Activation of CD137 using multivalent and tumour targeted ......G ra n B IF N b IF N g IL -2 IL -6 T...

Activation of CD137 using multivalent and tumour targeted bicyclic peptides

Punit Upadhyaya

Peptide Congress 2019

• Internal focus on oncology

• BT1718 – Phase 1/2a (Cancer Research UK)

• 2nd Generation Bicycle Toxin Conjugates® in pre-clinical development

• Bicycle® T cell modulators and Bicycle® targeted innate immune activators

in lead optimization

• Key strategic partnerships outside oncology

Bicycle Therapeutics

• Founded by Sir Gregory Winter & Prof. Christian Heinis

• UK & US based (Cambridge, UK; Boston, USA)

Peptide Congress April 20192

3

Highly constrained: high affinity, exquisite selectivity, excellent stability

Large binding footprint: disrupt protein-protein interactions

Fully synthetic: NCE classification and synthetic control

Highly flexible modality: modular building blocks retain pharmacology

Adjustable PK: excellent tissue penetration, renal elimination, tuneable T1/2

COOH

X

NH2

Linear peptide

NH2 COOH

Bicycle

Chemical modification with scaffold

Bicycles®: a new therapeutic modality

Loop 1

Loop 2

Scaffold

=


Bicycle Toxin Conjugates® : hit and run delivery of toxins to tumour cells

4 Peptide Congress April 2019

Retained in the

tumour

Rapidly eliminated

BT8009 affords rapid and long lasting MMAE retention with rapid plasma clearance of toxin and parent.

Mouse PK

BT8009

Toxin MMAE Cleavable linker Sar10 spacer Nectin-4 Bicycle Binder

0 4 8 12 16 20 241

10

100

1000

10000

h

[MM

AE]

pm

ol/

ml o

r p

mo

l/g

MMAE Tumour

MMAE Plasma

BT8009 Plasma

Bicycle Toxin Conjugates® : hit and run delivery of toxins to tumour cells

5 Peptide Congress April 2019

Retained in the

tumour

Rapidly eliminated

BT8009

BT8009 shows excellent efficacy in large and small MDA-MB-468 xenografts

Toxin MMAE Cleavable linker Sar10 spacer Nectin-4 Bicycle Binder

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

1 2 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 ic le

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

Signal 2:Costimulatory/

Coinhibitory

Signal 1:MHC-Antigen

T cell receptor (TCR)+

Exploring Bicycles® as T cell agonists

6

Adapted from Mellman et al. Nature 480, 480-489 (2011)

Short acting Bicycles have been validated as toxin conjugates, will they offer advantages as T cell modulators?


Antigen Presenting Cell: T cell Interaction Costimulatory/Coinhibitory Signal

Agonistic Molecules

BlockingMolecules

Bicycle® CD137 multimers

Receptor complexity fits Bicycles’® tolerance for multimerization

• CD137 is member of TNF superfamily, requires trimerisation for activation

8

Loop 1 Loop 2

CD137 Lead Bicycle

8

Phage campaign identifies leadCD137 Bicycle

Naïve phage peptide KD = 1.4uM

Affinity matured phage peptide KD = 67nM (Wild Type: WT)

Chemically optimised KD = 5nM (High Affinity: HA)

Human SpecificNo Rodent

X-reactivityCRD1

CRD2

CRD3

CRD4

CD137

TM

CD

CD137L

Antigen presenting cell

ActivatedT cell

CD137 agonism by trimeric ligand

Proposed CD137 agonism by

multimeric Bicycle


Adapted from Chin et al. Nat Commun 9, 1-13 (2018)

Chemically enabled optimization of CD137 multimers

9 9Peptide Congress April 2019

Reporter cell based screening of CD137 multimers

10 10Peptide Congress April 2019

CD137

Jurkat reporter

NF-kB Luciferase

-14 -12 -10 -8 -60

2

4

6

8

Log (concentration [M])

Fo

ld in

du

cti

on

BCY7838

BCY8960

BCY8958

BCY8947

CD137 multimers are active in primary immune cell assays

11

Primary human T cells(cytokine release)

Primary human immune cells(tumour cell killing)

