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Novel, Improved Cell-Based Assays to Enable Immunotherapy Drug Development for Checkpoint Receptors

Abstract

Regulation of immune responses is tightly controlled through a balance of co-stimulatory and inhibitory checkpoint receptors, often exploited by many cancers. Therefore, therapeutics that block inhibitory receptors or activate immunostimulatory checkpoint receptors have proved to be powerful agents to restore anti-tumor immune responses. However, developing drugs targeting these checkpoint proteins has proved to be quite challenging as cell-based assays used to screen for functional drugs are often difficult to create, involve the use of human primary cells, and have long, complicated protocols. Here, we present data for several new PathHunter® Checkpoint assays that target clinically relevant co-inhibitory and co-stimulatory checkpoint receptors using the industry-validated enzyme fragment complementation (EFC) technology. The PathHunter assays measure receptor activation and signaling for co-stimulatory receptors and co-inhibitory receptors. These assays enable the development of relevant therapeutics, enabling rapid and sensitive screening of biologics and small molecules. Further, the robustness and repro-ducibility of these assays lend themselves well to use in lead optimization, relative potency and QC lot release testing of immunotherapy drugs. These mechanism of action-based, biologically relevant, cell-based assays do not require human primary cells and have an easy-to-use protocol, providing a highly sensitive response in less than 5 hours. Here, we present data for assays targeting a number of clinically important immuno-therapy targets, including PD-1 (with PD-L1 and PD-L2), CTLA4, OX-40, 4-1BB and CD40.

Targeting Co-Stimulatory and Co-Inhibitory Checkpoint Receptors

Activatingreceptors

CD28

OX40

GITR

CD137

CD27

HVEM

PD-1

LAG-3

Blockingantibodies

Agonisticantibodies

T-cellstimulation

VISTA

BTLA

TIM-3

CTLA-4

Inhibitoryreceptors

T-cell

Image adapted from Nature 480: 480-489.

SHP Recruitment Assay for PD-1 Checkpoint Target: Assay Concept

Infectedcells/tumor

Effector T cell (CD8+)

Attenuation ofTCR signal

Reducedproductionof autocrineparacrine cytokines

PD-L1/PD-L2 PD-1

TCRMHC

SHP1/SHP2

SHP

EA

PD-L1or

PD-L2

PD-1

Jurkat Cells

U-2 OS Cells

Substrate

Light

A B

A. Many inhibitory checkpoint receptors (e.g. PD-1, TIGIT) harbor immunoreceptor tail tyrosine (ITT)-like and ITIM motifs in their cytoplasmic tails (motifs that recruit SH2 domain proteins to phosphorylated tyrosines). These SH2 domain proteins, such as the SHP phosphatases are critical in mediating the downstream pathway responses for the receptor. B. Full-length PD-1 receptor was engineered with a small β-gal fragment (PK in red) fused to its C-terminus, and the SH2-domain of SHP-1 was engineered with the complementing β-gal fragment (EA). These constructs were stably expressed in Jurkat cells, while untagged full length PD-L1 or PD-L2 were stably expressed in U-2 OS cells (ligand-presenting cells). Ligand engagement, through co-culture with ligand-presenting cells, results in phosphorylation of PD-1-PK fusion protein, leading to the recruitment of SHP-1-EA which forces complementation of the EFC components to create an active β-gal enzyme. This active enzyme hydrolyzes substrate to create chemiluminscence as a measure of receptor activity. Figure A derived from Okazaki et al., 2013. Nature Immunology 14, 1212-1218.

Assay Outline

Plate Jurkat PD-1 cells & add anti-PD-1 antibody

Add Detection Reagents Read on benchtop Luminometer

Add U-2 OS PD-L1 or PD-L2 presenting cells

Incubate for 1 hr Incubate for 1-2 hrs Incubate for 1 hr

PD-1 Assay Is Suitable for Detection of Biologic and Small Molecule Inhibitors

10-10 10-9 10-8 10-7 10-6 10-5 10-4

0

20000

40000

60000

80000

PD-1 Signaling Cell Line

Keytruda / Opdivo [g/mL]

RLU

Keytruda

Opdivo

OpdivoKeytrudaIC50 6.63 ng/mLS/B ratio 15.9

9.40 ng/mL15.4

α-PD1 AntibodyPD1/PD-L1 inhibitorDasastinibLapatinib

-1.515-1.601-0.9441

23.1 ng/mL575 nM2.79 nM

HillSlope EC50

A B

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -30

50000

100000

150000

200000

Jurkat PD1 Signaling Cell Line Co-culture with PD-L1

Inhibitor [M] or [g/mL]

