Sensitivity of
89Zr-labeled
anti-CD8 minibody for PET imaging of infiltrating CD8+ T cells
Tove Olafsen, Ziyue Karen Jiang, Jason Romero, Charles Zamilpa, Filippo Marchioni, Green Zhang, Michael Torgov, Daulet Satpayev, Jean Gudas
ImaginAb Inc., Inglewood, CA, USA
Summary and Conclusion
Abstract

89Zr-Df-IAB22MC2
A
 Ongoing preclinical imaging, safety and toxicology studies will enable the the start of clinical studies in Q3
2016
Lung CD8 (10X)
~1 million CD8+ T
cells in ~500 mg
lung tissue
~100,000 CD8+ T
cells in ~50 mg
spleen
Ex vivo labeling of CD8+ T cells
Intramuscular Injected Cells
4h
24h
No
T cells
0.04M
heart
liver
spleen
0.4M
Tumor cells were mixed with the indicated numbers of CD8+ T IHC of S.C. Matrigel Plugs
CD8
CD3
cells and implanted I.M. without Matrigel (left panel) or S.C. in
4M
Saline:Matrigel (1:1) (lower panel). 2 days after (IM) and 6
days after (SC) cell implantation 89Zr-Df-IAB22M2C was
injected I.V. and imaging performed. The highest signal I.M.
1.6M
was seen with 4 million CD8+ T cells. S.C. Matrigel plugs a
weak signal could be detected in the plugs containing ≥3.2
million CD8+ T cells in vivo. Ex vivo imaging of the plugs
0.4M
clearly showed uptake in plugs containing ≥3.2 CD8+ T cells.
Drawn regions of interest (ROIs) at 24h and 48h images
confirmed a significant difference in activity in plugs
0M
containing ≥3.2 CD8+ T cells. Anti-CD8 and anti-CD3 staining
of matrigel plugs containing 4, 1.6 and 0.4 million CD8+ T 10x magnification
cells is shown on the right (different study).
In vivo imaging of S.C. Matrigel Plugs at 48 hours
1.8
2.4
5.2
5.6
6.3
7.7 %ID/g
Matrigel plugs
CH3
No
T cells
0.8M
3.2M
5.6M
8M
P < 0 .0 0 0 1
10000
12.8M
P = 0 .0 0 0 1
ns
4000
2000
0
0 .0
0 .8
3 .2
5 .6
8 .0
1 2 .8
m illio n C D 8 + T c e lls
Df-IAB22M2C Conjugated Mb (~80 kDa)
48h R O I N=2
None
8.0M
12.8M
5.6M
8.0M
3.2M
5.6M
0.8M
3.2M
None
0.8M
10000
to ta l r a d ia n c e in R O I
Figure 1:
(A) Schematic structure of 89Zr-Df-IAB22M2C. (B) Binding to cell surface CD8 demonstrates that
binding was retained following conjugation with Df (EC50 0.64 vs 0.83 nM)
12.8M
8000
6000
4000
2000
0
0 .0
The mean radioactive uptakes (%ID/g) at 48h are shown above
0 .8
3 .2
110
~1 million
CD8+ T cells
4
%ID/organ
5
30
25
20
15
10
0
3
21
Liver
control
D
ay
Blood
ay
14
110
Spleen
week 1
week 2
5 .6
8 .0
m illio n C D 8 + T c e lls
1 2 .8
Lungs
week 3
PET images reveal higher radioactive uptakes in lungs, spleen and liver with GvHD progression that
correlates with increased numbers of CD8+ T cells measured by FACs analysis of extracted T cells and
IHC. After 3 weeks, approximately 1 million CD8+ T cells were present in 0.5 g lung tissue that could
readily be visualized by PET. As expected, the percent dose accumulation in the lung at 24h p.i.
increased with the severity of GvHD.