BCY7839 = Trimer WT affinity Bicycle

BCY7842 = Tetramer WT affinity Bicycle

BCY8945 = Tetramer High affinity Bicycle


0 20 40 60 80 100 120

0

250

500

750

1000

Hours

Casp

ase 3

/7 A

cti

vit

y

(GC

U x

um

2p

er

imag

e)

DMSO

Urelumab

Monomer

BCY8945

BCY78

42

BCY89

45

BCY78

39

Mon

omer

Ure

lum

ab

0

1

2

3

4

5

IL-2

rele

ase f

old

ch

an

ge

(no

rmali

ze

d t

o C

D3

sti

m a

lon

e)

CD137 multimers have prolonged receptor engagement and tunable PK

12

Prolonged activity Cyno PK: Agonists with range of exposure

BCY7839 BCY7842

BCY8945 Projected Target Coverage at 2 mg/kg


CD13

7L

BCY78

39

BCY78

42

BCY89

45

0

50

100

150

200

% o

f m

ax i

nd

ucti

on

at

10n

M

No washout

30 min

60 min

120 min

Anti-tumour activity of CD137 multimers correlates with increased tumour infiltrating lymphocytes

13 13

T cell infiltration vs. activityTumour Volume in MC38 huCD137 C57Bl/6

D0 Start treatment~100mm3 tumour

D21 TerminateFACS -analysis

MC38

huCD137 C57Bl/6


Bicycle® CD137 bispecifics

Bispecific tumour/CD137 binding Bicycles® as potent and targeted T cell activators

CD137 is member of TNF superfamily & requires clustering for activation

15 15

TumourAntigen

CD137

Tumour Cell

ActivatedT cell

CD137 binder

Antigen binder

Linker

Fully synthetic molecules comprising CD137 and tumour antigen targeting Bicycles could achieve potent CD137 activity through receptor cross-linking across the immune synapse

CRD1

CRD2

CRD3

CD137

TM

CD

CD137L

Antigen presenting cell

ActivatedT cell

CRD4

EphA2Nectin-4PD-L1


Chin et al. Nat Commun 9, 1-13 (2018)

Proof of concept with the first EphA2/CD137 molecule

16 16

CD137 reporter assay co-culture Primary human immune cells-A549 co-culture (Tumour cell killing)

CD137 Bicycle

EphA2 Bicycle


Nectin-4/CD137 bispecific as an exemplar(concept is generalizable)

17

Nectin-4

CD137

Tumor cell

Immune cell

Cancer cell expressing high levels of Nectin-4

BCY8854


PD-L1/CD137 : 3rd bispecific exemplified

18

PD-L1 Bicycle binds to epitope that is directly competitive with PD1

PD1

Bicycle

PD-L1

CD137 reporter assayPD-1/PD-L1 blockade bioassay

PD-L1 Bicycle blocks PD1/PD-L1 Interaction between PD1 expressing T cells and CHO-

K1 stable expressing PD-L1

PD-L1/CD137 bispecifics induce agonism in CD137 reporter assay only when cocultured with PD-L1

expressing RKO cells.


CD137 bispecific chemistry: rapid progress from POC

19

4321

First EphA2

Bispecific

8 months5 6

First PD-L1

Bispecific

Initiated PD-L1

Optimisation

7

-14 -12 -10 -8 -60

5

10

15

20

25


Fo

ld i

nd

uc

tio

n

BCY7985EphA2-CD137/WT

BCY9173EphA2-CD137/HA

BCY7845 Tetramer

-12 -10 -8 -6 -40.1

1

10

100

1000


fold

ind

uct

ion

BCY8854 in T47D

BCY8854 in NCI-H292

no cells

CD137L

-11 -10 -9 -8 -7 -60

500

1000

1500

2000


IL-2

(p

g/m

L)

BCY8854 (Nectin4+)

BCY10000 (Nectin4+)

Nectin4+ = Nectin-4 overexpressing 4T1

Nectin4- = Parental 4T1

BCY10000 (Nectin4-)

BCY8854 (Nectin4-)

