zPREDICTA Disease Models

1For Internal Use Only© 2019
April, 2019
Next Generation Organ-Speciﬁc 3D Culture
Technology Enabling Accurate Efﬁcacy Testing
Of Anticancer Agents

What makes a successful
anti-cancer drug?
1

KEY CHARACTERISTICS OF "
A SUCCESSFUL ANTI-CANCER DRUG
DRUG
effective
safe

How do we ﬁnd compounds
that will be effective in patients?
2

Overcomes environment-mediated
drug resistance driven by:
•  Extracellular matrix (ECM)
•  Tumor-tumor interactions
•  Stromal elements
-  Secreted factors (cytokines, etc.)
-  Tumor-stroma interactions
-  Immune-protection
IDEAL CLINICAL CANDIDATE

zPREDICTA performs services for testing of anticancer compounds
WHAT WE DO
Unique advantages of zPREDICTA technology:
•  Long-term culture of primary human tumor cells
•  Organ-speciﬁc extracellular matrix
•  Compatible with multiple drug types (small molecules, antibodies, etc.)
•  Compatible with multiple readouts (FACs, imaging, etc.)
* Also available co-culture and cell line models

zPREDICTA’S TECHNOLOGY RECAPITULATES "
THE COMPLEXITY OF HUMAN TISSUES
Human
Organ-specific
3D culture system
1-to-1 reconstruction of human tissue
microenvironment
•  Organ-specific ECM
•  Disease-specific medium
supplements

EXTRACELLULAR MATRIX ARCHITECTURE OF r-BONE "
IS INDISTINGUISHABLE FROM IN VIVO TISSUE
In vivo bone marrow
r-Bone
(reconstructed bone marrow)
50μm
50μm
Cell cluster
Cell cluster
Cryo-electron microscopy (cryo-EM) of bone marrow organ reconstructed in the r-
Bone model compared to in vivo bone marrow.

UNIQUE ADVANTAGES OF zPREDICTA TECHNOLOGY
PREDICTIVE:

prediction of clinical outcomes based on the 1-to-1 reconstruction of
human tissues

ORGAN-SPECIFIC:
accurate reconstruction of organ-speciﬁc environment enabling
evaluation of therapeutic agents under conditions mimicking human
physiology, a capability currently lacking in pharma

VERSATILE:
high throughput format for large scale drug screening (10,000+
compounds) and high complexity format for lead optimization
single and multi-compartment models compatible with various cell
types from tumor and healthy tissues
(epithelium, stroma, endothelium, immune cells, etc.)
compatible with all drug classes tested
(small molecules, antibodies, antibody-drug conjugates (ADC), biologics,
cellular and immunotherapies, including CAR-T, etc.)
compatible with all standard readout methods
(in situ imaging, immunohistochemistry, ﬂow cytometry, cell-based assays,
genenomics, proteomics, metabolomics, etc.)

SIMPLE 4 STEP SET-UP
1. Coat plate w/ ECM
(bone endosteum/submucosa/
basement membrane)
2. Mix cells w/ organ-
speciﬁc ECM-2
3. Add cell/ECM mix to
pre-coated plate &
allow to polymerize
4. Overlay cell/ECM matrix w/
medium supplemented w/
disease-speciﬁc factors
bioassays imaging
READOUT
SET-UP
5. Add therapeutic agents
(small molecules, antibodies,
ADCs, biologics, CAR-Ts)
TREATMENT
in vivoFACs -omics
6. Isolate cells from ECM
ISOLATION

zPREDICTA MODELS SUPPORT WIDE RANGE OF TISSUES
Model Tissue
Cell lines
Multi-compartment
co-culture*
Primary
cells
HTS**
Custom
r-Brain in progress
Custom models can
be set-up for any
tissue of interest
utilizing the readout of
choice
r-Breast ✔ ✔ in progress
r-Bone (bone marrow) ✔ ✔ ✔#
in progress
r-Colon ✔ ✔
r-Lung ✔ ✔ in progress ✔
r-Ovary ✔ ✔
r-Prostate ✔ ✔
r-Skin ✔ ✔
r-Stomach ✔ ✔
Recommended
readout
CellTiter-Glo,
ELISA, imaging
FACs, ELISA,
imaging
FACs, ELISA,
imaging
CellTiter-Glo
*Tumor cell lines co-cultured with primary cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), or non-malignant
fibroblasts with or without immune components.
**Currently available High Throughput (HTS) platform for evaluation of small molecule efficacy using tumor cell lines
#r-Bone system has been validated for 3D culture of primary cells from multiple myeloma and AML

