H. LEE MOFFITT CANCER CENTER & RESEARCH INSTITUTE, AN NCI COMPREHENSIVE CANCER CENTER – Tampa, FL

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Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity via High Mobility Group Box 1

Hao Liu, Pasquale Patrick Innamarato, Krithika Kodumudi, Amy Weber, John L Robinson, Satoshi Nemoto, Georgina Crago, Timothy McCardle, Erica Royster, Amod A Sarnaik*, and Shari Pilon-Thomas*

H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL

Abstract

Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. Rose Bengal (RB) is a staining agent that was originally used by ophthalmologists and in liver function studies. Previously, IL injection of RB induced regression of injected and uninjected tumors in murine models. However, the relevant mechanism is yet unknown. In this study, we used an OVA-expressing B16 melanoma murine model and found that IL RB treatment led to increased tumor-specific T cells with memory characteristics. IL RB therapy also increased antigen-specific T cell proliferation and enhanced tumor regression. In addition, IL RB facilitated dendritic cells (DCs) infiltrating lymph nodes draining from tumor. Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs. The blockade of HMGB1 significantly reduced the antigen-presenting ability of DCs. To determine whether this mechanism was relevant in patients treated with IL RB, we performed a pilot clinical study in melanoma patients (NCT01760499). IL RB led to tumor regression in both RB-injected and uninjected lesions, associated with an increase in circulating T cells. Increased tumor-specific response was found from those circulating T cells of 5 out of 7 tested patients after IL RB treatment. HMGB1 levels in patient sera were also elevated. Together, these results reveal a clinically relevant immunoadjuvant pathway triggered by tumor cell death secondary to ablation with RB.

IL PV-10 elicits a tumor-specific immunity

IL PV-10 leads to DC infliation and maturation

Increased serum HMGB1 levels in patients after IL PV-10 therapy

Background IL PV-10 facilitates the proliferation of tumor-specific CD8+ T cells

IL PV-10 leads to the release of HMGB1 from necrotic melanoma cells Conclusion

1 0 1 5 2 0 2 5 3 00

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

D a y s a fte r tu m o r in o c u la tio n

Tu

mo

r a

rea

(m

m2

)

P V 1 0

P B S

MO5 s.c.

0 7 day

IL PV-10

Tumor

10 15 20 25 300

200

400

600

800

Days after tumor inoculation

Tum

or a

rea

(mm

2 )

IL PBSIL PBS+OT-1IL PV-10IL PV-10 + OT-1

*

1:40 1:160 1:6400

20

40

60

c.p.

m. (

x 10

4 ) TS (PBS)TS (PV-10)w/o TS

***

***

DC:T

MO5 s.c.

0 7 day

IL PV-10 IL FITC – OVA i.t.

8 LNs

BMDC day 0 2

Supernatants (TS) + OT-1 cells

+3H-thymidine

B16 PV-10 or PBS

DLN NDLN0

5

10

15

# Li

ve F

ITC

+ DC

s pe

r LN

(x10

3 )

PV-10PBS

n.s.

*

pre D7-14 0

100

200

300

IFN

-g p

g/m

l

*

Biopsy Biopsy of 2 lesions PBMC, Serum Pretreatment

IL PV-10 into one lesion; the uninjected one is bystander lesion

PBMC, Serum Post treatment

D0 D7-14 D21-28

Autologous tumor

pre D7-14 D21-280

5

10

15

20

HM

GB1

(ng/

ml)

in s

era

*n.s.

matched mismatched0

50

100

150

200

250

IFN

-g (p

g/m

l)

preD7-14

**

D21-28

*

n.s.n.s.

HMGB-1

PV-10

Lymph node

IFN-g

Tumor cells

CTLs

Tumor lesion

Activated DC

DCs

T cell priming

Naïve T cells

Necrotic tumor

IL PV-10 led to the necrosis of melanoma cells and release of HMGB1 to activate DCs and elicit a systemic anti-tumor immune response. Hypothetic Model:

Acknowledgement: This study was supported by the Cancer Center Support Grant P30 CA076292 from the National Cancer Institute and NCI 5K23CA178083-02 (AAS). PV-10 was provided by Provectus Biopharmaceuticals Inc.

Draining LN

PV-10 ILPBS IL

PV-10 s.c.

PBS s.c.0

20

40

60

80

100

Div

ided

OT-

1 (%

)

** *

PBS PV-10 0

5

10

15

20

25

% O

VA te

tram

er+ /

CD

8

**

HLA- matched PBMCs

Enrich CD8+T

ELISA

+Tumor

Safety: •  Not metabolized •  Short circulatory half-life (ca 30 min) •  Excretion via bile

Late 1990’s

Rose Bengal (PV-10)

1884 1923 1914 Mid- 2000’s

Rose Bengal

Question: How does PV-10 induce a systemic anti-tumor response?

Mouse B16 melanoma expressing OVA (M05)

PV-10 can directly kill tumor cells and also elicits a systemic response

B6 mice

s.c. Model

MO5 s.c. OT-1 T cells i.v. IL PV-10

0 13 day 17 Tumor growth

B 1 6 3 T 3 P 2 9 3 T0

1 0

2 0

3 0

% n

ecr

osi

s

P B S

P V -1 0

Flow cytometry analysis of B16, 3T3 fibroblasts, primary human melanoma cells (P) and human embryonic 293T cells were incubated with 50 uM RB for 48 hours. Increased necrotic cells (DAPI+ Annexin V-) were observed.

TS: tumor supernatant

BMDC 0 2

TS OT-1 cells B16

PV-10

HMGB-1 blockade

+3H-thymidine

1 :1 0 1 :4 0 1 :1 6 0 1 :6 4 00

5

1 0

1 5

2 0

c.p

.m (

x10

4)

Is o

a n ti-H M G B 1**

**

*

n .s

D C :T

B 1 6 8 8 80

1 0

2 0

3 0

4 0

5 0

HM

GB

1 (

ng

/ml)

0 u M

1 0 0 u M

2 0 0 u M

****

**

Data showed the increased level of HMGB1 in the supernatant from B16 and human melanoma 888 cells after RB incubation.

Increased HMGB1 were observed in patient’s sera after IL PV-10 (n=14)

Early- 2000’s

Early- 2010’s