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Real-Time Monitoring of CAR-T Potency Assays and its Modulation by
Checkpoint Inhibitors
Yama A. Abassi, Ph.D.
Vice President, R&D
ACEA Biosciences, Inc.
Presentation Outline • Potency assays for cellular therapies • The xCELLigence Real-Time Cell Analysis System • The utility of xCELLigence System for assessment of genetically modified T cells and NK cells
– CAR-T – CAR-NK – TCR
• Important quantitative potency parameters that can be derived from real-time data • Assessment of combinations with checkpoint inhibitors • Development of potency assays for other immunotherapy approaches • The xCelligence potency assay as a patient simulator • Summary
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Potency Assay as a Critical Quality Attribute for Cellular Therapy Development and Manufacturing
3
Regulatory Rapporteur – Vol 12, No 5, May 2015
xCELLigence Real-Time Cell Analyzer
4
xCELLigence Real-Time Cell Analyzer
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Cellular Impedance
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Single Well
(side view)
well bottom
(glass or PET)
culture
medium
electric current flow
Key features:
o Simple workflow
o Standard media
o Label-free (physiological conditions)
o Adhesion/growth on electrodes is
normal
o Real-time monitoring
o Orthogonal assays
negative
terminal
positive
terminal o Signal reported as:
Cell Index = (Impedancet=n) (Impedancet=0)
impeded current flow
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Ce
ll In
dex
Time (h)
Proliferation
100% Confluence
Microelectrodes
+ Adherent Tumor Cells
Ce
ll In
dex
Time (h)
No Adhesion Minimal Signal
+ Non-adherent Effector Cells
Immune Cell-Mediated Killing
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Adherent Tumor Cells
Dead Target Cells
+ Non-adherent
Effector Cells
+ Effector Cells
0.1:1
0.4:1
2:1
Ce
ll In
dex
Time (h)
Effector : Target Ratio
0:1 (negative control)
Immune Cell-Mediated Killing
Key benefits:
o No 51Cr
o Time range: minutes to days
o Continuous data vs. end point (nothing is
missed)
Developing the Right CAR for Targeting the Right Tumor
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Establishment of Real-Time Potency Assays for CAR-T
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0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 80 100 120
No
rmal
ize
d C
I
Time (Hours)
CAR-T
100
50
12.5
6.25
3.13
1.56
0
0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 80 100 120
No
rmal
ize
d C
I
Time (Hours)
Mock-T 100
50
12.5
6.25
3.13
1.56
0
Effector
Effector
E:T
X:1
E:T
X:1
Data generated in collaboration With Dr. Prasad Adusumilli at MSK
E:T 1:100
Non-specific cytolysis
Specific cytolysis
Optimization of CAR-T E:T Ratio Using Real-Time
Cell Analysis
E:T 1:50
Non-specific cytolysis
Specific cytolysis
Optimization of CAR-T E:T Ratio Using Real-Time
Cell Analysis
E:T 1:12.5
Non-specific cytolysis
Specific cytolysis
Optimization of CAR-T E:T Ratio Using Real-Time
Cell Analysis
E:T 1:6.25
Non-specific cytolysis
Specific cytolysis
Optimization of CAR-T E:T Ratio Using Real-Time
Cell Analysis
E:T 1: 3.13
Non-specific cytolysis
Specific cytolysis
Optimization of CAR-T E:T Ratio Using Real-Time
Cell Analysis
E:T 1: 1.56
Non-specific cytolysis
Specific cytolysis
Optimization of CAR-T E:T Ratio Using Real-Time
Cell Analysis
CAR-T Variations
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Fesnak et al. (2016) Nature Reviews Cancer, 16, 566-580
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Designing CARs Targeting Glioblastoma
Chimeric Antigen Receptor
T Cell
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CARpool biCAR TanCAR
J Clin Invest. 2016 Aug 1;126(8):3036-52.
