stomach and bladder.

• Member of Nectin family and close relative to Nectin-like family

• Other family members more widely expressed through body

• Over expression in numerous tumours; highest frequency in bladder, breast, and pancreatic, but also in lung and esophageal cancers

• Nectin-4 targeting ADC, enfortumab vedotin, in Phase 2 - 3 trials, for metastatic urothelial carcinoma, with “Breakthrough Therapy Designation”

Cancer type

% s

am

ple

s b

y e

xp

ressio

n level

Lung SQ/A

deno

Lung met

asta

tic

Bre

ast

Pan

crea

tic

Ova

rian

Bla

dder

Hea

d/Nec

k

Oes

ophagea

l

0

20

40

60

80

100negative

low

moderate

Strong

62% 60% 78% 71% 57% 83% 59% 55%

Challita-Eid et al Cancer Res 76: 3003-3013 (2016)

Rabet et al Annals of Oncology 28:769-776 (2017)

Nectin-4 Protein expression

4

Normal BreastTNBC

Bicycle AACR 2019 #4479

Bicycle optimization from lead to Bicycle® Toxin Conjugate; BT8009

Stabilised Bicycle

Amino Acids Important For Target Engagement

Optimised Bicycle

Ki (nM) cLogP T1/2 (plasma)

18.4 -6.98 1.3h

Parent Bicycle

Ki (nM) cLogP T1/2 (plasma)

13 -6.74 >24h

Ki (nM) cLogP T1/2 (plasma)

3.21 ‐13.32 >24h

+ affinity + hydrophilicity

Improve stability, hydrophilicity

Improve half-life and hydrophilicity, whilst retaining binding AAs

Increase affinity and improve hydrophilicity. Selected as candidate peptide binder for Bicycle Toxin conjugate, BT8009

Poor solubility, short half-life. AAs required for binding identified

Ac C P F G C M K N W S W P I W C

Ac C P F d C M hArg N W S W P I W C

Ac C P 1Nal d C M hArg D W S T P Hyp W C

+ affinity,hydrophilicity

BT8009; Bicycle Toxin conjugate

Cys

Pro

1Nal

D-Asp

Met

Cys

TrpSer

Thr

Pro

HyP

Trp

Cys

AspHArg

N-terminus

C-terminusSarSarSarSarSarSarSarSarSarSarΒ-Ala

Toxin MMAE Cleavable linker Sar10 Spacer Bicycle Binder5

Solubility = 4.9 ug/ml, LogD = -0.7

Solubility = 420 ug/ml, LogD < -2.2

Bicycle AACR 2019 #4479

Human Nectin KD (nM) Human Nectin-like KD (nM)

Subtype 1 2 3 4 1 2 3 4 5

BT8009No

bindingNo

bindingNo

binding3.02 No

bindingNo

bindingNo

bindingWeak @

5 uMNo

binding

BT8009 shows excellent selectivity over the close family members of Nectin and Nectin-like family

Parameter Human Cynomolgus Rat Mouse

Affinity for native Nectin-4 (KD) 3.02 1.14 4.42 Not tested

PPB(% unbound) 20.7 20.7 19.4 11.8

Blood/plasma partition (recovery %) 0.64 0.37 0.65 Not tested

Plasma stability (T1/2 h) >57.8 >57.8 60.7 4.4

Blood stability (T1/2 h) 26.1 28.3 8.5 5.0

Hepatocyte stability (T1/2 h) >3.61 >3.61 >3.61 Not tested

BT8009; overall in vitro characteristics

BT8009 shows appropriate cross-species characteristics to enable safety studies

6

0 7 1 4 2 1 2 8 3 5 4 2 4 9 5 6 6 3 7 0 7 7 8 4 9 1

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

D a y s a f t e r s t a r t o f d o s i n g

Tu

mo

r V

olu

me

(m

m3

)

V e h i c l e

B T 8 0 0 9 5 m g / k g q 2 w

B T 8 0 0 9 3 m g / k g q w

Bicycle AACR 2019 #44797

BT8009 binds Nectin-4 on MDA-MB-468 cells, and shows efficacy in xenograft model

ICC of cells using anti-MMAE antibody. After preincubation with BT8009, a non-binding Bicycle Toxin Conjugate (BTC) or MMAE demonstrates only BT8009 is retained on cell surface