HOW TO WORK WITH US?
1.  Fee-for-service
2.  Integrate zPREDICTA’s technology into your workﬂow
3.  Custom models/development partnerships

SINGLE-COMPARTMENT MODELS:

Reconstructed Bone (r-Bone™)
Multiple myeloma (MM)
Acute myelogenous leukemia (AML)

BONE STRUCTURE

Matrigel
day 14
Collagen I
day 14
Plastic
day 14
Non-physiological environments: poor survival & lack of proliferation of primary multiple myeloma cells
NON-PHYSIOLOGICAL ENVIRONMENT DOES NOT SUPPORT "
SURVIVAL OF PRIMARY TISSUE

r-BONE SUPPORTS LONG-TERM SURVIVAL OF PRIMARY BONE MARROW FROM
MULTIPLE MYELOMA AND AML PATIENT, AND HEALTHY DONORS
r-BONE: robust long-term survival and proliferation of primary multiple myeloma
Osteoblasts

Adipocytes
Osteoclasts
Stromal
compartment

r-BONE SUPPORTS PROLIFERATION "
OF PRIMARY BONE MARROW CELLS
Proliferation of primary bone marrow cells from patients with multiple myeloma was measured by ﬂow
cytometry as a function of dilution of CFSE label. B cells exhibited high rates of proliferation while
plasma cells divided slowly, consistent with their proliferation patterns in vivo.

COMPOSITION OF THE HEMATOPOIETIC COMPARTMENT"
IS RETAINED IN r-BONE
Blast
1%
PMN
46%
Lymphocyte
20%
Monocyte
4%
Plasma cell
18%
NRC
11%
Blast
1%
PMN
48%
Lymphocyte
20%
Monocyte
2%
Plasma cell
21%
NRC
8%
PMN, polymorphonuclear cells
NRC, nucleated red blood cells
Ex vivo bone marrow
r-Bone

PHYSIOLOGIC ENVIRONMENT IS INDISPENSABLE FOR SURVIVAL "
AND GROWTH OF AML BONE MARROW
osteoclasts
osteoblasts
stromal
cells Live /Dead
Vehicle control Cytarabine
Live /Dead
Non-physiologic environments: poor survival & lack of proliferation of primary AML cells
rBONE AML Model: robust long-term survival and proliferation of primary AML cells
Matrigel
day 14
Collagen I
day 14
Plastic
day 14
r-BONE
day 14

r-BONE SUPPORTS LONG-TERM SURVIVAL AND "
PROLIFERATION OF PRIMARY AML
Hematopoietic compartment
r-Bone
Live/Dead

day 28
Live/Dead

day 4
2D (conventional culture)
2D (conventional culture)

SINGLE-COMPARTMENT MODELS:
Reconstructed Lung (r-Lung)
Non-small cell lung cancer (NSCLC)

r-LUNG SUPPORTS PROLIFERATION AND LONG TERM SURVIVAL"
OF PRIMARY NSCLC TUMOR EXPLANT TISSUE
7 days
plastic
Live/Dead
r-Lung
27 days
27 days
stroma
ex vivo
r-Lung
Ki67 staining
Viably cryopreserved primary NSCLC
tumor tissue was diced into small
pieces and cultured in r-Lung or on
the surface of a tissue culture plastic.
Cultures were grown for the indicated
number of days. Cell viability was
assessed using Live (calcein AM)/
Dead (ethidium homodimer staining)
and cell proliferation was assessed
by immunohistochemistry. 7days

r-LUNG SUPPORTS LONG TERM SURVIVAL OF PRIMARY NSCLC
DISSOCIATED TUMOR CELLS & INFILTRATING IMMUNE CELLS
Live/Dead

day 28
Live/Dead

day 33
Live/Dead

day 33
Primary NSCLC were dissociated from the
viably cryopreserved tumor tissue and
cultured in r-Lung. Cultures were grown for the
indicated number of days and cell viability was
assessed using Live/Dead staining.
The extent of the immune cell inﬁltration cells
was measured by ﬂow cytometry.
day 28
T cells (CD3+)
Myeloid cells
(CD33+)
15%
10%

