PM

EL

DP-1

5 PM

EL

DP-1

2 PM

EL

Com

binat

ion

0

5×102

1×103

1×104

2×104

3×104

4×104

5×104

IFN-g secretion Day 5 (E:T = 10:1)

IFN

-g (

pg

/mL

)

********

PM

EL

DP-1

5 PM

EL

DP-1

2 PM

EL

Com

binat

ion

0

5×103

1×104

5.0×104

1.0×105

1.5×105

PMEL counts Day 5 (E:T = 10:1)

Liv

e P

ME

LT

cells

**

Conclusions

• Combination of T cells loaded with DeepIL-12 and Deep IL-15 optimized T cellactivity in both human and murine invitro models, consistent with thecombined benefits of each monotherapy:

• Persistent T cell activation

• Enhanced cytotoxicity

• Increased IFN-γ secretion

• Increased expansion of T cells

• Co-administration of Deep IL-12 loadedand Deep IL-15 loaded PMEL T cells toB16-F10 melanoma-bearing mice

• Elicited superior anti-tumor activity

• Resulted in increased engraftment oftransferred PMEL T cells

• Was well-tolerated, with minimal andreversible body weight loss

• Resulted in minor increases ofsystemic cytokines relative to DeepIL-12 loaded PMEL T cells

• A Phase I clinical trial of Deep IL-15 MTCs(TRQ15-01) in solid cancers andlymphoma is currently enrolling(NCT03815682). Evaluation of Deep IL-12primed MTCs (TRQ12-01) is planned toinitiate in 2020, including a combinationarm with TRQ15-01.

Complementary biology of IL-15 and IL-12

A68Presented at the AACR

Tumor Immunology and

Immunotherapy

November 17-20, 2019

● Boston, MA

Combining Deep IL-12 Primed and Deep IL-15 Primed T cells induces potent antigen-

dependent in vitro cytotoxicity and in vivo anti-tumor activityElena Geretti, Katharine Sackton, Pengpeng Cao, Shawn Carey, Xiaoyan Liang, Jonathan Nardozzi, Zishu Gui, Alicia Worthylake, Becker Hewes, Tap Maniar, Jonathan Fitzgerald, Andy Rakestraw, Douglas

Jones, Karsten Sauer, Thomas Andresen Torque Therapeutics, Cambridge, MA

IntroductionInterleukin-15 (IL-15) and Interleukin-12 (IL-12) play complementary roles asimmunomodulators. IL-15 induces T cell memory and supports survival,activation and proliferation of CD8 T and NK cells. IL-12 promotes T cellcytotoxicity and innate immune responses in the tumor microenvironment(TME). Both cytokines have been explored as cancer immunotherapies, butsevere side effects have limited clinical success. Torque has developed theDeep-Primed™ T cell therapy technology to direct the stimulatory activity ofthese cytokines to the TME, to prime and boost the immune response of T celltherapies with limited systemic exposure and toxicity. Multi-targeted T cells(MTC) specific for multiple tumor antigens are generated from patientapheresis. Cytokines are tethered to MTCs to support MTC persistence andactivity following adoptive transfer into patients, while limiting systemiccytokine exposure. This study evaluates the combination of Deep IL-15Primed™ and Deep IL-12 Primed™ T cells to leverage their complementarybiology for superior efficacy.

The Torque platform harnesses natural T cell biology in an ex vivo process

Multitargeted, antigen-primed T cells• For efficacy against heterogeneous tumors

Surface-anchored cytokines / immunomodulators• To overcome immunosuppression in the tumor

microenvironment

High margin of safety• Outpatient, without myeloablative chemotherapy• Enables repeat dosing

Torque Deep PrimedTM T Cell

Deep IL-15 and Deep IL-12

De

ep

IL

-15

Lo

ad

ing

De

ep

IL

-12

Lo

ad

ing

Combination of Deep IL-12 and Deep IL-15 maximizes PMEL T cell activation, and effector function

