Adenoviruses armed with TNFa and IL-2

increase efficacy of adoptive cell therapy

in the absence of lymphodepleting

preconditioningSantos JM1,2, Cervera-Carrascon V1,2, Havunen R1,2, Zafar S2, Siurala M1,2, Sorsa S1,2,

Anttila, M3, Hemminki, A1,2,4.1TILT Biotherapeutics Ltd, Helsinki, Finland;´2Cancer Gene Therapy Group, Department of Oncology, Faculty of Medicine, University of Helsinki,

Helsinki, Finland; 3Pathology Unit, Finnish Food Safety Authority (EVIRA), Helsinki, Finland; 4Helsinki University Hospital Comprehensive Cancer

Center, Helsinki, Finland.

Figure 2 – Schematics of the treatment regimens. (A) Tumor-infiltrating lymphocyte (TIL, 8x106 cells per animal i.t.) infused syrianhamsters with day 7 established HapT1 tumors, received preconditioning chemotherapy (Cyclophosphamide+Fludarabine) or saline andwere administered intratumorally 5 times with TILT-123 (oncolytic Ad5/3-E2F-D24-hTNFa-IRES-hIL2) or with PBS; (B) OVA specific T-cell(OT-I, 1x106 cells) infused C57BL/6 mice with day 9 established B16.OVA tumors received preconditioning chemotherapy(Cyclophosphamide+Fludarabine) or saline and were administered once with non-replicative Ad5-CMV-mIL2 and Ad5-CMV-mTNFa in aratio of 1:1 or with PBS.

Background

Conclusions

Adenovirus Improves the Efficacy of ACT in the Absence of Preconditioning

• Our oncolytic adenovirus plattform successfullyreplaces high dose preconditioning chemotherapyregimens;

• Adenovirus and preconditioning therapeuticregimens induce a similar changes in themicroenvironment status and immunepopulations of tumors from animals treated withadoptive cell therapy;

• Decreased toxicity and similar efficacy justify thereplacement of preconditioning chemotherapy byadenovirus therapy to support ACT;

• A phase I clinical trial is underway to validate thishypothesis.

• Lymphodepleting chemotherapy improves adoptive cell transfer (ACT) but it is very toxic

• TILT-123, an oncolytic adenovirus expressing Interleukin-2 (IL-2) and Tumor Necrosis Factor a (TNFa) improves the efficacy of adoptive celltransfer (ACT) in animal models

• Oncolytic adenoviruses have a better safety profile• Here we determine the safety and efficacy of TILT-123 with ACT in the

absence of lymphodepleting preconditioning

Methods

d1 d2 d13

0.5x108 VPs Ad5-CMV-mIL20.5x108 VPs Ad5-CMV-mTNFa/PBS i.t.

1x106 OT-I cells/RPMI i.p. Endpoint

d0d-1

Cyclophosphamide/Salinei.p.

B16.OVA inoculatedCB57B/L6 Mouse

Fludarabine/Salinei.p.

d1 d2 d28

1x108 VPs TILT-123/PBSi.t.

4x106 TILs cells/RPMI i.t. Endpoint

d0d-1

Cyclophosphamide/Salinei.p.

Fludarabine/ Salinei.p.

HapT1 inoculatedSyrian Hamster

Virus injection (day 5, 9, 14 and 20)

A

B

ResultsLymphocytes and Antigen Presenting Cells (APCs) are

infiltrated in Th1 cytokine enriched tumormicroenvironments

Adenovirus therapy does not induce significanttoxicity in hamsters and mice

Ve

hi c

l e

OT

- I

Ad

5- m

TN

Fa

/mI L

2 +

OT

- I

Pr e

co

nd

+ O

T- I

Pr e

co

nd

+ A

d5

- mT

NF

a/m

I L2

+ O

T- I

0

2

4

6

8

1 0

C D 3 + T c e l l s

CD

3+

T c

ell

s

(%

of C

D1

9- c

ell

s)

*

*

*

* *

*

Ve

hi c

l e

OT

- I

Ad

5- m

TN

Fa

/mI L

2 +

OT

- I

Pr e

co

nd

+ O

T- I

Pr e

co

nd

+ A

d5

- mT

NF

a/m

I L2

+ O

T- I

0

1

2

3

4

5

C D 8 + T c e l l s

CD

3+

CD

8b

+ C

ell

s

(%

of c

ell

s)

