OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy

Paul-Joseph Aspuria, Michele Bauer, Sandro Vivona, Steve E. Kauder, Scott McCauley, Romina Riener, Rene de Waal Malefyt, Deepti Rokkam, Jan Emmerich, Rakesh Verma,

Patrick J. Lupardus, Rob A. Kastelein, and Martin Oft

Synthekine, Inc. Menlo Park, California

Disclosures

• Employee: Synthekine, Inc.

2

Orthogonal IL-2 Program Selectively Activates CAR-Ts In Vivo

3

hoRβHuman orthogonal IL-2Rb

uniquely expressed on CAR-T

STK-009 Orthogonal IL-2 Ligand

SYNCAR-001• Lentiviral vector• CD19 CAR • Ortho receptor (hoRβ)• Bicistronic expression via T2A

cleavage peptide

Incorporated into:

Efficacy of STK-009 and SYNCAR-001 In a Disseminated Raji NSG Model

Cells Treatment Mice- PBS 8

SYNCAR-001 - 8

SYNCAR-001 STK-009 1ug (q.o.d.) 8

4

SYNCAR-001 cell transfer1E6 CAR+ cells/mouse

d5 d12 d19d0

Raji-lucimplant (i.v. 5E5 cells)

d26

Last dose (d17)

STK-009 dosingStudy Design

Treatment Groups

5 10 15 20 25 30 35 40 45105

106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

PBS

STK-009 Drives Complete Responses Even With a Sub-Efficacious CAR Dose

SYNCAR-001 + PBS

SYNCAR-001 + STK-009 (1 µg)PBS

5

Relapse

Last Dose

5 10 15 20 25 30 35105

106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

CD19_28z_T2A_hORB + STK009-1ug

5 10 15 20 25 30 35105

106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

CD19_28z_T2A_hORB + PBS

STK-009 Treatment Increases CAR-T Levels With Elevated Long Lived Memory TSCM Populations

15 20 25 30 35102

103

104

105

106

107

108

Time (days)

Cel

ls/m

l of b

lood

TSCM

15 20 25 30 35102

103

104

105

106

107

108

Time (days)

Cel

ls/m

l of b

lood

TEM

15 20 25 30 35102

103

104

105

106

107

108

Time (days)

Cel

ls/m

l of b

lood

TCM

15 20 25 30 35102

103

104

105

106

107

108

Time (days)

Cel

ls/m

l of b

lood

TEMRA

CD19 orthoCAR + STK-009

CD19 orthoCAR + PBS

15 20 25 30 35104

105

106

107

108

Time (days)

hCD

3+ (c

ells

/ml o

f blo

od)

hCD3+ cells

6

SYNCAR-001 + PBSSYNCAR-001 + STK-009

Last Dose

Last Dose

Last Dose

Last Dose

Last Dose

Study to Address Whether Retreatment of Mice with “Resting” SYNCAR-001 Cells with STK-009 Increases T Cell Levels

7

Group Treatment Mice

1 PBS 4

2 STK-009 1 µg q.o.d., s.c. 5

3 STK-009 10 µg q.o.d.,s.c. 5

STK-009 Admin

d7 d15 d29-40d(est)

Days

Last dose of STK-009

d0

All mice clear of tumor

burden (IVIS)

-100d(est)

SYNCAR-001 injection

/ /

Study Design

Treatment Groups

/ /

Retreatment Phase

Disseminated Raji-luc studies

0 10 20 30102

103

104

105

106

107

108

Time (days)

hCD

3+ (c

ells

/ml o

f blo

od)

TSCM

0 10 20 30101

102

103

104

105

106

107

108

Time (days)

hCD

3+ (c

ells

/ml o

f blo

od)

TEM

0 10 20 30101

102

103

104

105

106

107

108

Time (days)

hCD

3+ (c

ells

/ml o

f blo

od)

TCM

0 10 20 30101

102

103

104

105

106

107

108

Time (days)

hCD

3+ (c

ells

/ml o

f blo

od)

TEMRA

0 5 10 15 20 25 30103

104

105

106

107

108

Time (days)

hCD

3+ (c

ells

/ml o

f blo

od)

hCD3+

PBS

STK-009 (10 ug)

STK-009 (1 ug)

STK-009 Induces Re-expansion of CAR-T Cells Following Complete Response And 40+ day Drug Holiday

T-cell phenotype

8

Last dose Last dose

Last dose Last dose

STK-009dosing

STK-009 STK-009

STK-009 STK-009

Efficacy of STK-009 and SYNCAR-001 in a Subcutaneous Raji NSG Model

Group CAR T Cells Treatment Mice

1 - PBS 82 SYNCAR-001 - 83 SYNCAR-001 STK-009 10ug (q.o.d.) 84 SYNCAR-001 STK-009 1ug (q.o.d.) -> 10 ug on Day 50 (q.o.d) 8

SYNCAR-001 transfer4E5 CAR+ cells/mouse

STK-009 dose (s.c. q.o.d.)

