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Specifically Measure Target Cancer Cell Death in

Co-CulturesKILR™ Cytotoxicity Assays

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• How to determine target cell death specifically in the presence of multiple cell types in a well?

• How to get sensitive detection of target cell death for ADC’s?

Cancer Immunotherapy Development Challenges

Specificity and Sensitivity

CAR-T Bispecific AbADCsADCPADCC

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• Dye-based assays are radioactive and/or “leaky”

• General cell death measures are not specific for target cells

• Assays are complicated and long

• Low throughput of assays

Challenges with Existing Cytotoxicity Assays

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PathHunter® Assay Technology

Enzyme Fragment Complementation (EFC)

Split β-galactosidase Enzyme

ProLabel (ePL) Enzyme Acceptor (EA)

Inactive Fragments Active Enzyme

~40 aa peptide Large fragment Complemented Enzyme

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• Housekeeping gene engineered with ePL (high affinity for EA)

• KILR construct stably engineered into target cell line

• Detection reagent contains EA

Introducing the KILR™ Cytotoxicity Assay

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• Unparalleled Specificity

• Signal from dead and lysed target cells only

• Not dye-based

• Easy to Use

• Simple add and read protocol with chemiluminescent readout

• Highly Sensitive

• Detect as few as 75 dead cells/well

• Flexibility

• Ability to run assay from 30 minutes to 72 hours

Assay Highlights and Advantages

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KILR™ Cytotoxicity AssaysSimple Add and Read Protocol Comparison to Chromium51 Release Protocol

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KILR™ Cytotoxicity Assays

Unparalleled Sensitivity

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KILR™ Cytotoxicity Assays

Ultimate Flexibility – Run Assay from 30 minutes to 72 hours

• Protein reporter stable for

72 hours in media

• Low background, non-leaky

protein reporter

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KILR™ Cytotoxicity Assay Applications

CAR-T Bi-specific AbADCsADCPADCC

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KILR™ Cytotoxicity Assay Applications

Antibody Dependent Cellular Cytotoxciity (ADCC)

1 0 -1 2 1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5

0

1 0

2 0

3 0

4 0

E rb B 2 O v e r-e x p re s s in g C e ll P o o ls

4 :1 N K : ta rg e t ce lls

T ra s tu z u m a b [g /m L ]%

ly

sis

S /BHillSlope EC50

SKBR3 2.113 2.654e-009

MCF7 1.330 8.779e-009

NCI N87 0.7026 1.110e-008

1 2 .2

6 .1

1 0 .8

1 0 -1 2 1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5

0

3 0 0 0

6 0 0 0

9 0 0 0

U 2 O S -E G F R e P L

A n tib o d y [g /m L ]

RL

U

S /B

8 .6

HillSlope EC50

Cetuximab 1.021 4.696e-009

Rituximab ~ 19.81 ~ 5.119e-011 --

ADCC Assay Verified with Marketed Drugs

ADCC

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• Key Benefits

• True MoA of ADCC molecule

• Accuracy

• Precision

• Reproducibility

• Robust

KILR™ Cytotoxicity Assay ApplicationsADCC for QC Lot Release

IC50 (ng/mL) Relative Potency R2

Ref. Std. 0.485 1 1

100% Ctrl. 0.519 0.961 0.99960% 0.897 0.574 0.99980% 0.581 0.83 0.996

130% 0.421 1.21 0.997160% 0.324 1.64 0.998

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KILR™ Cytotoxicity Assay Applications

Antibody Dependent Cellular Phagocytosis (ADCP)

ADCP with with Primary Macrophages

Plate-based true ADCP assay

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KILR™ Cytotoxicity Assay Applications

Bi-specific AbPlate Target Cells in 384-well plate

Add non-activated primary T cells

Add antibody

Add KILR Detection reagent

Read signal

41 hrs

1h

Bi-specific Antibody for T-cell Redirection

Data provided courtesy of big pharma partner

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• Highly specific measure of target cell death in co-culture

• Easy to use with a simple add and read protocol

• Highly sensitive detection of as few as 75 dead cells per well

• Broad applications for immuno-oncology drug development

Benefits of KILR™ Cytotoxicity Assays

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• View updated assay list and learn more at:

• www.discoverx.com/kilr

Available KILR™ Cytotoxicity Assays

Cell Line or Pool Cell Line Part No.

KILR ARH-77 Cell Line 97-1001C017

KILR SKBR3 Cell Pool 97-1002P018

KILR H322 Cell Pool 97-1003P020

KILR NCI-N87 Cell Pool 97-1004P021

KILR Ramos Cell Pool 97-1006P022

KILR Raji Cell Pool 97-1012P026

KILR A549 Cell Pool 97-1005P015

KILR SKOV3 Cell Pool 97-1007P023

KILR NCI-H292 Cell Pool 97-1008P024

KILR Daudi Cell Pool 97-1009P025