A humanized mouse model for translational assessment of targeted immune checkpoint blockade
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Gilson S. Baia , David Vasquez-Dunddel , Daniel Ciznadija , David Sidransky , Amanda Katz , and Keren Paz
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Champions Oncology, USA. Johns Hopkins University School of Medicine, USA.
Introduction
Results
Results
The blockade of immune checkpoints is a promising therapeutic avenue for cancer therapy, with durable objective
2. Tumor engraftment and growth in humanized mice and immune activation
4. Anti-tumor responses engaged in ImmunoGrafts after a
-CTLA4 treatment
responses observed in patients with a variety of solid tumors. Despite these successes, current animal models do not
human immune system), are combined in a single platform. We now describe our results employing the ImmunoGraft to
assess the effectiveness of immunotherapy on tumor growth and immune activation.
Methods
4000
Humanized
(n = 9)
3000
2000
1000
0
0
10
20
30
Day
n.s
40
20
Before
treatment
50
huCD8+ cytotoxic T cells
Autologous
CD34+ HSCs
After 5
treatments
(at 21 days)
At sacrifice
(28-53 days)
PBMCs
Spleen
Flow cytometry
on reconstituted
animals
Marrow
Removal of tumor
Vehicle
a
-CTLA4
a
-CTLA4
NSCLC PDX models grow equally well in humanized (immunografted) and non-humanized animals (A)
a
-CTLA4 treatment of NSCLC ImmunoGrafts results in an increase in circulating human CD3+ T cells (B, **p<0.05, Student’s t-test)
huCD19+ B cells
Implant tumor fragments
* Tumor growth inhibition
* Activated huCD4+/CD69+ T cells
a
-CTLA4 antibodies enhance infiltration of human CD3+ and CD8+ T cells and CD68+ macrophages into NSCLC tumors (C, D)
3. Anti-CTLA4 treatment induces regression of ALK+ NSCLC ImmunoGrafts
*Proliferating huCD4+/CD71+ T cells
Screen against
a
-CTLA4 antibodies
(immunotherapy)
IMMUNOGRAFT
a
-CTLA4 dosing
1. Human immune cell profile in reconstituted immunodeficient mice
Whole blood (n = 18)
% of live cells
60
20
peripheral
blood
80
100
n=4
40
huCD45+
(naive NOG)
25
Crizotinib
20
Vehicle
a
-CTLA4
+ Crizotinib
D
Activated huCD4+ Th1 cells
20
15
10
n.s
5
12
2.0
Human Ki-67
Crizotinib
a
-CTLA4
+ Crizotinib
Regulatory huCD4+ Th1 cells
n.s
n.s
Crizotinib
a
-CTLA4
+ Crizotinib
9
6
3
0
Crizotinib
a
-CTLA4
+ Crizotinib
Vehicle
Levels of proliferating and activated human T cells (CD4+ and CD8+) in ImmunoGrafts are highest following treatment with a
CTLA4 antibodies (A-C, **p<0.05, Mann-Whitney)
Levels of human CD25+ regulatory T cells are unchanged after any treatment (D)
Vehicle
1.5
Conclusions
1.0
Crizotinib
Human immune components were successfully reconstituted and activated in immunodeficient animals (humanization)
a
-CTLA4
0.5
a
-CTLA4/Crizotinib
PDX models successfully engrafted and grew in humanized mice (the ImmunoGraft model)
a
-CTLA4 antibody treatment induced human immune cell activation, anti-tumor responses, and substantial tumor regression
10
20
30
40
Days post treatment initiation
bone
marrow
mCD45+
Vehicle
0
20
huCD45+
40
0
spleen
0
Fractional tumor volume (relative to Day 0)
Results
0
n.s
0
Vehicle
ImmunoGraft spleen
* Tumor-infiltrating huCD8+ T cells
% of huCD45+ cells
40
60
4
Crizotinib dosing
2.5
B.
n.s
60
0
* huCD4+/CD25+ regulatory T cells
A.
8
huCD68+ macrophages
C
Vehicle
12
Vehicle
Reconstitute with
CD34+ hematopoietic
stem cells (HSCs)
10-12 weeks
16
0
% huCD4/CD69+ (% of huCD3+)
Commercial
CD34+ HSCs
a
-CTLA4
whole blood
ImmunoGraft tumor site
?
Impaired B,T, and NK cells
huCD3+ T cells
40
D
?
NOG mice (truncated IL-2Rg
)
huCD45+ leukocytes
Vehicle
60
80
Proliferating huCD4+ Th1 cells
huCD3+ T cells
80
0
NSCLC from patients or
banked tumor fragments
Chemical myeloablation
( immune suppression)
100
20
% huCD4/CD71+ (% of huCD3+)
Champions TumorGraft (patient-derived xenograft, PDX) and humanized mice (immunodeficient mice reconstituted with a
Tumor volume (mm3)
systems. To overcome this challenge, we developed the ImmunoGraft, whereby two innovative technologies, the
Vehicle
(n = 9)
a
-CTLA4
(n = 9)
B
huCD8+ cytotoxic T cells
% huCD4/CD25+ (% of huCD3+)
Non-humanized
(n = 9)
C
A
% huCD8+ (% of huCD3+)
5000
B
huCD3+ (% of huCD45+)
reliably identify immune targets with the greatest clinical potential due in part to differences between human and murine
A
ImmunoGraft tumor site
Treatment of NSCLC ImmunoGrafts with a
-CTLA4 antibodies induces definitive regression of tumor implants (left panel)
huCD3+
(T cells)
huCD19+
(B cells)
huCD56+
(NK cells)
a
-CTLA4 treatment leads to increased proliferation of human cells within the spleen of immunografted animals (right panel)
Acknowledgments
We thank Taconic for supplying immunodeficient NOG animals reconstituted with human CD34+ cells for this work and for important
guidance during these experiments.