PET Imaging of CD8+ TILs with a Murine Surrogate Probe
OX40 single treatment
OX40/PD-1 combo
8% ID/g
24 hrs
CD8 10x
700
600
500
400
300
200
100
0
8% ID/g
24 hrs
CD8 10x
Tumor Growth Curve
Tumor Growth Curve
1.5% ID/g
3 4 7 9 11 14 16 18 21 24
3.0% ID/g
700
600
500
400
300
200
100
0
3 4 7 9 11 14 16 18 21 24
Days
P < 0 .0 0 0 1
6000
110
6
5
0
6.2% ID/g
P < 0 .0 0 0 1
8000
200
1.5% ID/g
24h R O I N=6
Ex vivo imaging at
48 hours
~ 500 mg
tissue
110
D
• (A) Human T cells were expanded ex vivo, incubated with >2 molar excess
and subsequently
aliquoted into tubes to yield 12, 4, 1.2, 0.4, 0.12 and 0.04 million CD8+ T cells. Gamma counting of each aliquot
generated a standard curve that correlated with the number of radiolabeled CD8+ T cells present. PET images of tubes
showed that the lower limit of detection ex vivo was approx. 0.4 million CD8+ T cells.
• (B) Serial dilutions of 4, 1.6, 0.4 and 0.16 million “hot” T cells were admixed with 2 million Raji cells, resuspended in
Saline:Matrigel (1:5) and implanted either subcutaneously (S.C.) or intramuscularly (I.M.) into mice. PET imaging showed
that four (4) million CD8+ T cells could readily be visualized at 4h and 18h. 0.4 million CD8+ T cells were detectable at 0
hour due to very low background activity in normal tissues.
400
35
7
0.16M
40
ay
30
0.16M
0.4M
45
7
D
10
20
# of T cells (million)
0.4M
110
per Lung
0
1.6M
4M
o f M ic e w ith G v H D
600
T C e l ls
0
0.04×106
1.6M
No. CD8+
500000
0.12×106
4M
21
1000000
i.m.
50 µL
PERCENT DOSE/ORGAN
C D 8 + T C e ll C o u n t s in L u n g s
L u n g M a s s ( m g ) w it h T im e
ay
1500000
0.4×106
D
2000000
0.16M
14
R² = 0.9964
0.16M
0.4M
1.2×106
ay
2500000
0.4M
D
3000000
4M
2%
ID/g
1.6M
1.6M
7
3500000
s.c.
300 µL
4×106
4M
ay
4000000
D
4500000
12×106
(m g )
T cell standard curve
(gamma counting)
Liver CD8 (10x)
B
M ass
A
4M
scFv
VH
18%
ID/g
Week 3 GvHD
Sensitivity of PET for Detecting CD8+ T Cells
Subcutaneous Matrigel Plugs (48 hours)
VL
Control
Spleen CD8 (10x)
-- IAB22M2C
-- Df- IAB22M2C
89Zr 4+
Week 3
Week 1
Tumor volume (mm3)
B
Imaging at 4 and 24 hrs
post 89Zr-Df-IAB22M2C
is a sensitive and promising tracer for detecting human CD8 positive immune cells in vivo
Tumor volume (mm3)
IAB22M2C is an approximately 80 kDa minibody (Mb) comprised of humanized anti-CD8
VL and VH sequences assembled into scFvs and fused to the human IgG1 CH3 domain
via a proprietary hinge sequence H (Figure 1). IAB22M2C Mb is conjugated with Df on
lysine residues and radiolabeled with 89Zr (t1/2 3.3 days) to yield 89Zr-D-IAB22M2C.
3-4 weeks
Imaging at 4 and 24 hrs
post 89Zr-Df-IAB22M2C
 CD8+ TILs could be visualized in syngeneic mouse tumors following treatment with check-point inhibitors
using a surrogate anti-murine CD8 Mb
to ta l r a d ia n c e in R O I
Anti-CD8 Probe-
GVHD clinical signs
1 week
 In a GvHD model, rapid expansion of CD8+ T cells occurred from 1 to 3 weeks with tissue infiltration that
allowed detection of approximately 1 million CD8+ T cells in 0.5 g lung and as few as 0.1 million CD8+ T cells
in 50 mg of spleen tissue
In vivo detection of CD8+ T cells with an intravenously administered probe
89Zr-Df-IAB22M2C
No clinical
signs
 CD8+ T cells implanted in the muscles or in Matrigel plugs indicated that the lower limit of detection was
between 1.6 and 4 million CD8+ T cells in the presence of normal tissue background activity
89Zr-Df-IAB22M2C
Conclusion- These studies show that the lower limit of CD8+ T cell detection by 89Zr-DfIAB22M2C is between 1.6-4.0 million cells in the presence of normal tissue background
activity and that the probe can be used to monitor CD8+ T cell trafficking in a GvHD model
in vivo. 89Zr-Df-IAB22M2C has sensitivity properties that may enable the detection of
CD8+ T cells in human tumors. Clinical trials with 89Zr-Df-IAB22M2C in melanoma patients
will commence later this year.