ORGAN-SPECIFIC 3D MODELS FOR DRUG DEVELOPMENT
Evaluation of efficacy and toxicity in a single assay
screening/efficacy testing/
combination treatments
drug-resistance
bystander effect
off-target toxicity
target/biomarker discovery
patient stratification:
responders vs. non-responders
companion diagnostics
immuno-oncology
attrition management/
rescue failed drug candidates

zPREDICTA MODELS ARE:
A.  Compatible with any drug class
•  Small molecules
•  Antibodies
•  Antibody-drug conjugates (ADC)
•  Biologics
•  Immunomodulatory agents
•  CAR-T
B.  Can be utilized to evaluate efﬁcacy and toxicity
in a single assay

IDENTIFICATION OF RESPONDERS VS. NON-RESPONDERS
0.01 0.1 1 10
0
25
50
75
100
125
Naive
Refractory
Pomalidomide [µM]
%viability%viabilityCD138+cells

PIPELINE PRIORITIZATION
r-Bone mirrors the eﬃcacy
response seen in human trials,
outperforming current methods for
evaluation of clinical response, and
thus, reducing the rates of clinical
failure during drug development
and treatment regimen selection.
HEETpTMIxchel Scientific
conventional, plastic (2-D)
extracellular matrix only (3-D)
r-Bone
conventional (2D)
ECM only
FDA approved Advanced to clinical trials Advanced to in vivo PK/PD studies
AbandonedFailed efficacy (phase II) trial

ATTRITION MANAGEMENT & PIPELINE PRIORITIZATION
0.01 0.1 1 10
0
50
100
150 A
B
C
D
E
H
K
G+H
A+H
E+H
Patient I-P
µM
%viabilityCD38+cells
(normalizedtoDMSOcontrol)
Patient A1
2
3
4
5
6
7
8
9
10
%tumorcellviability
individual
compounds
combinations

EVALUATION OF COMBINATION TREATMENTS
BI2536/dexamethasone
combination demonstrates eﬃcacy
under the conditions of
environment-mediated drug
resistance of r-Bone™ model.
Cultured under physiological
conditions of r-Bone, BI2536/
bortezomib combination induces
tumor cell growth
BI2526 + Velcade BI2536 + Dex
0
50
100
150
200 2D
r-Bone™
%viability
(normalizedtovehiclecontrol)
BI2536 + Bortezomib

EVALUATION OF EFFICACY AND TOXICITY IN A SINGLE ASSAY
A.  Viability of tumor cells was evaluated in
response to bortezomib and INV
(investigational) compound in r-Bone,
revealing a dose and time dependent eﬀect
of treatment.
0
0.01
0.05
0.1
0
0.1
0.5
4
0
0.1
0.5
4
0
50
bortezomib (µM) GENE (µM)
day 7 day 14day 7
%
(normalizedt
0
0.01
0.05
0.1
0
0.1
0.5
4
0
0.1
0.5
4
0
50
100
150
day 7 day 14day 7
%CD38+
Patients 1-3A. Viability of tumor cells
INV
0
0.01
0.05
0.1
0
0.1
0.5
4
0
0.1
0.5
4
0
50
day 7 day 14
day 7
%C
(normalizedto
0
0.01
0.05
0.1
0
0.1
0.5
4
0
0.1
0.5
4
0
50
100
150
day 7 day 14day 7
%CD38-
0
0.01
0.05
0.1
0
0.1
0.5
4
0
0.1
0.5
4
0
50
day 7 day 14day 7
%C
(normalizedto
0
0.01
0.05
0.1
0
0.1
0.5
4
0
0.1
0.5
4
0
50
100
150
day 7 day 14day 7
%CD38-
Patient 3 Patients 1-3
B. Viability of non-tumor cells
INV
B.  Viability of non-tumor cells was evaluated in
response to bortezomib and INV
compound in r-Bone, showing that neither
compound is toxic against non-tumor cells.

