1

Vincenzo De Laurenzi

BAG3 a potential target for Pancreatic adenocarcinoma treatment

2http://atlasgeneticsoncology.org/Genes/Images/BAG3Fig3.png

Intracellular BAG3

PLC-g

CASA Complex

Chaperone-assisted

selective autophagy

-BAG3 protein belongs to a family of co-chaperones that interact with the ATPase domain of the heat shock protein Hsp70 through the BAG domain

-It is constitutively expressed, in normal muscle cells, and in several tumor types (melanomas, glioblastomas, pancreatic adenocarcinomas, others)

-Can be induced in most cell types in response to stress

BAG3 expression in tumors

Ovarian carcinoma

Lung cancers 62-100%

BAG3

ALLCLL

Astrocytomas (20-50%)

Melanomas (67-70%)

Colorectal carcinoma

Liver (100%)

Breast cancer

PDAC (100%)

Thyroid carcinomas (20-50%)Prostate carcinomas

BAG3 expression is increased in tumors

4

• 346/ 346 analyzed PDACs expressed BAG3.

• In a cohort of 66 patients who underwent radical resection (R0), patients’ survival was not correlated to age,sex, tumor grade, tumor stage, nodal stage, but was inversely correlated to BAG3 levels.

IHC RT-PCR

BAG3 is expressed in PDACs and

correlates with poor prognosis

Rosati et al., Am. J. Pathol., 2012

Pancreatic ductal adenocarcinoma

PDAC

Pancreatic ductal adenocarcinoma (PDAC; 12.4 new

cases per 100,000 men and women per year) is the

fourth leading cause of cancer death in Western

countries.

Pancreatic adenocarcinoma is characterized by a

poor prognosis after diagnosis, with a <8% survival

at five years.

Pancreatic Cancer

BREAST

Pancreas

5 year Survival

BAG3 is released by PDAC cells

BAG3 co-localizes with Rab7a, a cytosolic marker for endosomes,

BAG3 is released by PDAC cells

BAG3 can be detectedin patient serum

BAG3 (ng/ml)

N Total M F Age Average Mean 95% Conf Interval

T test vs PDAC

Subjects

T test vs Healthy Subjects

Healthy Subjects 329 90 239 53 (+9.7) 268 228-314 < 0.001

Pancreatic Ductal Adenocarcinomas

94 35 59 63 (+ 9.7) 727 520-1016

Chronic Pancreatitis 106 70 36 53 (+ 12.6) 299 219-406 < 0.001 0.514

Bile Duct Obstructions* 25 6 19 71 (+ 7.1) 207 111-382 < 0.001 0.399

Colon Carcinomas 45 8 37 66 (+ 8.3) 180 103-315 < 0.001 0.103

Stomach Carcinomas 44 13 31 67 (+ 8.1) 209 121-359 < 0.001 0.311

BIOUNIVERSA’S BAG3 ELISA Can Detect PDAC in a Pre-Symptomatic Phase

This cohort consisted of PDAC (n=29) and HS (n = 151) samples which were tested using a multiple logistic regression model for least two consecutive years. At 95% specificity, in the general population (PDAC risk: 1.3%), the post-test probability after four yearly repeated measurements is:

o at 1-2 years pre diagnosis 7.6-fold higher than the pre-test risk

o at 2-3 years pre diagnosis 5.2-fold higher than the pre-test risk

The increased post-test probability can allow early diagnosis

10

BAG3 binds monocytes/macrophages

100 101 102 103 104

FL2-H

Data.024

Quadrant Statistics

File: Data.024 Log Data Units: Linear Values

Sample ID: Patient ID:

Tube: Untitled Panel: Untitled Acquisition Tube List

Acquisition Date: 19-Sep-13 Gate: No Gate

Gated Events: 10000 Total Events: 10000

X Parameter: FL2-H (Log) Y Parameter : FL1-H (Log)

