1

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

2

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

5

Cellular Impedance

6

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

7

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

8

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

9

Establishment of Real-Time Potency Assays for CAR-T

10

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

17

Fesnak et al. (2016) Nature Reviews Cancer, 16, 566-580

18

Designing CARs Targeting Glioblastoma

Chimeric Antigen Receptor

T Cell

19

CARpool biCAR TanCAR

J Clin Invest. 2016 Aug 1;126(8):3036-52.

HER2

IL13R2

Designing CARs Targeting Glioblastoma

CAR-NK Cell Mediated Cytolysis

20

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

21

Dargel et al. (2015 ) 149(4):1042-52 Gastroenterology

xCELLigence B Cell Tethering Kit

22

- Tethering Kit - (catalog #: 8100005)

CAR-T-Mediated Cytotoxicity (Liquid Tumors)

23

B cells

CAR-T

Data credit: Biao Xi

anti-CD40

Development of a Real-Time Potency Assay for Assessment of Checkpoint Inhibitors

24

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

25

SEB

PD-1

PD-1 Expression

Anti-PD-1 Modulation of PBMC- mediated PC3 Tumor Cell Killing

26

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

28

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

29

BiTE/Bi-Specific ADCC

9/14/2017 ACEA Biosciences Confidential

Oncolytic Virus

Attributes of xCELLigence RTCA Functional Potency Assay for CAR-T Manufacturing

30

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

31

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

32

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

35