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
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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
te
A5
49
HC
T1
16
NC
I -H
52
6
Pa
nc
2. 1
3
Lu
- 01
- 00
07
NC
I -H
29
2
MD
A- M
B- 4
68
MD
A- M
B- 4
68
La
r ge
NC
I -H
32
2
Lu
- 01
- 04
12
CT
G- 1
77
1
CT
G- 1
17
1
CT
G- 1
10
6
CT
G- 0
89
6
- 2 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
% 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
mo
ur
Vo
lum
e (
mm
3)
^ ^ ^^
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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