Characteristics
•  80% of CAR-T cells infiltrate r-Bone within 4 days
•  Antigen-specific CAR-T cells activation
•  CAR-T proliferation and activation over a period of
>8 days in culture
•  Suitable for evaluation of CAR-T-mediated tumor
kill and CAR-T cell dynamics
EVALUATION OF IMMUNO-ONCOLOGY COMPOUNDS
Tumor spheroids were established in r-Bone model prior to addition of CAR-T cells. Once added, CAR-T cells were allowed
to infiltrate the r-Bone. CAR-T cell activation and proliferation were measured by flow cytometry and tumor cell kill was
evaluated after Live/Dead staining by microscopy and flow cytometry. Target-specific T cells were activated in the presence
of cancer cells and induced tumor cell death.
Live/Dead
Mock CAR-T
Live/Dead
Target CAR-T
T cell activation
Tumor cell death
Target CAR-T 17% 73%
Control CAR-T 1% 22%
No CAR-T n/a 15%
r-Bone system incorporates tumor and immune components in a single assay, and
thus, is an ideal platform for evaluation of CAR-T and other immuno-oncology agents.

zPREDICTA’S r-BONE SYSTEM DEMOSTRATES HIGH CORRELATION "
WITH CLINICAL RESPONSE: CLINICAL STUDY SET-UP
Cancer
patient
1. Biopsy
2. Clinical treatment
B. Lab treatment
A.  zPREDICTA system
3. Clinical outcome
response
resistance
sensitive
resistant
ESTABLISH CORRELATION
between clinical outcome &
response in r-Bone
A patient’s biopsy is used to determine clinical treatment
and to set-up r-Bone culture. The culture is then treated
the clinical regimen and response to treatment in r-Bone
is compared with the clinical outcome.

r-BONE PLATFORM DEMONSTRATES "
HIGH CORRELATION WITH CLINICAL RESPONSE
Using the r-Bone system 19 out of 21 cases* were predicted correctly, in
contrast with current clinical failure rates of 19 out of 20 new drugs.
*This is an open study currently accruing patients
0.001 0.01 0.1 1 10 100
0
50
100
150
VGPR
PD
PR
µM
%viability(CD38+)
progressive disease (PD)
partial remission (PR)
very good partial remission
(VGPR)
%plasmacell
viabilityinr-Bone
Clinical
response

r-BONE DEMONSTRATES HIGH CORRELATION "
WITH CLINICAL RESPONSE
r-Bone
11/13
8/8 2/13
Clinical outcome
Using the r-Bone system
19 out of 21 cases* were
predicted correctly,
in contrast with current
clinical failure rates of 19
out of 20 new drugs.
*This is an open study currently
accruing patients

MULTI-COMPARTMENT MODELS
Co-culture
epithelial tumor cell line + primary cancer-associated ﬁbroblast (CAFs)
epithelial tumor cell line + primary non-malignant ﬁbroblasts
epithelial tumor cell line + primary mesenchymal stem cells (MSCs)
+/-
primary immune components
(PBMCs, B cells, T cells, macrophages, dendritic cells (DCs), etc.)

SIMULTANEOUS TESTING OF THERAPEUTIC AGENTS "
ON TUMOR AND NON-MALIGNANT POPULATIONS
Epithelium in
organ-specific ECM
Growth
medium
Stromal niche
(1) co-cultures can be set-up with immune cells added to growth medium to mimic their infiltration into the
tissues or (2) added directly to the ECM mimicking resident tissue immune compartment (various
populations can be added, i.e. PBMCs or purified B, T, dendritic (DCs) cells, macrophages, etc.).
(3) co-cultures can be set-up with various stroma elements including cancer-associated fibroblasts
(CAFs), non-malignant primary fibroblasts, mesenchymal stem cells (MSCs), endothelial cells, etc.
Evaluate effects of treatment on
tumor spheroids and/or stroma
Add test agents

r-STOMACH MODEL MIMICS "
THE MULTI-LAYER NATURE OF THE TISSUE
epithelium
lamina propria
stroma
DOI: 10.1158/1541-7786.MCR-12-0307

