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