HER2
IL13R2
Designing CARs Targeting Glioblastoma
CAR-NK Cell Mediated Cytolysis
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NK
CAR-NK Targeting CD133
Klapdor et al. (2017) Improved killing of cancer stem cells by combining novel
CAR-based immunotherapy and chemotherapy. Human Gene Therapy. DOI 10.1089/hum.2017.168
Gastroenterology. 2015 Oct;149(4):1042-52
T Cells Engineered to Express a TCR Specific for Glypican
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Dargel et al. (2015 ) 149(4):1042-52 Gastroenterology
xCELLigence B Cell Tethering Kit
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- Tethering Kit - (catalog #: 8100005)
CAR-T-Mediated Cytotoxicity (Liquid Tumors)
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B cells
CAR-T
Data credit: Biao Xi
anti-CD40
Development of a Real-Time Potency Assay for Assessment of Checkpoint Inhibitors
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Day 1
Seed target Cells Thaw frozen PBMC and activate separately
Add activated sPBMC in the wells of E-Plate containing the target cells -/+ checkpoint inhibitor
Day 2
Day 2-5 Continuous monitoring on the xCELLigence
sPBMC
Induction of PD-1 Expression in PBMC Population by SEB Treatment
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SEB
PD-1
PD-1 Expression
Anti-PD-1 Modulation of PBMC- mediated PC3 Tumor Cell Killing
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0
0.5
1
1.5
2
2.5
3
0 20 40 60 80 100 120 140 160
No
rmal
ized
Ce
ll In
dex
Time (Hr)
PC3 only
PD-1- 0.0
PD-1-66.7 nM
0
10
20
30
40
50
60
70
80
90
80 100 120 140 160
% C
yto
lysi
s
Time (Hr))
PD-1-66.7 nM
PD-1- 0.0
PBMC only
PC3+PBMC PC3+ sPBMC PC3+ sPBMC+ 67 nM PD1 Ab
PC3+sPBMC+ 67 nM PD1 Ab PC3+sPBMC PC3+ PBMC
xCELLigence Immunotherapy Software
• % Cytolysis, KT50, EC50
• Oncolytic virus, combination therapies, etc.
Assessment of Time-Dependent Cytolysis: KT50 Parameter
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Target alone
E:T
1:1
0.5:1
0.3:1
The % of Cytolysis plot is used to measure the Killing Time at 20%, 40%, 50%, 60% and 80%
Validated RTCA Potency Assays for Various Immunotherapy Approaches
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BiTE/Bi-Specific ADCC
9/14/2017 ACEA Biosciences Confidential
Oncolytic Virus
Attributes of xCELLigence RTCA Functional Potency Assay for CAR-T Manufacturing
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Potency Assay Attribute xCELLigence RTCA System
Representative of the mechanism of action
Accurate
Fast
Sensitive and specific
Ensure batch to batch consistency
Covering all of the product constituents
Quantitative
Predictive of clinical efficacy ?
9/14/2017 ACEA Biosciences Confidential
Examples of How xCELLigence Could be used as a Functional Diagnostic Assay
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Patient B tumor
Patient A tumor
Patient B tumor
Patient A tumor
Patient A TIL
Patient B TIL
Patient with metastatic adenocarcinoma Containing EGFR mutation E476-T751 delinsA, exon 19
Patient with metastatic adenocarcinoma Containing ras mutation
Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine. PLoS ONE 11(8): e0160807. doi:10.1371/journal
9/14/2017 ACEA Biosciences Confidential
Pleural Effusions
Tumor
Development of In Vitro Patient Simulator Assays
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Summary
• The xCELLigence system allows for label-free and real-time monitoring of potency assays
• The system can be used as a potency assay for different immunotherapy approaches including CAR-T, CAR-NK, engineered TCR and checkpoint inhibitors
• While the system is primarily used in R&D stage, we are currently exploring its utility during cellular therapy manufacturing
• The system is also currently utilized to test patient’s tumor cells along with CAR-T generated from the same patient
Acknowledgements
Nabil S. Ahmed MD Associate Professor Pediatrics-Hem-Onc Cell & Gene Baylor College of Medicine Houston, TX, US
Prasad S. Adusumilli, MD, FACS Deputy Chief Thoracic Service Memorial Sloan Kettering
ACEA Biosciences Biao Xi, PhD Fabio Cerignoli, PhD Lincoln Muir
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