• Model enables evaluationof antigen-specific T cellactivity in fullyimmunocompetent mice

• PMEL T cell in vitroexpansion is comparableto that of human multi-targeted T cells

A mouse model for Deep PrimedTM T cells

PMEL-1 mice:

Transgenic TCR specific for

gp100 antigen in B16-F10

melanoma

RECIPIENT

CD8+ T (PMEL) cell

Isolation

(spleen / lymph node, LN)

PMEL T cell

Activation / Expansion

B16-F10

melanoma

B16-F10 tumor-

bearing C57BL/6 mice

PMEL T cell

Loading with

Deep IL-12 and Deep IL-15

DONOR

ADOPTIVE CELL

TRANSFER (ACT)

IN VITRO TESTING

Proliferation, Activation,

Phenotype, Cytotoxicity

Combination of Deep IL-15 and Deep IL-12 primed T cells in vivo

Figure 5. C57Bl/6 mice were inoculated with B16-F10 melanoma cells (1 x 106, s.c.).When tumors reached an average volume of 80 mm3, mice were treated withcyclophosphamide (4 mg/mouse, Day -1), followed by administration of vehiclecontrol (HBSS), Deep IL-12 Primed PMEL T cells (DP-12 PMEL; 2.5 x 106), or Deep IL-15 Primed PMEL T cells (DP-15 PMEL; 10 x 106) alone or together with either PMEL Tcells (2.5 x 106) or Deep IL-12 Primed PMEL T cells (DP-12 PMEL; 2.5 x 106). (A) Anti-tumor activity. (B) Blood was drawn at the indicated time points for quantification oftransferred PMEL T cells (CD90.1+). (C) Changes in body weight. (D) Systemic IFN-γand IP-10 were measured by Luminex on mouse serum (D1).

Superior anti-tumor activity Enhanced engraftment Well tolerated, mild and

reversible body weight loss

CA B

Results

Figure 1. Torque’s cell process for MTCgeneration was adapted to train human T cells againsta single peptide from the MART-1 antigen. Deep IL-15 drives enhanced expansion and activation ofMART1 – targeted T cells upon antigen exposure. Tcells ± Deep IL-15 cultured alone or in 1:1 co-culturewith MART1neg target cells (RPMI6666) or MART1pos

target cells (RPMI6666 loaded with MART126-35peptide). Deep IL-15 promotes activation (CD25+) inMART1 – targeted T cells through Day 5 of co-culturewith MART1pos target cells.

0 1 2 3 4 50

10

20

30

0 1 2 3 4 50

10

20

30

0 1 2 3 4 50

10

20

30

0 1 2 3 4 50

10

20

30

T c

ell

count (f

old

day 0

)

MA

RT

1 –

targ

ete

d T

cells

MA

RT

1 –

targ

ete

d T

cells

+ D

eep

IL

-15

MART1 – T cells MART1 – T cells +

MART1neg targetMART1 – T cells +

MART1pos target

0 1 2 3 4 50

10

20

30

T c

ell

count (f

old

day 0

)

Activated CD3

Total CD3

Non-activated CD3

Deep IL-15 improves human T cell expansion and antigen-specific activation

Combination of Deep IL-12 and Deep IL-15 optimizes human T cells activity

Improved cytotoxicity Enhanced IFNɣ production

C

Figure 3. (A) Combination of Deep IL-12 and Deep IL-15 treatment improved cytotoxicity towards SK-MEL-5 cancer cells atan E:T ratio of 1:10 on Day 3 relative to mock or individually loaded MART-1 CTLs. (B) Combination treatment increasedIFNɣ production at all E:T ratios tested on Day 3. (C) Combination increased cytotoxicity post re-challenge at E:T ratio of 1:10.

A

Robust Activation T cell expansionEnhanced cytotoxicity

Figure 4. Murine PMEL CD8+ T cells were activated/expanded in vitro and loaded with Deep IL-12, Deep IL-15 or both, followed byevaluation of in vitro cytotoxicity (A), IFN-γ production (B), activation (C) and expansion (D) in co-culture with B16-F10 target cells.

