Sensitizing Protective Microenvironments to Antibody

Sensitizing Protective
Microenvironments to Antibodymediated Therapy
7.3.2014 | Dr. Christian P. Pallasch
The Microenvironment in Lymphoma
and Leukemia
Therapy
Lymphomagenesis
Progression
Seite 2
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
Macrophages determine antibody
therapy outcome in the hMB
humanized Lymphoma Model
Human
Cord Blood
derived
CD34+ HSC
Eµ/CD19-Pro
GF
P
MY
C
BCL2
Control
+
Antibody
Treatment
Leskov et al.Oncogene 2012
n.s.
***
**
107
GFP+ cells / organ
GFP+ cells / spleen
107
106
105
104
Control
Alem.
Clod.
Clod
+ Alem.
**
***
**
106
105
104
103
102
Alem.:
+
+
+
+
Peripheral Spleen Liver Bone
Blood
marrow
Pallasch et al. CELL 2014
Seite 3
***
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
Development of resistence towards
Alemtuzumab in the Bone Marrow
Day 4
Day 9
Day 12
Day 15
• Lymphoma cells in the bone marrow are susceptible
to Alemtuzumab treatment until they reach a
threshold of ~5% infiltration
Seite 4
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
The tumor microenvironment
determines therapeutic response
Antibody-mediated
phagocytosis of
lymphoma cells
Primary resistance
to therapeutic antibody
???
Seite 5
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
An in vivo RNAi approach towards
macrophage inactivating mechanisms
shRNA library
(MLSmCHERRY)
PCR amplify
hairpins
Control
Control
PTGES3
High-throughput
sequencing
FCGR2B
CD52
MIF
Harvested
hMB cells
Alemtuzumab treated
Quantify shRNA
sequences
Antibody
treatment
?
PGE2
FCGR2B
% Alem. specific killing
**
100
**
80
60
40
20
R2
B
sh
PT
G
ES
3
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
sh
FC
G
Seite 6
sh
CD
52
Co
nt
ro
l
0
G
Un
tre
at
ed
Co
A
nt
M
l
e
ro
-C
m
l
t
SF
uz
um
+
Al
ab
em
tu
zu
Irr
m
ad
ab
ia
Irr
t io
ad
n+
ia
Al
t io
em
n
t
uz
Cy
um
cl
op
ab
ho
sp
Cy
ha
cl
m
op
id
+
e
Al hos
em ph
tu am
zu id
m e
ab
Combinatorial Chemo-immunotherapy
overcomes site-specific resistance
Seite 7
Pallasch et al. CELL 2014
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
Cyclophosphamide induced Cytokine
response ehances macrophage dependent
lymphoma cell depletion
**
**
200
**
75
**
**
**
17.5
**
100
50
20
10
TNFα pg/ml
VEGF pg/ml
15.0
150
IL8 pg/ml
CCL4 pg/ml
30
50
25
50
-
10.0
2.5
5.0
d1 - - d1 d2
-
40
d1 - - d1 d2
***
80
***
***
**
60
30
40
20
20
10
0
an
Blocking antibody
in conditioned media
L4
CC
GF
VE
Fα
TN
IL
8
tiVE
GF
0
M
ed
ia
Seite 8
d1 - - d1 d2
*
*
Co
nt
ro
CT C
l
X ond
Pr .
et Me
re d
at ia
ed
BM
an
tiTN
Fα
% Alem. specific killing
*
-
% Alem. specific killing
M
ed
ia
Co
CT C
n
X ond tro
l
Pr .
et Me
re d
at ia
ed
BM
d1 - - d1 d2
12.5
2.5
0
Irradiation CTX
-
***
Recombinant
cytokines
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
GFP+ leukemic cells / femur
Finding a dosing regimen synergy is time dependent
107
*
*
106
105
104
103
102
-4
-2
-1
0
+1
+2
Interval Alemtuzumab to
Cyclophosphamide / days
Pallasch et al. CELL 2014
Seite 9
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
+4
untreated
% F4/80+ macrophages / femur
Cyclophosphamide Treatment rewires
the Tumor Microenvironment
*
8
*
6
4
*
2
0
d0
d1
Untreated
non-leukemic
Cyclo.
GREEN
RED
GFP+ lymphoma cells
70kD Dextrane-TexasRed/
Macrophages
leukemic
24 h
FC
Leuk vs.
72h CTX
72 h
FC
Non-Leuk.
vs. Leuk.
2.8 *
1.4 ***
-1.5 ***
1.4 *
CD86
CXCL9
TNF
CXCR4
CD16/32
Lyve-1
CD80
CD14
CD206
TLR2
Arginase1
FcgR1a
CCL2
CD68
IL4
Dectin-1
IL10
IL12
1.5 *
6.0 **
2.6 *
-2.5 *
-1.6 ***
-3.2 ***
1.4 *
2.1 *
log2 MFI
2.5
Seite 10
d5
Cyclophosphamide
6
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
10
***
100
80
60
40
20
0
Seite 11
***
*
hCD19+ leukemia cells / femur
% Rituximab spec. cell killing
Cytokine Release determines
Cyclophosphamide/Rituximab Resistance
in Patient derived B-ALL
**
*
108
**
106
104
102
0
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
n.s.
Chemotherpy in ALL patients increases
macrophage frequencies in the bone
marrow
Seite 12
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
Cyclophosphamide induced acute
Cytokine Release enables
macrophage-dependent tumor cell
depletion
VEGF
TNF
IL8
CCL4
Differentiation
Migration
Activation
Cyclophosphamide
Treatment
Pallasch et al. CELL 2014
Seite 13
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
Tumor Microenvironment and Therapy Perspectives and further plans…
1. Microenvironmentdependent
resistance
?
?
2. Mechanism of
the Acute
secretory
activating
response
?
4. Development of
3. Therapeutic
Microenvironment-tailored
Implications of
therapeutic
strategies
Macrophages
Seite 14 | Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch
Acknowledgements
AG Therapie und
Mikromilieu Maligner
Lymphome
Daniela Vorholt
Yvonne Meyer
Nadine Kutsch
Janine Schwamb
Andreas Roth
Jana Baller
Steffen Hoppe
On Yu Kwon
Seite 15
Uniklinik Koeln
Lukas Frenzel
Karl Kreuzer
Lukas Heukamp
Clemens Wendtner
Michael Hallek
VJM Amsterdam
Nico van Rooijen
David H. Koch
Institute for
Integrative Cancer
Research at MIT
Luke Gilbert
Christian Braun
Yadira Soto
Ilya Leskov
Jianzhu Chen
Frank Gertler
Herman Eisen
Mike Hemann
| Sensitizing protective microenvironments to antibody-mediated therapy | Dr. Christian P. Pallasch

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