zPREDICTA CO-CULTURE MODELS SUPPORT "
EPITHELIAL, STROMAL, AND IMMUNE COMPARTMENTS
Epithelium in
organ-speciﬁc ECM
Growth
medium
CAFs in
lamina propria ECM
r-Lamina propria
r-Stomach
PBS
Ab-202
(10nM
)
R
Ab-202
(2nM
)
Ab-003
(10nM
)
R
Ab-003
(2nM
)
Ethanol
ribulin
(1.7nM
)
ibulin
(0.34nM
)
ribulin
(0.2nM
)
0.0
0.5
1.0
1.5
2.0
2.5
αSMAintensitypercell
normalizedtountreatedcontrol
PBS-202
(10nM
)b-202
(2nM
)-003
(10nM
)b-003
(2nM
)
Ethanolulin
(1.7nM
)lin
(0.34nM
)ulin
(0.2nM
)
0.0
0.5
1.0
1.5
2.0
2.5
Vehicle ADC
PBS
Ab-202
(high)R
Ab-202
(low
)Ab-003
(high)R
Ab-003
(low
)
Ethanolribulin
(1.7nM
)ibulin
(0.34nM
)ribulin
(0.2nM
)
0
50
100
150
200
Pericyte panel
%ofvehiclecontrol
Pericytemarkers
Vehicle ADC
PBSR
Ab-202
(high)R
Ab-202
(low
)R
Ab-003
(high)R
Ab-003
(low
)
Ethanolribulin
(1.7nM
)ibulin
(0.34nM
)ribulin
(0.2nM
)
0
50
100
150
200
250
Adipocyte panel
%ofvehiclecontrol
Adipocytemarkers
Vehicle ADC
êactivated stroma

é adypocytic phenotype

zPREDICTA CO-CULTURE MODELS SUPPORT"
EVALUATION OF STROMAL REMODELING
(0.2nM
)
αSMA (co-culture)
PBS
R
Ab-202
(10nM
)
O
R
Ab-202
(2nM
)
R
Ab-003
(10nM
)
O
R
Ab-003
(2nM
)
Ethanol
Eribulin
(1.7nM
)
Eribulin
(0.2nM
)
0
50
100
150
Intensitynormalizedtocellnumber
(%ofvehiclecontrol)
PBS
M
O
R
Ab-202
(10nM
)
M
O
R
Ab-202
(2nM
)
M
O
R
Ab-003
(10nM
)
M
O
R
Ab-003
(2nM
)
Ethanol
Eribulin
(1.7nM
)
Eribulin
(0.34nM
)
Eribulin
(0.2nM
)
0.0
0.5
1.0
1.5
2.0
2.5
ol
ribulin
(1.7nM
)
Eribulin
(0.34nM
)
Eribulin
(0.2nM
)
Vehicle ADC
BREAST CO-CULTURE MODEL (r-Breast): êactivated stroma / é endothelial phenotype
PBS
M
O
R
Ab-202
(10nM
)
M
O
R
Ab-202
(2nM
)
M
O
R
Ab-003
(10nM
)
M
O
R
Ab-003
(2nM
)
EthanolEribulin
(1.7nM
)Eribulin
(0.2nM
)
0
100
200
300
400
500
CD31+
(%ofvehiclecontrol)
PBS
M
O
R
Ab-202
(10nM
)
M
O
R
Ab-202
(2nM
)
M
O
R
Ab-003
(10nM
)
M
O
R
Ab-003
(2nM
)
EthanolEribulin
(1.7nM
)Eribulin
(0.2nM
)
0
100
200
300
400
500
CD31+
(%ofvehiclecontrol)
PBS
M
O
R
Ab-202
(10nM
)
M
O
R
Ab-202
(2nM
)
M
O
R
Ab-003
(10
0
100
200
300
400
500
CD105+
(%ofvehiclecontrol)
PBS
M
O
R
Ab-202
(10nM
)
M
O
R
Ab-202
(2nM
)
M
O
R
Ab-003
(10nM
)
M
O
R
Ab-003
(2nM
)
EthanolE
0
100
200
300
400
500
CD31+
(%ofvehiclecontrol)
PBS
M
O
R
Ab-202
(10nM
)
M
O
R
Ab-202
(2nM
)
M
O
R
Ab-003
(10nM
)
M
O
R
Ab-003
(2nM
)
EthanolE
0
50
100
150
200
250
CD13+
(%ofvehiclecontrol)
Endothelialmarkers
Vehicle ADC Vehicle ADC Vehicle ADC
CD31 CD13 CD105
Epithelium
Growth
medium
CAFs in
interstitial ECM