FACS shows Nectin-4 surface

expression

BT8009 shows excellent efficacy in MDA-MB-468 xenografts, even against

larger tumours

7

1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

- 5

5

1 5

B T 8 0 0 9 K d = 1 2 n M

n o n b i n d i n g B T C

C o n c e n t r a t i o n [ n M ]

RF

U

1 uM BT8009 1 uM Non-binder 1 uM MMAE

BT8009: In vivo PK

BT80091 mg/kg

CLp(ml/min/kg)

Vss(L/Kg)

T1/2 (h)

BT8009 MMAE

Cmax(uM)

AUC(uM. h)

Cmax(uM)

AUC(uM. h)

Mouse 3.5 0.25 0.98 1.401 1.131 0.065 0.103

Rat 9.4 0.44 0.86 1.114 0.432 0.013 0.022

BT8009 affords rapid and long lasting MMAE retention in MDA-MB-468 tumour, with rapid plasma clearance of toxin and parent.

Lasting tumour retention also demonstrated in other models.

BT8009 shows high Cmax with a short plasma half-life, reflective of rapid clearance from systemic circulation, characteristic of Bicycles and unlike ADCs

Bicycle AACR 2019 #4479

0 4 8 1 2 1 6 2 0 2 4

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

h

[M

MA

E]

pm

ol/

ml

or

pm

ol/

g

M M A E T u m o u r

M M A E P l a s m a

B T 8 0 0 9 P l a s m a

BT8009 3 mg/kg

8

An

aly

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HC

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NC

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52

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Pa

nc

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6

- 2 0 0

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6 0 0

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% c

ha

ng

e i

n t

um

ou

r s

ize

v e h i c l e D a y 1 4 3 m g / k g B T 8 0 0 9 D a y 1 4

3 m g / k g B T 8 0 0 9 D a y 2 8v e h i c l e D a y 2 8

Bicycle AACR 2019 #4479

BT8009 efficacy correlates with expression in CDX/PDX xenografts

Xenografts with low/no Nectin-4 expression show reduced tumour growth. Xenografts expressing Nectin-4 show good tumour regression

nullIncreasing protein

expression High RNA

Full regression-100

0 1 4 2 8 4 2 5 6 7 0 8 4 9 8 1 1 2

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

V e h i c l e , i v , q w

B T 8 0 0 9 , 1 / 3 / 5 m g / k g , q w

B T 8 0 0 9 , 3 m g / k g , q w

D a y s a f t e r s t a r t o f d o s i n g

Tu

mo

ur

Vo

lum

e (

mm

3)

^^ ^ ^^ ^ ^^

0 7 1 4 2 1 2 8 3 5 4 2 4 9 5 6 6 3 7 0 7 7 8 4 9 1 9 8

0

2 0 0

4 0 0

6 0 0

V e h i c l e , i v , q w

B T 8 0 0 9 , 1 m g / k g , q w

B T 8 0 0 9 , 3 m g / k g , q w

^ ^ ^^D a y s a f t e r s t a r t o f d o s i n g

Tu

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mm

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Lung adenocarcinoma PDX

Lung squamous carcinoma PDX

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BT8009: A Nectin-4 targeting Bicycle Toxin Conjugate, for the treatment of Solid Tumours

Bicycle AACR 2019 #4479

• Nectin-4 is highly expressed on tumour cell surface in a wide range of solid tumours

• BT8009 was developed as a Bicycle Toxin Conjugate to target Nectin-4

• High affinity binding, highly selective for Nectin-4

• Short plasma half–life, with renal elimination

• Hit and run delivery of toxin to tumour cells, minimising systemic exposure

• BT8009 shows excellent efficacy in multiple PDX and CDX models, with rapid regression in small and large tumours

• Efficacy in NSCLC, TNBC, Esophageal and bladder PDX models

• Efficacy is dose related and correlates with expression of the Nectin-4 target

• PK shows retention of toxin in tumour, well in excess of systemic clearance

• IND enabling toxicology studies with BT8009 are ongoing

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