Quad Location: 29, 10

Quad Events % Gated % Total X Mean X Geo Mean Y Mean Y Geo Mean

UL 2576 25.76 25.76 9.99 8.44 37.28 26.99

UR 3109 31.09 31.09 1687.04 1149.23 46.87 28.25

LL 3991 39.91 39.91 5.74 4.20 4.33 3.71

LR 324 3.24 3.24 332.06 130.94 6.31 5.72

CD14

rBAG3

CD14+rBAG3+

FL

1-H

10

01

01

10

21

03

10

4

0

4

0

80

12

0

1

60

20

0

BAG3 binding activates macrophages

J774.A1 cells

Primary human monocyts

J774 A.1 Cells

+ rBAG3 - rBAG3

Membrane fraction purification

LC-MSMS

9 proteins present only in rBAG3+

Affinity chromatography

Identification of BAG3 receptors by affinity chromatography and LC-MSMS analysis

IFTM-2 is the receptor for BAG3

Functions:-Inhibits entry of viruses in the host cell-Induces caspase dependent, p53 independent celldeath

Interferon-Induced TransMembrane protein-2 (IFITM-2)

BAG3 binding to IFITM-2 induces

the phosphorylation of p38 and Akt

BAG3 activated Macrophages

induce PDAC cell growth

BAG3R480A, a mutant that lost its ability to

interact with Hsp70, is still capable of binding

and activating monocytes/macrophages

YM-1 an inhibitor of BAG3 /Hsp70 Interaction

does not affect monocyte activation

An anti BAG3 antibody can block

BAG3-dependent macrophage activation

p values were calculated by Student’s t: *, p < 0.05 > 0.01 ; **, p < 0.01 > 0.001; ***p < 0.001

Anti-BAG3 antibody reduces tumour growth

20p values were calculated by Student’s t: *, p < 0.05 > 0.01 ; **, p < 0.01 > 0.001; ***p < 0.001

heterotopic xenografts of patient- derived tumor tissues (PDXs) in nu/nu mice (murine anti-BAG3 mAb AC-2)

immune-deficient mice

tumor tissue

21

Anti-BAG3 antibody reduces tumour growthand metastatic spreading

orthotopic xenografts of MIA PaCa-2 cells in nu/nu mice: inhibition of tumor growth and metastatic process ( murine anti- BAG3 mAb AC-2)

Immune-deficient mice

Anti BAG3 tretment results in reduction of:number of tumor- infiltrating macrophages

and of cytokine content in tumor tissue

22orthotopic xenografts of MIA PaCa-2 cells in nu/nu mice: ( murine anti- BAG3 mAb AC-2)

Immune-deficient mice

Anti BAG3 antibody mantains its effects in the presence of a fully competent immune system

23Heterotopic allografts of a murine pancreatic cancer cell line (mt4-2D) in syngeneic, immunocompetent mice (C57BL6) (murine anti- BAG3 mAb AC-2)

mt4-2D

immunocompetent mice

We generated a Humanizedanti-BAG3 monoclonal antibody

IN VITRO BINDING (Elisa)

INHIBITION OF BAG3 BINDING TO MACROPHAGES (FACS)

LEAD COMPOUND/CANDIDATE SELECTION

mAC-2 cAC-2 hAC-2

Humanized anti-BAG3 monoclonalAccumulates in the tumor

Immune-deficient mice

MIA PaCa-2

Blu: Draq5 (nuclei)Green: anti-human Alexa Fluor 488

PBS hAC-2 (H2L4)

72hrs after i.v. Treatment

MiaPaca-2

PBS

hAC-2

0

500

1000

1500

2000

2500

% t

um

or g

ro

wth

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

Day 22Day 27Day 30Day 34Day 37Day 41Day 44Day 48Day 51Day 55

% o

f tu

mo

r gr

ow

th

PBS

hAC-2

A Humanized anti-BAG3 monoclonalantibody shows similar effects

heterotopic xenografts of MIA PaCa-2 cells in nu/nu mice. Humanized anti-BAG3 monoclonal antibody: hAC-2

Immune-deficient mice

MIA PaCa-2

Start of treatment(20mg/Kg 3q/w)