Co

ntr

ol

Ure

lum

ab

- 3

µM

Ure

lum

ab

- 1

µM

Ure

lum

ab

- 0

.3µ

M

an

ti-P

D1

- 1

00

µg

/mL

an

ti-P

D1

- 1

0µ

g/m

L

an

ti-P

D1

- 1

µg

/mL

BC

Y1

00

00

- 3

µM

BC

Y1

00

00

- 1

µM

BC

Y1

00

00

- 0

.3µ

M

IP -1 0

G r a n B

IF N b

IF N g

IL -2

IL -6

T N F a

Im m u n e m a rk e r m o d u la t io n

P a tie n t # 1 0 0 0 3 3 4 5

> 3 0 %

in c re a s e

> 3 0 %

d e c re a s e

N o ch a n g e

fro m c o n tro l

Activity in 1

Patient Tumour

T cell: Tumour

Cell Kill

0 20 40 60 80 100 120

0

1000

2000

3000

4000

Hours

Casp

ase 3

/7 A

cti

vit

y

(GC

U x

um

2p

er

imag

e)

DMSO

Urelumab

BCY9173

POC Target

ExpansionFirst Activity on

Human PBMC

First Nectin-4

Bispecific

In Vivo Expt

Initiated

POC Phase Early Med Chem Phase


Higher affinity CD137 Bicycle® increases potency of Nectin-4 bispecific in reporter and human PBMC assay

20

Human PBMC-4T1(Nectin-4+) co-culture (cytokine release)

Compound Molecular DescriptionKD(nM)

Nectin-4KD(nM) CD137

BCY8854 Nectin-Sar10-Peg12-CD137(WT, C-term) 2.76 108

BCY10000 Nectin-Sar10-Peg12-CD137(HA, C-term) 2.26 6.19

BCY10572 Nectin-Peg5-CD137(HA, dLys4) ND 5.00

Potent Activation of

human PBMCs

CD137 Reporter Assay Coculture with H1376 Cells


21

Nectin-4/CD137 bispecific Bicycles® induce target dependent cytokine release in ex vivo cultures of patient-derived lung tumours

• Tumour cells• Lymphocytes• Other associated

cells

Patient derived tumour cells ex vivo for 48h

10X

ex vivo patient derived tumour cells form 3D spheroids within 4h in culture


ID CD137+ T cells (%)

Nectin-4+ cells (%)

PT1 19.8 4.4

PT2 15.1 25.8

PT3 30.0 15.1

40 pM 120 pM

0

2

4

6

T Cell Proliferation

% K

i67

+ o

f C

TL

(Ba

ck

gro

un

d s

ub

tra

cte

d) PT1, Nectin-4 Low

PT2, Nectin-4 Med

PT3, Nectin-4 Med

BCY10572

22

Nectin-4/CD137 bispecific Bicycles® induce target dependent cytokine release in ex vivo cultures of patient-derived lung tumours

• Tumour cells• Lymphocytes• Other associated

cells

Patient derived tumour cells ex vivo for 48h

10X

ex vivo patient derived tumour cells form 3D spheroids within 4h in culture


% change in immune markers between BCY10572 vs Vehicle

ID CD137+ T cells (%)

Nectin-4+ cells (%)

PT1 19.8 4.4

PT2 15.1 25.8

PT3 30.0 15.1

40 p

M

120

pM

40 p

M

120

pM

40 p

M

120

pM

IFN

IL-2

granzyme B

IL-6

TNF

IL-8

CCL2

CCL4

IP-10

IL-10

Nectin-4Low

(PT1)

Nectin-4 Med(PT2)

Nectin-4 Med(PT3)

0

25

50

75

Bicycle® T cell agonists have tunable PK properties

23Peptide Congress April 2019

CD137 Multimer Agonist PK Nectin-4/CD137 Bispecific Agonist PK

Summary

• First fully synthetic Bicycle® multimeric T cell activator and Bicycle®

bispecific T cell activator platform.

• In vivo anti-tumour activity in humanized mouse models with Bicycle®

CD137 Multimers.

• Profound agonist activity in primary human T cell assays, and in human tumours ex vivo with Nectin-4/CD137 Bicycle® bispecifics.

• Promising in vitro activity with EphA2/CD137 and PD-L1/CD137 Bicycle®

bispecifics.

24Peptide Congress April 2019

• Team at Bicycle UK & US

Acknowledgements

25

LinkedInTwitter (@Bicycle_tx)

#NotWaiting


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Activation of CD137 using multivalent and tumour targeted ......G ra n B IF N b IF N g IL -2 IL -6 T...

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