Increased IFN-ɣ production

E:T = 10:1 E:T = 1:100

50

100

150

200

B16-F10 Viability

B16-F

10 v

iab

ilit

y (

%)

PMEL

DP-15 PMEL

DP-12 PMEL

CombinationPM

EL

DP-1

5 PM

EL

DP-1

2 PM

EL

Com

binat

ion

0

20

40

60

80

CD25+ CD69+ PMEL Day 5

(E:T = 10:1)

CD

25

+ C

D6

9+ (

% o

f P

ME

L)

**

****

CA B D

Activated CD3

Total CD3

Non-activated CD3

Total CD3 CD3+ CD25- CD3+ CD25+

Activated CD3

Total CD3

Non-activated CD3 Activated CD3

Total CD3

Non-activated CD3

D

Deep IL-12 improves human T cell effector function

Cyto

toxic

ity

B

MART-1 T cells

Deep IL-12 Primed

MART-1 T cells

A B

SKMEL-5 coculture A375 coculture

Study report figure, SEM

1:1 1:5 1:10 1:15

0

200

400

600

800

1000

Effector:Target

pg

/ml IF

N-g

Transpose of SKMEL All

mock

mock+1 nM rIL-12

344 ng Deep IL-12 /1e6 cells

172 ng Deep IL-12 /1e6 cells

86 ng Deep IL-12 /1e6 cells

43 ng Deep IL-12 /1e6 cells

21.5 ng Deep IL-12 /1e6 cells LLOQ

A B

SKMEL-5 coculture A375 coculture

Study report figure, SEM

1:1 1:5 1:10 1:15

0

200

400

600

800

1000

Effector:Target

pg

/ml IF

N-g

Transpose of SKMEL All

mock

mock+1 nM rIL-12

344 ng Deep IL-12 /1e6 cells

172 ng Deep IL-12 /1e6 cells

86 ng Deep IL-12 /1e6 cells

43 ng Deep IL-12 /1e6 cells

21.5 ng Deep IL-12 /1e6 cells LLOQ

ASK-MEL-5

(MART-1 expressing)

A375(not MART-1 expressing)

1:1 1:5 1:10 1:150

20

40

60

80

100

120

140

Effector:Target

% liv

e S

K-M

EL

-5 c

ells

Single SK-MEL5-Day 4

mock

344ng Deep IL-12 /

1e6 cells

1:1 1:5 1:10 1:150

20

40

60

80

100

120

140

160

Effector:Target

% liv

e A

375 c

ells

Single A375-Day 4

mock

344ng Deep IL-12 /

1e6 cellsImproved

antigen-specific

cytotoxicity

Figure 2. (A) Deep IL-12increases cytotoxicitytowards the MART-1-expressing cancer cell lineSK-MEL-5 (Day 4),particularly at low E:Tratios. (C) Deep IL-12increases IFNɣ productionfollowing incubation withSK-MEL-5 cells (Day4). Cytotoxicity (B) andIFNɣ production(D) following incubationwith MART-1 negativeA375 cells is not increasedby Deep IL-12,demonstrating antigen-dependence.

C

IFN

-ɣp

rod

ucti

on D

LLOQLLOQ

1:1 1:5 1:10 1:150

200

400

600

800

1000

Effector:Target

pg

/ml IF

N-g

Single SKMEL All

mock

344 ng Deep IL-12 /

1e6 cells

LLOQ

1:1 1:5 1:10 1:150

200

400

600

800

1000

Effector:Target

pg

/ml IF

N-g

Single A375 Plate

344 ng Deep IL-12 /

1e6 cells

mock

LLOQ

Enhanced IFNɣ

production

Systemic IFN-γ and IP-10

Day 3Collect T cells from supernatant

Tumor cells

T cells

+

Re-challenge T cells

with new Tumor cellsDay 3 post

re-challenge

Persistent cytotoxicity

B