Engraft
huPBMCs
Lung
Results- CD8+ T cells implanted in the muscles of mice were imaged one day later and
SC implanted Matrigel plugs imaged 6 days later. Both approaches yielded similar results
and indicated that the lower limit of detection was between 1.6 and 4 million CD8+ T cells
in a volume of ~480 mm3 in the presence of normal tissue background activity. The
sensitivity of detection increased 10-fold when ex vivo radiolabeled CD8+ T cells were
implanted SC with Matrigel. NSGTM mice engrafted with human PBMCs provide a reliable
model for xenogeneic T cell driven Graft versus Host Disease (GvHD). Human CD8+ T
cells were readily detectable in the spleens of mice 1 week post PBMC engraftment using
89Zr-Df-IAB22M2C. As GvHD progressed 4 weeks later, expansion and trafficking of the
engrafted T cells to extra-lymphoid tissues including lungs could be followed. Terminal
biodistribution showed a 2-3 fold increase in radioactivity uptake in lungs by week 4 postengraftment; a result that was confirmed by IHC analysis. T cell enumeration and IHC
analyses are in progress to further define the sensitivity range using an optimal dose and
specific activity of 89Zr-Df-IAB22M2C.
Df-IAB22M2C was radiolabeled with 89Zr to yield a specific activity of ~18 µCi/µg. Approximately 80 µCi
was administered i.v. to visualize CD8+ T-cells in NSG mice engrafted with human PBMCs by PET.
 Ex vivo and in absence of normal tissue background activity the lower limit of detection was 0.4 million CD8+ T
cells
Number of CD8+ cells per tube is indicated
Methods- IAB22M2C, a humanized anti-CD8 minibody, was conjugated with
desferrioxamine (Df) and radiolabeled with 89Zr. NOD scid mice were implanted with
varying ratios of CD8+ T and tumor cell admixtures either intramuscularly (IM) without
Matrigel or subcutaneously (SC) with Matrigel. One or six days later, CD8+ T-cells were
visualized with 89Zr-Df-IAB22M2C. The same probe was used to detect CD8+ T cells in
NSGTM mice engrafted with human PBMCs for 1 and 4 weeks to monitor the temporal
progression of Graft versus Host Disease (GvHD).
 A humanized anti-CD8 Mb (IAB22M2C) was generated, conjugated with Df and shown to retain high affinity
and specificity for binding to human CD8 positive T cells
CPM
Background- The ability to monitor CD8 positive tumor infiltrating lymphocytes (TILs) in
vivo is important for evaluating response to immunotherapies and assisting in the
development of more effective immune cell targeted single and combination therapies.
“ImmunoPET” imaging of tumor infiltrating T cells can provide a specific and sensitive
modality to aid selection of patients for specific immunotherapy regimens and determine
whether the therapy is working. Here, we report initial results to define the number of
CD8+ T cells that can be detected with 89Zr-Df-IAB22M2C, an anti-CD8 immunoPET
probe, using different animal models
CD8+ T Cell Detection in a GvHD Model
• Surrogate anti-mouse CD8 Mb (89Zr-Df-IAB42M) was used to
evaluate CD8+ T cell infiltration in Balb/c mice bearing CT26 tumors
following treatment with the immune modulating antibodies OX-40
and PD-1
• The left and right panels above show the PET image, anti-CD8 IHC
staining and tumor growth curve (blue line) of a responding and nonresponding mouse respectively, following treatment. Dotted line =
isotype control treated tumor growth curve.
• A stronger PET signal corresponding to a greater number of CD8+ T
cells detected by IHC was observed in the treatment responsive
tumor. Biodistribution confirmed a ~2 fold higher radioactive uptake in
the responding tumor and in isotype treated tumor (lower right panel).
Days
Isotype control treated
8% ID/g
CD8 10x
1.5% ID/g
2.6% ID/g