thor-707: an engineered il-2 for the treatment of solid tumors … · 2019-04-19 · ex vivo...

Marcos E. Milla; Jerod L. Ptacin; Carolina E. Caffaro; Hans R. Aerni; Lina Ma; Kristine M. San Jose; Michael J. Pena; Robert W. Herman;

with superior pre-clinical efficacy and safety evidenceTHOR-707: An engineered IL-2 for the treatment of solid tumors

Yelena Pavlova; David B. Chen; Laura K. Shawver; Lilia K. Koriazova; Ingrid B. Joseph. Synthorx Inc. La Jolla, CA

mmilla@synthorx.com

Binding Affinity of THOR-707 to Human L-2 Receptor aand b Chains

0 . 0 0 0 1 0 . 0 1 1 1 0 0

0

4 0 0 0

8 0 0 0

1 2 0 0 0

1 6 0 0 0

0 . 0 0 0 1 0 . 0 1 1 1 0 0

0

3 0 0 0

6 0 0 0

9 0 0 0

1 2 0 0 0

THOR-707 Pharmacokinetics and In Vivo Activation of Mouse and

Non-human Primate (NHP) CD8+ T Cells

• Stable pegylation confers to THOR-707 high plasma AUC in both

NHP and mouse

• Full PD effect on peripheral CD8+ T cell IL-2R proximal activation

marker pSTAT5, and early proliferation marker Ki67

THOR-707 Single IV Dosing in Mouse: Induction of Peripheral

CD8+ T and NK Cell Extravasation and Expansion

0 .0 0 .5 1 .0

0

2 0

4 0

6 0

8 0

1 0 0

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

T im e (h )

pS

TA

T5

+%

in

CD

8+

T c

ell

s

V e h ic le

A ld e s le u k in

T H O R -7 0 7

THOR-707: long PD

effect due to longer

half-life

Protein EC50 nM

IL-2 0.0022

THOR-707 1.95

Protein EC50 nM

IL-2 1.47

THOR-707 5.71

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0

0 .1

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

T im e (h )

TH

OR

-70

7 (

ng

/ml)

M o u s e

C y n o m o lg u s

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

0

1 0

2 0

3 0

4 0

5 0

T im e (h )

NK

%

V e h ic le

T H O R -7 0 7

• THOR-707 does not engage IL-2R a chain yet binds the b chain

similarly to IL-2

• As expected for a not-alpha IL-2, THOR-707 activates with similar

potency the IL-2R abg and bg complexes in human primary T cells

Agent / PK speciesDose

mg/kgAUC0-t

ng*h/mLt1/2

hCD8+ pSTAT5

%

Aldesleukin mouse 0.3 432 0.576 80%

THOR-707 mouse 0.3 45600 13.3 100%

THOR-707 NHP 0.3 76,100 11.0 100%

0 2 8 1 2 2 4 4 8 7 2 1 2 0

0

2 0

4 0

6 0

8 0

Tre

g%

in

CD

3+

T

V e h ic le

T H O R -7 0 7

T im e (h )

THOR-707 Activation of Human CD4+ Tregand CD8+ TeffCells

Ex vivo (pSTAT5)

Human IL-2 Pharmacology is Comparable Between Human and

NHP, but not Between Human and Mouse IL-2R

• Binding studies show that human IL-2 has similar affinity for the human and

Cynomolgus IL-2R β chains. No detectable binding to the mouse b chain

• Cell-based studies (pSTAT5) show that human IL-2 potency depends on the

cell type:

▪ Comparable potency across species for Treg cells expressing IL-2 abg chain

complexes

▪ Dramatic drop in potency at mouse CD8+ T cells relative to human (75x) and NHP

(154x)

• CD8+ T cells are the target cell for the anti-tumor pharmacological effect of IL-

2. Therefore, THOR-707 is dosed in mice at relatively high levels, to

compensate for IL-2’s decreased potency at the mouse IL-2 receptor b chain

THOR-707 Induces Durable CD8+ T Cell Infiltration in Mouse

B16F10 Tumors

THOR-707 Shows Dose-dependent Single Agent

Efficacy in CT-26 Tumor-bearing Mice

Da

y 0

Da

y 1

Da

y 2

Da

y 3

Da

y 5

Da

y 7

Da

y 1

0

0

2 0

4 0

6 0

CD

8+

% in

C

D3

+T

V e h i c l e

3 m g / k g

P r e - d o s e

* * * * * *

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

D a y s

Me

an

tu

mo

r v

olu

me

(m

m3

S

EM

)

Many regressions

observed in THOR-707

+ anti mPD-1 treated

mice

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0

0 . 0 1

0 . 1

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

1 0 0 0 0 0

0 . 0 1

0 . 1

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

1 0 0 0 0 0

T i m e ( h )

Pla

sm

a P

K (

ng

/m

L)

Tu

mo

r/S

ple

en

P

K (

ng

/g

)