6 12 180

Raji-luc + Matrigelimplant (s.c. 5E5 cells)

75Last treatment

(d75)

//

9

Study Design

Treatment Groups

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

PBS

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

orthoCAR T + PBS

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

orthoCAR T + STK-009 (1ug)

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

orthoCAR T + STK-009 (10ug)

STK-009 Delivers Complete Responses With a Non-Efficacious Dose of CARs

10 µg STK-009

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

Time (days)

Surv

ival

(%)

PBS

CD19_28z orthoCAR + PBS

CD19_28z orthoCAR + STK-009 (1 ug)

CD19_28z orthoCAR + STK-009 (10 ug)

10

SYNCAR-001 + STK-009 (1 µg) (10 µg)

PBSSYNCAR-001 + PBS

SYNCAR-001 + STK-009 (10 µg)

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

Time (days)

Surv

ival

(%)

PBS

CD19_28z orthoCAR + PBS

CD19_28z orthoCAR + STK-009 (1 ug)

CD19_28z orthoCAR + STK-009 (10 ug)

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

PBS

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

orthoCAR T + PBS

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

orthoCAR T + STK-009 (1ug)

10 20 30 40 50 60 70 80 90106

107

108

109

1010

1011

1012

Time (days)

Tota

l flu

x (p

/s)

orthoCAR T + STK-009 (10ug)

PBS SYNCAR-001 + PBS

SYNCAR-001 + STK-009 (1 µg) (10 µg) SYNCAR-001 + STK-009 (10 µg)

Last dose

Last dose

20 40 600

500

1000

1500

2000

2500

3000

Time (days)

Tum

or v

olum

e (m

m3 )

Tumor Volume

PBS

orthoCAR + PBS

orthoCAR + STK-009 (1ug)

orthoCAR + STK-009 (10 ug)

10µg STK-009

PBS

SYNCAR-001 + PBS

SYNCAR-001 + STK-009 (10 µg)

SYNCAR-001 + STK-009 (1 µg) (10 µg)

10µg STK-009

10µg STK-009

STK-009 Significantly Increases Peripheral T Cell Numbers and All T Cell Memory Subtypes Including TSCM Cells

11

Last dose

Last dose

Last dose

Last dose

10 20 30 40 50 60 70 80 901×102

1×103

1×104

1×105

1×106

Time (days)

hCD

3+ (c

ells

/ml o

f blo

od)

hCD3+

orthoCAR + PBS

orthoCAR + STK-009 (1ug)

orthoCAR + STK-009 (10ug)

Last dose

10µg STK-009

10µg STK-009

10µg STK-009

SYNCAR-001 + STK-009(1 µg) (10 µg)

SYNCAR-001 + PBS

SYNCAR-001 + STK-009 (10 µg)

10 20 30 40 50 60 70 80 901×102

1×103

1×104

1×105

1×106

Time (days)

hC

D3+

(cel

ls/m

l of b

loo

d)

hCD3+

orthoCAR + PBS

orthoCAR + STK-009 (1ug)

orthoCAR + STK-009 (10ug) 20 30 40 50 60 70 80 901×101

1×102

1×103

1×104

1×105

1×106

Time (days)

Cel

ls/m

l of b

lood

TSCM

20 30 40 50 60 70 80 901×101

1×102

1×103

1×104

1×105

1×106

Time (days) C

ells

/ml o

f blo

od

TEM

20 30 40 50 60 70 80 901×101

1×102

1×103

1×104

1×105

1×106

Time (days)

Cel

ls/m

l of b

lood

TCM

20 30 40 50 60 70 80 901×101

1×102

1×103

1×104

1×105

1×106

Time (days)

Cel

ls/m

l of b

lood

TEMRA

STK-009 non-GLP tolerability and toxicology study in non-human primates

12

Group Test Material Dose Frequency

Dose Level (mg/kg)

Day 1 Day5 Day 10

1 Proleukin(control)

single dose 0.037 - -

2 STK-009 q5d 0.06 0.06 0.06

3 STK-009 q5d 0.12 0.12 0.12

4 STK-009 q5d 0.02 0.12 -

5 STK-009 q5d 0.06 0.25 -

Study design

STK-009 has a favorable PK profile in cynomolgus monkeys

130 48 96 144 192 240 288 336

0.1

1

10

100

1000

Time, hours

STK

-009

, ng/

ml

0.06 / 0.06 / 0.06 mg/kg

dose

0.037 mg/kg

0.12 / 0.12 / 0.12 mg/kg

0 48 96 144 1920.1

1

10

100

1000

Time, hours

STK

-009

, ng/

ml

0.02 / 0.12 mg/kg

0.06 / 0.25 mg/kg

dose

Proleukin

STK-009

Dose Dose

Dose Dose Dose

STK-009 exposure was very stable with minimal clearance between doses.

In the absence of an hoRbexpressing cell, STK-009 does not undergo target mediated clearance.

