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Novel, Improved Cell-Based Assays to Enable Immunotherapy Drug

Development for Checkpoint Receptors

Jane E. Lamerdin, PhD

Director of R&D, DiscoverX

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Target & Phenotypic Platforms

Enabling Cancer Immunotherapy Drugs – From Screening to Clinics

Target-Based Platform Phenotypic Platform

PathHunter Cellular Assays BioMAP Human Primary Cell Assays

APPLICATIONS

• Screening & lead optimization

• Bioassays for potency & stability testing

• NAb assays for immunogenicity testing

APPLICATIONS

• Efficacy & biomarker selection

• Pre-clinical safety studies

• Combination studies

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Target & Phenotypic Platforms

Enabling Cancer Immunotherapy Drugs – From Screening to Clinics

Target ID and Validation

Screening & Hit

Identification

Lead Optimization & Selection

Efficacy and Biomarker Selection

Safety & Pre-clinical Studies

Clinical Combination

Studies

PathHunter® Checkpoint Assays PathHunter® Bioassays for QC Lot Release Testing

BioMAP® Oncology Systems

BioMAP® Diversity PLUS™

BioMAP® Combo ELECT

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• Harnesses the immune system to battle tumors

• Selectively activate or inhibit T cells

• Based on clinical success of molecules targeting two inhibitory receptors, CTLA4 and PD-1

• Combination therapy, personalized medicine

• Customize treatment depending on patient’s tumor type

Cancer Immunotherapy

“Scientific Breakthrough of the Year” – 2013, Science Magazine

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Targeting T Cell Activation and Inhibitory Checkpoints

Tools Are Needed to Screen for and Develop New Therapeutics

Figure from: Nature 480 480–489 (22 December 2011) doi:10.1038/nature10673

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Challenges with Checkpoint Receptors

• Difficult to create cell-based assays for checkpoint receptors

• Often needs human blood tissue

• Difficult to handle human samples

• Donor variability

• Long, complicated protocols

• Assay not specific for target receptor

Pembrolizumab

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PathHunter® Assay Technology

Enzyme Fragment Complementation (EFC)

Split β-galactosidase Enzyme

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ProLink™ (PK) Enzyme Acceptor (EA)

Inactive Fragments Active Enzyme

~40 aa peptide Large fragment Complemented Enzyme

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• PD-1 contains inhibitory motifs

• Phosphorylation by Src kinases

• SHP proteins recruited to phosphorylated motifs

• SHP-2 attenuates TCR activation

PD-1 Signaling BiologyAssay based on native receptor biology

PathHunter PD-1 assay quantifies this early activation event

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PathHunter® PD-1 Signaling Assay

Target early step in PD-1 receptor activation

Plate Jurkat PD-1 cells;

add anti-PD-1 antibodyAdd U2OS PD-L1 or

PD-L2-presenting cells

Incubate 1hr

Add Detection Reagents

Incubate 1hr

Read on Benchtop

Luminometer

Incubate 1-2hr

Simple Add and Read Protocol

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PathHunter® PD-1 Signaling Assay

Responds to Cell-presented Ligand (Co-culture with PD-L1 or PD-L2)

Co-culture with PD-L2

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PathHunter® PD-1 Signaling Assay

Rapid and Robust Response to Anti-ligand or Anti-PD-1 Antibodies

Robust inhibition in < 2hr

Inhibition with anti-PD-1 or

anti-PD-L1 antibodies

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PathHunter® PD-1 Signaling Assay PerformanceHighly Specific and Reproducible Response

RSD <4%

Highly Specific Response Excellent Reproducibility

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PathHunter® Assay Applications

Supports development of biologics and small molecules

SMALL MOLECULES• Screening & lead optimization

BIOLOGICS• Screening & lead optimization• Characterization Assays• Development of QC Lot Release Assays

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PathHunter® vs. Reporter Gene Assay

15X More Sensitive and 4X Faster

PathHunter Assay Competitor Assay

Data generated with same commercial anti-PD-1 antibody (BioLegend Cat # 329709)

Total Assay Time <5 hrs >22 hrs

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PathHunter® Checkpoint Assay Advantages

• Biologically relevant response

• Without handling difficult and donor-variable human tissue

• Easy Protocol With Fast Results

• Simple add and read protocol and results in less than 5 hours

• Multiple Applications

• Supports development of biologics and small molecules

• Highly Sensitive Response

• 15X more sensitive than other assays

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Targeting T-cell Activation and Inhibitory Checkpoints

Modulate immune response to destroy cancer cells

Activating

Receptors =

TNFR superfamily

membersSignal through

canonical and non-

canonical NF-kB

pathways

Nature 480 480–489 (22 December 2011) doi:10.1038/nature10673

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• A number of co-stimulatory receptors have been reported to signal through both canonical and non-canonical NF-kB pathways:

• BAFF

• 4-1BB

• OX40

• GITR

• Interrogate non-canonical signaling by quantifying NIK stabilization

TNFR Superfamily Receptor SignalingSignaling Through the Non-canonical NF-κB Pathway

Immunol Rev. 2012 Mar; 246(1): 125–140

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NIK is Stabilized in Response to Ligand Engagement of Endogenous TWEAK Receptors in U-2 OS Cells

• TWEAKR (Fn14) is a TNFR

family member that is up-

regulated in response to

tissue damage

• TWEAKR is a therapeutic

target in multiple inflammatory

diseases (e.g. RA, MS,

atherosclerosis) and cancer

(melanoma, glioma, etc.)