RL

U PD-1/PD-L1 inhibitor

A. Jurkat PD-1 cells were treated with serial dilutions of Keytruda (blue) or Opdivo (light blue) for 1hr prior to stimulation with U-2 OS PD-L2 cells for 2hr at RT. Expected rank order of the two therapeutic antibodies is observed in the assay with low ng/ml sensitivity for the two marketed drugs. Keytruda® and Opdivo® are registered trademarks of Merck and BMS, respectively. B. Testing of the PathHunter PD-1 assay with small molecule inhibitors that are known to inhibit several known Src family kinases has shown inhibition of SHP-1 recruit-ment to PD-1. Additionally, a low molecular weight inhibitor of the PD-1/PD-L1 interaction (PD-1/PD-L1 inhibitor) also inhibits the assay, although not as potently as the anti-PD-1 antibody. These data demonstrate that the assay can be used to identify novel small molecule inhibitors of PD-1, including kinase inhibitors and disruptors of PD-1 interaction with its ligands, PD-L1 and PD-L2.

PathHunter PD-1 Assay Has 15X Greater Sensitivity than Reporter Gene Assay

PathHunter Assay Competitor Assay8.0

6.0

4.0

2.0

0.0-9 -8 -7 -6 -5 -4

Log [PD-1 Ab], g/ml

IC50=927 ng/mLIC50=54 ng/mL

Total Assay Time <5 hrs >22 hrs

-10 -9 -8 -7 -6 -5 -40

3

6

9

12

Log [PD-1 Ab], g/ml

Fold

Inhi

bitio

n

Fold

IInd

uctio

n

Comparison of PathHunter Jurkat PD-1 signaling bioassay to commercially available PD-1 reporter gene bioassay. Using the same commercially available anti-PD-1 antibody (Bi-oLegend Catalog Number 329912), we were able to compare the assay performance of our PathHunter PD-1 signaling assay to a commercially available reporter gene assay, and observed that the PathHunter PD-1 assay has 17-fold better sensitivity than the reporter gene assay, with a slightly better assay window and a much quicker response.

PathHunter Jurkat CTLA4 Signaling Assay Is Robust and Sensitive

CD86 response in CTLA4 cells Anti-CTLA4 inhibits CTLA4 Signaling

PathHunter CTLA4 Signaling Assay 140 ng/ml 7.26,641 ng/ml 4.5Competitor Assay

Assay IC50 of Ipilimumab S/B

10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1

0

100000

200000

300000

400000

Log Ratio U2OS CD86:Jurkat CTLA4 cells

10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -40

50000

100000

150000

Ipilimumab [g/mL]

A B

RLU

RLU

CTLA4 is an inhibitory checkpoint receptor that is localized in the endoplasmic reticulum. Activation of the TCR is required to transiently mobilize the receptor to the cell surface therefore the generation of a stable assay has traditionally been difficult. The PathHunter CTLA4 signaling assay bypasses this problem with Jurkat cells that are engineered to stably express a chimeric CTLA4-PD-1 fusion protein, with the extracellular domain of CTLA4 fused in frame to the cytoplasmic tail of PD-1. Activation of CTLA4 leads to the recruitment of SHP-1 to the chimeric receptor and causing the formation of an active β-gal enzyme, resulting in chemiluminescence similar to the PathHunter PD-1 Signaling assay. A. Increasing the amount of CD86 expressing cells with a constant number CTLA4 expressing cells leads to an increase in chemiluminescence. B. Adding an anti-CTLA4 antibody such as Ipilim-umab into the co-culture assay inhibits the activation of the CTLA4 receptor and results in a decrease in chemiluminescence.