3 m g / k g T u m o r

3 m g / k g P l a s m a

3 m g / k g S p l e e n

707A tumor half-life was ~2X plasma half life (24.6 vs 12.6 h): Synthorin

penetrates the tumor compartment and is retained there

Based on AUC, 8% of plasma levels are in the tumor (spleen: 3.6%):

increased tumor distribution

THOR-707 Shows Increased Distribution and Retention in

C57Bl6 Mouse B16F10 Tumors

Binding KD (SPR) nM IL-2R EC50 (pSTAT5) pg/mL

Species IL-2Ra IL-2Rb CD4+ Treg Cell CD8+ T Cell

Human 1.71 360 6.50 4,884

NHP 2.90 270 9.15 2,372

Mouse 23.5 N.D.A 12.7 366,276

A: No detectable binding

C: Concentration-response curves lack upper baseline

THOR-707: A Reprogrammed IL-2 That Expands Lymphocytes

Without Inducing Eosinophil Proliferation

THOR-707 Single Dose

Leukocyte Subpopulations0.3 mg/kg IV

THOR-707 Single Dose

Leukocyte Expansion 0.1 or 0.3 mg/kg IV

Unlike aldesleukin, THOR-707 Shows Strong Bias for Expanding

Tumor-Fighting Lymphocytes vs. Eosinophils Responsible for VLS;

no increase in lung weights up to 1 mg/kg

Repeat Dosing of THOR-707 in NHP Elevated CD8+ T Cells in the

Lymphocyte Population and Achieved Ki67 Expression (≥60%)

Peripheral CD8+ Teff Cell Activation and Proliferation

Peripheral CD8+ Teff Cells Ki67 Expression

Summary

-1 1 2 3 4 5 6 7 8 910

11

12

13

14

15

16

17

18

19

20

21

0

1 0

2 0

3 0

4 0

D a y s

ce

ll c

ou

nts

x1

03/

L

w h ite b lo o d c e lls

d o se

ly m p h o c y te s

e o s in o p h ils

-1 2 4 6 8

10

12

14

16

18

20

0

1 0

2 0

3 0

4 0

D a y s

x1

03

/

L

0 . 1 m g / k g I V

d o s e

0 . 3 m g / k g I V

• At the approved clinical dose

(0.037 mg/kg), aldesleukin (IL-

2) induces eosinophilia

concomitantly with lymphocyte

proliferation, leading to

vascular leak syndrome, a fatal

adverse event

At Doses Leading to Maximal PD, THOR-707 Expands Lymphocytes

Without Eosinophilia in NHP

• Synthorx Expanded Genetic Alphabet technology platform was applied to the

discovery of THOR-707, an engineered form of IL-2 with a stable, covalent PEG

bioconjugate at a single, targeted position

• THOR-707 displays a “not-alpha” pharmacological profile: it activates the IL-2R bg

complex of CD8+ T effector and NK cells with similar potency to IL-2. In contrast,

because of its lack of a chain engagement, THOR-707 does not have a high bias

for activation of the IL-2R abg complex of CD4+ regulatory T cells

• THOR-707 exhibits extended half-life and high AUC in both mouse and NHP,

resulting in IL-2R occupancy levels that drive maximal activation and proliferation of

CD8+ effector and memory cells, and NK cells, with little or no expansion of CD4+

T regulatory cells

• THOR-707 elicits durable infiltration of CD8+ T cells into CT-26 syngeneic mouse

tumors. In this model, it displays dose-dependent single efficacy in those tumors,

and additivity in combination with a PD-1 checkpoint inhibitor

• In NHP, maximal peripheral CD8+ T cell proliferation is observed at 0.1 mg/kg: the

same level and durability of expansion is seen at the 0.3 and 1 mg/kg dose levels.