STK-009

STK-009STK-009

STK-009 avoids any T cell or NK cell activation in cynomolgus monkeys

14

STK-009 has negligible effects on cells that express WT IL-2 receptors and are responsive to WT IL-2 (Proleukin)

Pre-dose8hr post-d

ose 1Proleukin (0.037 mg/kg)

#1501STK-009 (0.12 mg/kg)

#3501

Pre-dose8hr post-d

ose 1

CD4+ CD25+ T cells

IL-2 (37µg/kg)

STK-009 (MAD 60µg/kg)

STK-009 (MAD 120µg/kg)

STK-009 (MAD 20-120µg/kg)

STK-009 (MAD 60-250µg/kg)

CD4+ CD25+ T cells

0 5 10 150

20

40

60

80

100

Timepoint (Day)

pSTA

T5 (%

Pos

itive

)

CD4+CD25hi_Grouped

Grp1 (Proleukin) - 1x - 0.037 mpk

Grp2 (STK-009) - 3x - 0.06mpk

Grp3 (STK-009) - 3x - 0.12 mpk

Grp4 (STK-009) - 2x - 0.02 mpk / 0.12 mpk

Grp5 (STK-009) - 2x - 0.06 mpk / 0.25 mpk

-10 0 0 5 10 15

0

5

10

15

80100

Timepoint (Day)

pSTA

T5 (%

Pos

itive

)

CD4+CD25hi_Grouped

Grp1 (Proleukin) - 1x - 0.037 mpk

Grp2 (STK-009) - 3x - 0.06mpk

Grp3 (STK-009) - 3x - 0.12 mpk

Grp4 (STK-009) - 2x - 0.02 mpk / 0.12 mpk

Grp5 (STK-009) - 2x - 0.06 mpk / 0.25 mpk

pSTAT5 percentageIL-2 (37µg/kg)STK-009 (MAD 60µg/kg)STK-009 (MAD 120µg/kg)STK-009 (MAD 20-120µg/kg)STK-009 (MAD 60-250µg/kg)

0 5 10 150

20

40

60

Timepoint (Day)

Per

cent

KI6

7+

NK cells

Grp1 (Proleukin) - 1x - 0.037 mpk

Grp2 (STK-009) - 3x - 0.06mpk

Grp3 (STK-009) - 3x - 0.12 mpk

Grp4 (STK-009) - 2x - 0.02 mpk / 0.12 mpk

Grp5 (STK-009) - 2x - 0.06 mpk / 0.25 mpk

IL-2 (37µg/kg)STK-009 (MAD 60µg/kg)STK-009 (MAD 120µg/kg)STK-009 (MAD 20-120µg/kg)STK-009 (MAD 60-250µg/kg)

0 5 10 150

20

40

60

Timepoint (Day)

Per

cent

KI6

7+

NK cells

Grp1 (Proleukin) - 1x - 0.037 mpk

Grp2 (STK-009) - 3x - 0.06mpk

Grp3 (STK-009) - 3x - 0.12 mpk

Grp4 (STK-009) - 2x - 0.02 mpk / 0.12 mpk

Grp5 (STK-009) - 2x - 0.06 mpk / 0.25 mpk

Ki67+ percentage Pre-dose Proleukin STK-009(0.25mg/kg)

GzmB

Ki67

IL-2 (37µg/kg)STK-009 (MAD 60µg/kg)STK-009 (MAD 120µg/kg)STK-009 (MAD 20-120µg/kg)STK-009 (MAD 60-250µg/kg)

0 5 10 150

20

40

60

Timepoint (Day)

Per

cent

KI6

7+

NK cells

Grp1 (Proleukin) - 1x - 0.037 mpk

Grp2 (STK-009) - 3x - 0.06mpk

Grp3 (STK-009) - 3x - 0.12 mpk

Grp4 (STK-009) - 2x - 0.02 mpk / 0.12 mpk

Grp5 (STK-009) - 2x - 0.06 mpk / 0.25 mpk

GzmB

Ki67

GzmB

Ki67

NK cells

Conclusions

15

• STK-009/SYNCAR-001 platform drives improved efficacy• STK-009 rescues ineffective doses of SYNCAR-001 in subcutaneous and disseminated Raji models

• STK-009 dramatically increases SYNCAR-001 cell counts • 3-6 log effect depending on donor and tumor model

• SYNCAR-001 can be expanded at will by STK-009 dosing• Resting SYNCAR-001 cells in tumor burden free mice can be expanded by STK-009 after a 40 day drug

holiday

• SYNCAR-001 cells contract in the absence of tumor burden and STK-009 dosing• Enables ability for STK-009 dosing regimens to dictate increased or decreased SYNCAR-001 cell numbers

• STK-009/SYNCAR-001 cell platform expands both Tscm and Teff SYNCAR-001 T cells• STK-009 increases desired populations for anti-tumor efficacy and long term surveillance

• STK-009 is well tolerated and does not induce biological effects at the explored doses in non-human primates• STK-009 is highly specific for hoRb expressing cells

• Therefore, the STK-009/SYNCAR platform has the potential to overcome clinically relevant hurdles in cell therapy