Non-Canonical NF-kB Pathway Assay for Co-Stimulatory Receptors

No activation Pathway activation

TRAF3 TRAF2

cIAP1/2NIK NIK

NIK NIK

NIK degradation

TRAF3degradation

NIK de novosynthesis

Low NIKprotein levels

IKKα

IKKα

K48 Ub K63 Ub

K48 Ubp100processing

RelB

RelB

Cytoplasm

Nucleus

Cytoplasm

Nucleus

p100

p100

RelB p52

RelB p52

Activation leads to protein stabilization & increase in NIKprotein levels

NIK de novosynthesis

TRAF3 TRAF2

cIAP1/2

PP

P

P

A number of co-stimulatory receptors have been reported to signal through both canonical and non-canonical NF-kB pathways, including BAFF, 4-1BB, OX40 and GITR. NIK, the NFkB-inducing kinase, is a key regulator of the non-canonical NF-kB pathway. NIK protein levels are normally tightly controlled at a low level during steady state, but pathway ac-tivation leads to an increase in NIK protein levels, enabling us to interrogate NIK protein levels as measure of non-canonical NFkB pathway activation. In this assay, the NIK protein is tagged with the enhanced ProLabel (ePL) β-gal reporter fragment and stably expressed in U-2 OS cells. Stimulation of the cells with agonists for certain co-stimulatory receptors leads to stabilization of NIK, which is easily measured by the addition of detection reagent containing the EA fragment and susbtrate.

Measuring Activation of CD40 Receptor by Agonistic Antibodies and CD40 Ligand

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4

0

250000

500000

Oligomerized CD40L

Soluble CD40L

Agonist Anti-CD40 Antibody

CD40 Response with PathHunter NIK Signaling Assay

rhCD40L / anti-CD40 antibody [g/mL]

RLU

CD40L 87.32 ng/mL7.90 ng/mLCD40L+anti-His Ab

anti-CD40 antibody 311.4 ng/mL

EC50

Soluble CD40L

CD40L

CD40

LZ

Suboptimalactivation/maturation

Increased activity FcR-dependentactivity

Enforcedtrimerisation

Agonistic anti-CD40antibodies

For CD40L, as for many ligands in the TNF family, trimer stability and oligomerization status are crucial determinants of receptor activation. Indeed, activation of NF-kB signaling through CD40 is sub-optimal with soluble ligand, requiring either enforced trimerization (e.g. oligomerization with an antibody to a tag on the ligand) or with agonistic antibodies (which in turn require binding of FcgR). The data on the right shows the effects of soluble vs oligomerized CD40 ligand on NIK accumulation in U-2 OS cells. A commercial agonistic antibody was also tested, but was not as potent as oligomerized ligand. (Figure on the left adapted from Bremer, E., 2013. ISRN Oncology Vol. 2013, Article ID 371854)

Robust Assays for OX40, CD137 (4-1BB) and HVEM Co-Stimulatory Receptors Through NIK Signaling Response

OX40 Signaling Assay CD137 Signaling Assay HVEM Signaling AssayA CB

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5

0

50000

100000

150000

OX40L [g/mL]

RLU

RLU

+OX40

no OX40

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5

0

100000

200000

300000

LIGHT [g/mL]

EC50 27.43 ng/mLS/B 4.0 EC50 3.04 ng/mL

S/B 3.2

LIGHT

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -50

400000

800000

1200000

1600000

4-1BB Ligand / Urelumab (g/mL)

RLU

soluble ligand

Urelumab

S/B

Soluble 4-1BB Ligand

Urelumab

EC50

36.9 ng/mL 3.86.3140 ng/mL

OX40, CD137 (4-1BB) and HVEM are co-stimulatory receptors that are current targets for therapeutic development. We expressed OX40 and CD137 in the U-2 OS NIK Signaling Cell Line to generate the OX40 A. and CD137 B. Signaling assays, respectively. OX40 and CD137 assays produce a sensitive response to soluble ligand and agonistic antibodies in less than 6 hrs. C. HVEM is a TNFR family member that elicits either a co-stimulatory or inhibitory signal depending on the ligand that is activating the receptor. LIGHT delivers a co-stimulatory signal to HVEM cells, resulting in stabilization of NIK, as can be observed in the assay.

Summary

Benefits of PathHunter Checkpoint Signaling Assays

� No primary cells – Get biologically relevant responses without primary cells

� Easy-to-use protocol with fast results – Increase efficiency with an add and read protocol and results in 5-8 hours

� Highly Sensitive Response – 15X more sensitive than other assays

� Multiple Applications – Drive development of biologic and small molecule drugs

� Support broader drug program - Cell-based assay for functional screening, lead optimization and bioanalytical QC lot release applications

Abhi Saharia, Jennifer Lin-Jones, Hanako Daino-Laizure, Mimi Nguyen, Venkatesh Chari, and Jane E. Lamerdin

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