0

200

400

600

800

1000

1200

TUMOR GROWTH

control IgG1 anti-BAG3 anti-BAG3+anti-PD1 anti-PD1

Tum

or

volu

me

mm

3 +

SE

0 7 13 19

days from the first administration

anti-BAG3 20 mg/Kg 3 times a week

anti-PD-1 (clone J43–bioxcell) 10 mg/Kg twice a week

p vs anti-PD-1 0.002

p vs control IgG1 0.01

p vs control IgG1 0.05

control IgG1 (clone MOPC-21- bioxcell) 20 mg/Kg 3 times a week

20Iorio V et al. Gut. 2017

Combinationof anti-BAG3and anti-PD1 arrests tumor growthin pancreatic cancer allografts in immunocompetent mice

Anti-BAG3 and anti-PD1combination

mt4-2D

immunocompetent mice

Combination of anti-BAG3 and anti-PD1 Effect on macrophages

Control IgG

F4/8

0 //D

AP

I

Anti- BAG3 Anti- BAG3 / PD-1 Anti- PD-1 0

0,1

0,2

0,3

0,4

0,5

0,6

Rel

ati

ve

flu

ore

scen

t a

rea

F4

/80

/ D

AP

I

Control IgG

Anti- BAG3

Anti- PD-1

Anti- BAG3/ PD-1

**

** p < 0.001

n = 3

Field = 9

21Iorio V et al. Gut. 2017

Anti-BAG3 and anti-PD1combination

Control IgG Anti- BAG3 Anti- PD-1 Anti- BAG3 / PD-1

CD-8/DAPI

0

50

100

150

200

no

f p

osit

ive

cel

ls

Control IgG Anti- BAG3 Anti- PD-1 Anti- BAG3/PD-1

*

** p < 0.001

22Iorio V et al. Gut. 2017

Combination of anti-BAG3 and anti-PD1 Effect on CD8+ cells

Anti-BAG3 and anti-PD1combination

-PDAC cells release BAG3

-BAG3 binds to macrophages through a specific receptor and activates the PI3K and p38 signalling pathways

-BAG3 dependent activation of macrophages results in secretion of factors that promote PDAC cell growth.

-Blocking these paracrine loop through an anti-BAG3 antibody in vivo results in reduced tumor immune infiltrate and reduced tumourgrowth and metastatic spreading.

CONCLUSIONS

-PDAC cells release BAG3

-BAG3 binds to macrophages through a specific receptor and activates the PI3K and p38 signalling pathways

-BAG3 dependent activation of macrophages results in secretion of factors that promote PDAC cell growth.

-Blocking these paracrine loop through an anti-BAG3 antibody in vivo results in reduced tumor immune infiltrate and reduced tumourgrowth and metastatic spreading.

CONCLUSIONS

-PDAC cells release BAG3

-BAG3 binds to macrophages through a specific receptor and activates the PI3K and p38 signalling pathways

-BAG3 dependent activation of macrophages results in secretion of factors that promote PDAC cell growth.

-Blocking these paracrine loop through an anti-BAG3 antibody in vivo results in reduced tumor immune infiltrate and reduced tumourgrowth and metastatic spreading.

CONCLUSIONS

-PDAC cells release BAG3

-BAG3 binds to macrophages through a specific receptor and activates the PI3K and p38 signalling pathways

-BAG3 dependent activation of macrophages results in secretion of factors that promote PDAC cell growth.

-Blocking these paracrine loop through an anti-BAG3 antibody in vivo results in reduced tumor immune infiltrate and reduced tumourgrowth and metastatic spreading.

CONCLUSIONS

Thanks to:

• Gianluca Sala

• Daniela Barcaroli

• Erminia Carletti

• Daniela D’Agostino

• Antonella De Cola

• Verena Damiani

• Cosmo Rossi

• Caterina Turco• Alessandra Rosati• Anna Basile• Raffaella D’Auria• Morena D’Avenia• Margot De Marco• Antonia Falco• Vittoria Iorio• Liberato Marzullo• Maria Pascale

Our collaborators:• Michael Hahne

• Marteen Bijlsma

• Michael Karin

• David Tuveson

• Matt Firpo