MULTI-COMPARTMENT MODELS
Reconstructed Metastasis (r-Met)
breastprostate lung
bone lung
colon ovarianmelanoma gastric
Primarytumor
Metastaticsite

RECONSTRUCTED METASTASIS (r-MET) PLATFORM"
Modeling Multi-Step Nature of Tumor Progression
Primary tumor
Invasive
Metastatic
Primary site
microenvironment
Metastatic site
microenvironment
Circulatory “liquid”
environment
•  Simultaneous testing of therapeutics on tumor cell sub-populations
•  Population-speciﬁc target discovery

primarytumorinvasive
Failure to colonize Failure to colonize Colonization
r-MET MODEL RECAPITULATES TUMORIGENIC CAPACITY "
OF TUMOR CELLS
cell phenotype (input – breast cancer cell lines)
metastatic
cellphenotype(output)
non-malignant malignant metastatic
r-Bone
r-Breast

r-MET MODELS ACROSS MULTIPLE TUMOR SITES
primarytumorinvasivemetastatic
Colonization Colonization Colonization Colonization Colonization
The system was set-up with the environment of the primary tumor in the upper chamber and the r-Bone in
the bottom chamber. The assembled system represents the metastasis from lung, gastric, colon, ovarian,
and prostate cancer cell lines to the bone.
r-Lung r-Stomach r-Colon r-Ovary r-Prostate
Membrane pores

HIGH THROUGHPUT MODELS:

Reconstructed Lung (r-Lung™)
NSCLC cell lines

HTS WORKFLOW
Readout:
CellTiter-Glo
Treatment w/ small
molecules
Set-up r-Lung
Data
0.001 0.01 0.1 1 10 100
0
50
100
150
200
Erlotinib [µM]
Survival(%)
Manual workflow
Automated workflow
Response to treatment
Consistent with published data,
A549 spheroids demonstrate
erlotinib resistance.
Automated
Manual
Cell number plated
Luminescence(RLU)
Dynamic range
r-Lung demonstrates high dynamic
range (20 – 80,000 cells per well)
of CellTiter Glo readout.
Liquid handler
Liquid handler
Digital drug dispenser
Plate reader
Automatedset-up
Manualset-up
A549
Spheroid formation
NSCLC cell line, A549,
forms tumor spheroids
in r-Lung.

CUSTOM
MODELS
Blood cancers
Non-small cell lung cancer
Breast cancer
Triple negative breast cancer
Melanoma
Glioblastoma
Lymphoma
Spleen
Bladder
Kidney
Adrenal
Thyroid
Gastric
Colon
Testicular
Ovarian
Endometrial
Cervical
Prostate
Pancreatic
Liver

ADVANTAGES:
–  Speciﬁc models for the indication of your choosing
–  Development priority
–  Dedicated scientiﬁc and development team
–  Licensing/technology access
–  Discounts on future kit orders
CUSTOM MODELS

zPREDICTA MODELS SUPPORT WIDE RANGE OF TISSUES
Model Tissue
Cell lines
Multi-compartment
co-culture*
Primary
cells
HTS**
Custom
r-Brain in progress
Custom models can
be set-up for any
tissue of interest
utilizing the readout of
choice
r-Breast ✔ ✔ in progress
r-Bone (bone marrow) ✔ ✔ ✔#
in progress
r-Colon ✔ ✔
r-Lung ✔ ✔ in progress ✔
r-Ovary ✔ ✔
r-Prostate ✔ ✔
r-Skin ✔ ✔
r-Stomach ✔ ✔
Recommended
readout
CellTiter-Glo,
ELISA, imaging
FACs, ELISA,
imaging
FACs, ELISA,
imaging
CellTiter-Glo
*Tumor cell lines co-cultured with primary cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), or non-malignant
fibroblasts with or without immune components.
**High throughput (HTS) platform for evaluation of small molecule efficacy using tumor cell lines
#r-Bone system has been validated for 3D culture of primary cells from multiple myeloma and AML

Keith Williamson, MBA
Director of Business Development
keith@zpredicta.com
(408) 647-5523

Julia Kirshner, PhD
CEO
julia@zpredicta.com
(650) 739-5472
CONTACT
5941 Optical Court San Jose, CA, 95138
www.zpredicta.com

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