*

*

*

*

*

*

Ve

hi c

l e

OT

- I

Ad

5- m

TN

Fa

/mI L

2 +

OT

- I

Pr e

co

nd

+ O

T- I

Pr e

co

nd

+ A

d5

- mT

NF

a/m

I L2

+ O

T- I

0

5

1 0

1 5

2 0

D e n d r i t i c C e l l s

CD

11

c+

(%

of t

ota

l c

ell

s)

*

*

*

*

A B C D

Ve

hi c

l e

OT

- I

Ad

5- m

TN

Fa

/mI L

2 +

OT

- I

Pr e

co

nd

+ O

T- I

Pr e

co

nd

+ A

d5

- mT

NF

a/m

I L2

+ O

T- I

0

5

1 0

1 5

M a t u r e D e n d r i t i c c e l l s

CD

11

c+

CD

86

+

(%

of t

ota

l c

ell

s )

*

*

*

*

*

m I L - 2 m T N F a m F N g

0 . 0 0

0 . 0 1

0 . 0 2

0 . 0 3

0 . 0 4

T h 1 c y t o k i n e s

Le

ve

l (p

g/u

g)

V e h ic le

O T - I

A d 5 - m T N F a / m I L - 2 + O T - I

P r e c o n d + O T - I

P r e c o n d + A d 5 - m T N F a / m I L - 2 + O T - I

E

Figure 4 – Immune composition of the tumor microenvironment. (A) CD3+ T cells (B) CD3+CD8b+ T cells (C) CD11c+ Dendritic cells and(D) CD11c+CD86+ Mature Dendritic cells were obtained by staining extracted tumors with fluorescently conjugated antibodies andanalyzed by flow cytometry; (E) Th1 cytokine level production was assessed by cytometric bead array. All data is presented as meanplus SEM. Statistical significances were calculated using unpaired student´s T test with Welch´s correction. *p≤0.05,**p≤0.01

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

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

H a m s t e r

T I L T

1 2 3

T I L T

1 2 3

T I L s T I L T

1 2 3

T I L T

1 2 3

T I L T

1 2 3

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

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

M o u s e

A d O T - I

No

rm

ali

ze

d T

um

or

Gr

ow

th

(%

)

P r e c o n d t i t i o n i n g + T I L s

V e h ic le

T I L s

T I L T - 1 2 3 + T I L s

P r e c o n d i t i o n i n g + T I L T - 1 2 3 + T I L s

V e h ic le

O T - I

A d 5 - m T N F a / m I L - 2 + O T - I

P r e c o n d i t i o n i n g + O T - I

P r e c o n d i t i o n in g + A d 5 - m T N F a / m I L - 2 + O T - I

D a y s

* * * *

* **

* * * *

A B

* * * *

Figure 3 – Efficacy Data. (A) Hamster normalized tumor growth followed during 28 days; (B) Mice normalized tumor growth followedduring 11 days; All data is presented as mean plus SEM. Tumor growth percentages were obtained by normalizing the daily tumorvolumes to day 1 tumor volumes. significances between groups were acquired comparing normalized tumor volumes in linear-mixedmodels. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Model Timepoint Therapy Cardiac DegenerationExtramedullary

Hematopoiesis

Pulmonary Vascular

Degeneration

Ham

ste

rs

Day 4

Vehicle 0% 0% N/O

TILT-123 0% 0% N/O

Preconditioning 60% 0% N/O

Day 28

Vehicle 0% 0% N/O

TILs 60% 0% N/O

TILT-123

+ TILs20% 0% N/O

Preconditioning + TILs 0% 0% N/O

Preconditioning + TILT-123 + TILs 0% 25% N/O

Mo

use

Day 13

Vehicle 0% N/O 0%

OT-I 0% N/O 0%

Ad5-mTNFa/mIL2 + OT-I 10% N/O 10%

Preconditioing+ OT-I 10% N/O 67%

Preconditioning+ Ad5-mTNFa/mIL2 + OT-I

63% N/O 100%

Table 1 – Table evidences the degree of histopathological changes across the hematoxylin-eosin stainings from the extracted andprocessed organs from endpoint hamsters and mice. Percentages were obtained by normalizing the number of animals with significantfindings.

Cancer Patient

TT

T

T TT

TIncreased T cell infiltration/ Proinflammatory modulation

T cellInfusion

TILT-123 injection

TT

Tumor oncolysis and IL-2 and TNFa release

Figure 1 – Ilustrative mechanimand sequence of administration.Oncolytic adenovirus isadminstered locally in the lesionsand drives positive modulation ofthe tumor microenvironment.The tumor will now attract T cellsthat are infused afterwards.