• TWEAKR is endogenously

expressed in U-2 OS cells

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NIK is Stabilized in Response to Ligand Engagement of Endogenous HVEM Receptors in U-2 OS Cells

• HVEM is a TNFR family member that elicits either a co-

stimulatory or inhibitory signal depending on the ligand

• HVEM is endogenously expressed in U-2 OS cells

• LIGHT delivers a co-stimulatory signal to HVEM+ cells,

resulting in stabilization of NIK

APC (naiive T cells) T cell

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NIK is Stabilized in Response to Activation of Endogenous CD40 in U-2 OS Cells by Soluble CD40 Ligand

Bremer, E., ISRN Oncology 2013, Article ID 371854

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NIK is Stabilized in Response to Activation of Endogenous CD40 in U-2 OS Cells by Soluble and Oligomerized CD40 Ligand

Soluble CD40L

Oligomerized CD40L

Bremer, E., ISRN Oncology 2013, Article ID 371854

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NIK is Stabilized in Response to Activation of Endogenous CD40 in U-2 OS Cells by CD40 Ligand and Agonistic Antibodies

Soluble CD40L

Oligomerized CD40L

Agonistic CD40 Ab

Bremer, E., ISRN Oncology 2013, Article ID 371854

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NIK is Stabilized in Response to OX40 Ligand Engagement

• Exogenously expressed OX40 in

U-2 OS NIK Signaling Cell Line

• OX40 assay responds to soluble

ligand and agonistic antibodies

• Amenable to testing in 96-well or

384-well format (to conserve

antibodies)

96-well

384-well

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• Simple and biologically relevant assay

• Gain of signal assay

• Robust response over broad incubation time period (4-6hr)

• Compatible with biologics and small molecules

• Applicable to diverse TNFR family members

PathHunter® NIK Assay Benefits

Measures Activation of Endogenous or Exogenously Expressed TNFR Superfamily Receptors with Soluble Ligand and Agonistic Antibodies

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• Blockade of PD-1 and other inhibitory receptors

• CTLA-4, TIM-3, LAG3, TIGIT, CD244, CD160

• PD-1 blockade in combination with immunostimulators

• Anti-OX40, anti-CD137, ICOS, TLR ligands, IL-2

• PD-1 blockade in combination with small molecules or other targeted inhibitors

• e.g. angiogenesis inhibitors, HDAC or PARP inhibitors

• PD-1 blockade in combination with vaccines, CAR-T or oncolytic viruses

Immunotherapy’s Next Wave: Combination Therapy

Monospecific vs Bi-specific antibodies

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Assay Concept for Bi-specific Assays

Bi-specific Antibody

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Bi-specific Assays Developed for Immune Checkpoints

Immune Checkpoint Bi-specific Assay

• PD-1 / TIM3

• TIM3 / CEACAM

• PD-1 / LAG3

• PD-1 / TIGIT

• PD-1 / CTLA4

• PD-1 / 4-1BB

Examples of Available Bi-specific

Pools / Clones :

1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4

0

1 0 0 0 0 0

2 0 0 0 0 0

3 0 0 0 0 0

4 0 0 0 0 0

B is p e c if ic A n tib o d y [g /m L ]

Dim

eriz

ati

on

Sig

na

l (R

LU

)

A n tib o d y 1

A n tib o d y 2

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VISTA Dimerization Assay• VISTA is a negative checkpoint regulator that suppresses T-

cell activation and is highly expressed within the tumor micro-

environment.

• VISTA is expressed primarily on hematopoietic cells

• VISTA blockade may offer an immunotherapeutic strategy for

human cancer, especially in combination

• VISTA is related to PD-L1; currently the receptor for VISTA is

unknown

• Dimer assay provides tool to rank order antibodiesFigure from Cancer Immunol Res (2014); 2(6): 510-517

Highly Specific Response to

anti-VISTA antibodies

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TIM3 Dimerization Assay

• TIM3 is a negative checkpoint regulator expressed on

multiple hematological cells

• Recognizes ligands highly expressed on apoptotic cells,

leading to phagocytosis of dying cells

• Dimer assay provides tool to rank order antibodies

Figure from Science Webinar Series, part 5: Gordon J. Freeman, Ph.D.

APC T cell Highly Specific Response to

anti-TIM3 antibodies

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Assays for CSF1 (M-CSF) and GM-CSF

• Cell line responds robustly to M-CSF (CSF1) and IL-34

• Anti-M-CSF antibodies will lead to inhibition of ligand-induced dimerization

Cytokine immunotherapy with GM-CSF:

induces potent tumor-specific systemic immune

responses

~30 assays available for cytokines and interleukins

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• SH2 recruitment (signaling) assays for inhibitory receptors• Clone available for PD-1

• Assays for other targets coming soon

• Custom projects possible for additional novel checkpoint targets

• Assays for co-stimulatory receptors• Assays for OX40, TWEAKR, HVEM and CD40 are available

• Additional co-stimulatory receptors assays in progress

• Assays for Bi-specific molecules

Summary

Multiple Assay Formats for Immune Checkpoint Receptors

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Thank you for your attention!

Learn more at www.discoverx.com/checkpoint

Contact info@discoverx.com for additional information