For that, Ki67 induction must reach 60% of CD8+ T cells or above

• No eosinophilia or vascular leak syndrome was observed at any dose level. This

confirms that THOR-707 can be dosed at levels eliciting full pharmacodynamic

responses without VLS, a serious adverse event previously associated with

aldesleukin

-1 1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

0

2 0

4 0

6 0

8 0

1 0 0

D a y s

Ki6

7+

% in

C

D8

+T

c

ell

s

d o s e

V e h i c l e

0 . 0 3 m g / k g

0 . 3 m g / k g

0 . 1 m g / k g

d o s e d o s e

1 m g / k g

-1 1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

0

2 0

4 0

6 0

D a y s

CD

8+

% in

s

in

gle

ts

V e h i c l e

0 . 0 3 m g / k g

0 . 3 m g / k g

0 . 1 m g / k g

d o s e d o s e d o s e

1 m g / k g

Design Strategy for Not-alpha THOR-707 IL-2

IL-2 binds to the IL-2

receptor αβγ complex at

high affinity because of

the α chain

Targeted pegylation of

THOR-707 at the novel

amino acid blocks α

chain binding

• Improved Selectivity - Reduced CD4+ Treg bias with retained

stimulatory activity of CD8+ T and NK cells in preclinical studies

• Increased Therapeutic Index – No induction of vascular leak syndrome

in Cynomolgus non-human primate (NHP) up to 1 mg/kg

• Convenient dosing schedule - Expected Q2W dosing or less frequent

• Reduced Risk of Immunogenicity - Covalent attachment of stable,

“shielding” PEG; conjugation site devoid of MHC-II anchors

Single, stable PEG covalently attached to a novel amino acid site to

make a “not-alpha” IL-2 protein

Background

Screening: SAR for THOR-707 “Not-Alpha” IL-2 Synthorin

IL-2

IL-2Rb IL-2Rg

IL-2 drives Rbg

dimer formation

• IL-2/IL-2Ra crystallographic interface examined to identify potential sites

for bioconjugation

• Pegylated variants tested via DiscoveRx PathHunter™ assay (IL-2R bg or

abg recombinant U2OS cells)

• Identified three sites with not a IL-2R bg/abg profile. THOR-707 selected

based on expression yield

CD8+ T Cells Memory CD8+ T Cells

NK Cells CD4+ Treg Cells

• THOR-707 drives CD8+ tumor infiltration comparable to levels observed with a combination of CTLA-4 and PD-1 checkpoint inhibitor mAbs in this syngeneic melanoma tumor model*

• In contrast, THOR707 does not change over time Treg infiltration

*Curran MA et al (2010) PNAS 107: 4275

Day 2

Day 3

Day 5

Day 7

Day 1

0

0

2 0

4 0

6 0

Tre

g%

in

CD

3+

T

V e h ic le

3 m g /k g T H O R -7 0 7

T re g c e lls fro m D a y 0 s a m p le s w e re to o fe w to b e p lo tte d

IL-2 Synthorinbg

EC50 nM

abg

EC50 nMbg/abg ratio

IL-2 1.68 0.0740 23

Best possible 1.68 1.68 1

THOR-700 3.00 1.50 2

THOR-701 6.75 0.150 45

THOR-702 9.84 0.131 75

THOR-703 4.16 0.165 25

THOR-704 6.37 0.0489 130

THOR-705 6.09 0.515 12

THOR-706 7.70 0.08925 86

THOR-707 23.8 4.44 5

THOR-708 9.06 0.110 83

THOR-709 9.99 0.0830 121

IL-2 Dual Pharmacology Prevents Aldesleukin from Dosing for Maximal

CD8+ T cell Expansion

• IL-2 binds to two different receptor forms:

• At high affinity, it engages the IL-2 receptor (IL-2R) abg complex expressed on CD4+ immune suppressive regulatory T cells (Tregs).

• The IL-2R abg complex is also expressed on type 2 innate lymphoid cells and eosinophils. These cells mediate vascular leak syndrome (VLS), a severe clinical adverse event of IL-2 leading to hypotension, pulmonary edema, and liver and kidney shock

• At lower affinity, it engages the IL-2R bg complex expressed on CD8+ T and natural killer (NK) cells. These cells are critical for anti-tumor responses

• We sought to design an IL-2 drug with wild type affinity for the IL-2R bg complex yet no engagement of the a chain

Meyers FJ et al (1991) Clin. Pharmacol.

Ther. 49: 307

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

T re a tm e n t g ro u p s Q W x 3 IV

Tu

mo

r V

olu

me

(m

m3)

S

EM

* * * p < = 0 .0 0 1

Ve

hic

le

1 m

g/k

g

3 m

g/k

g

6 m

g/k

g

9 m

g/k

g

* * *

* * *

* * *

Day 17 Tumor Size Measurements

THOR-707 Shows Additive Efficacy with an Anti-PD-1

mAb in CT-26 Tumor-bearing Mice

• A single 0.3 mg/kg IV dose of THOR-707 induces the extravasation and expansion of CD8+ T cells, including memory cells. They are critical for tumor attack.

• THOR-707 also expands peripheral NK cells

• THOR-707 did not stimulate peripheral CD4+ Treg proliferation

7 2 9 6 1 2 0

0

1 0

2 0

3 0

4 0

T im e (h )

me

mo

ry C

D8

+ T

eff

% i

n C

D3

+T

V e h ic le

T H O R -7 0 7

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

T im e (h )

CD

8+

% i

n C

D3

+T

V e h ic le

T H O R -7 0 7

Day 14 Tumor Size Measurements

CD4+ Treg CD8+ Teff

pS

TA

T5

MF

I

nM cytokine

pS

TA

T5

MF

I

nM cytokine

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

Tu

mo

r V

olu

me

(m

m3

)

SE

M

v e h ic le (Q W x 3 + is o ty p e c o n tro l

1 0 m g /k g , B IW x 3

T H O R -7 0 7 (6 m g /k g - Q W x 3 , iv )

A n t i P D -1 (1 0 m g /k g - B IW x 3 , ip )

T H O R -7 0 7 (6 m g /k g - Q W x 3 , iv ) +

a n t i P D -1 (1 0 m g /k g , B IW x 3 , ip )3 5 .6 %

4 4 .1 %

7 4 .6 %

* *

* * *