www.promega.com

- 5 - 4 - 3 - 2 - 1 0 1

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

L o g [ a b a t a c e p t ] u M

Lu

min

es

ce

nc

e R

LU

IC50 =3x10-9 M

Hitting the Gas: Quantitative Cell-based Bioassays to Advance Immunotherapy

Programs Targeting Co-stimulatory Immune Checkpoint ReceptorsJun Wang, Michael Beck, Jamison Grailer, Jim Hartnett, Julia Gilden, Frank Fan, Mei Cong and Zhi-jie Jey Cheng

Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711

1. Introduction 4. FcRIIb-dependent Agonist Activity

2. Co-stimulatory Bioassay Design

3. Ligand-induced Agonist Activity

6. ICOS Bioassays for Agonist & Blocking Abs

8. Conclusions

A major challenge in the development of antibody-based biologics drugs isaccess to quantitative and reproducible functional bioassays. Existing methodsrely on primary cells and measurement of complex functional endpoints thatare cumbersome, variable, and often fail to yield data quality required for drugdevelopment in a quality-controlled environment. We have developed aportfolio of functional cell-based reporter bioassays to measure the activity ofbiologics drugs designed to target immune checkpoint receptors including co-inhibitory (e.g. PD-1, CTLA-4, LAG-3, TIM-3) and co-stimulatory (e.g. 4-1BB,GITR, OX40, CD40, ICOS, CD28) receptors. These bioassays consist of stable celllines that express luciferase under the precise control of receptor-mediatedintracellular signals. Here we describe the application of MOA-based immunecheckpoint co-stimulatory receptor bioassays for biologics drug discovery,development, potency and stability studies.

Cell-based reporter bioassays overcome the limitations of primary cell-based assays for functional characterization of antibody and other biologics drugs targeting individual or combination immune checkpoint receptors. Here we show a portfolio of MOA-based bioassays for co-stimulatory immune checkpoint receptors that can be used for antibody screening, characterization, potency and stability studies. These bioassays provide the following:

Biologically relevant measurement of antibody MOA

• Specific immune checkpoint regulated expression of luciferase that reflects the native biology of immune cells.

• Demonstrated critical role of FcRIIb in modulating antibody activity, consistent with published data using primary cells.

Consistent and reliable, easy to implement

• All assays can be used as “Thaw-and-use” cell format, no cell culture required

• Rapid and convenient workflow

• Amenable to standard 96-well and 384-well plate formats

co-stimulatory receptors

Co-stimulatory Bioassays without FcRIIb Crosslinking

Co-stimulatory receptor:

4-1BB (CD137)

GITR

OX40

CD40

CD28

ICOS

HVEM/LIGHT

CD27 (data not shown)

DR3 (data not shown)

Co-stimulatory Bioassays with FcRIIb Crosslinking

Increasing evidence suggests that crosslinking anti-TNFR agonist Abs in vivo via engagement of FcR, particularly FcRIIb, enhances receptor clustering and downstream signaling.

Therefore, functional analysis of Ab biologics in the context of FcR crosslinking is critical in drug development.

- White, et al. (2011) J Immunol, 187:1754-63- Wilson, et al. (2011) Cancer Cell, 19:101-13- Li and Ravetch (2011 Science, 1030-34

rh4-1BB Ligand (His tag) – R&D Systems (#2295-4L-025)anti-His tag Ab – BioLegend (#652502)

Antibody-based Biologic Drug

Bio-Glo™Reagent

GloMaxDiscover

General Protocol

Effector CellsCognate Cells

(where relevant)

+

Measure Luminescence

rhGITR Ligand (HA tag) – R&D Systems (#6987-GL-025)anti-HA tag Ab – R&D Systems (#MAB060)

4-1BB Bioassay GITR Bioassay

anti-hCD40L Ab – BioLegend (#310828)

OX40 Bioassay

rhOX40 Ligand – BioLegend (#555706)

4-1BB Bioassay(Biolegend #309811)

4-1BB Effector Cells + CHO-K1 Cells

4-1BB Effector Cells + FcRIIb CHO-K1 Cells

Log[anti-4-1BB Ab], g/ml

GITR Bioassay(R&D Systems #MAB689)

GITR Effector Cells + CHO-K1 Cells

GITR Effector Cells + FcRIIb CHO-K1 Cells

Log[anti-GITR Ab], g/ml

OX40 Bioassay(Biolegend #350002)

OX40 Effector Cells + CHO-K1 Cells

OX40 Effector Cells + FcRIIb CHO-K1 Cells

Log[anti-OX40 Ab], g/ml

CD40 Bioassay(R&D Systems #AF632)

*CD40 Effector Cells Only

*The anti-CD40 agonist Ab shown here has

agonist activity without FcRIIb crosslinking

Log[anti-CD40 Ab], g/ml

Anti-CD40 Agonist Ab #1

FcRIIb-independent

Anti-CD40 Agonist Ab #2

FcRIIb-dependent

Anti-CD40 Agonist Ab #3

Super-agonism

Panel of anti-CD40 Agonist Antibodies

Co-stimulatory Bioassay Specificity

Anti-GITR Agonist Antibody

Anti-4-1BB Agonist Antibody

Anti-OX40 Agonist Antibody

4-1BB Bioassay GITR Bioassay OX40 Bioassay

Log[antibody], g/ml Log[antibody], g/ml Log[antibody], g/ml

5. CD28 Bioassays for Agonist & Blocking Abs

EC50 = 1.1 x10-7 g/ml

Fold Induction = 121.9EC50 = 3.8 x10-8 g/ml

Fold Induction = 10.5

EC50 = 9.8 x10-7 g/ml

Fold Induction = 15.5

EC50 = 4 x10-7 g/ml

Fold Induction = 9

CD40 Bioassay

HVEM Effector Cells can be activated by:

• Agonist Ab

• Soluble LIGHT Ligand

• Membrane-bound LIGHT Ligand

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

0

51 0 0 5

11 0 0 6

21 0 0 6

21 0 0 6

31 0 0 6

lo g [L IG H T ], g /m l

Bio

lum

ine

sc

en

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(R

LU

)

EC50 =3x10-8 g/ml

Fold Induction = 13 fold

Log[LIGHT ligand], g/ml

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

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11 0 0 5

21 0 0 5

31 0 0 5

41 0 0 5

lo g [a n ti H V E M A b ], g /m l

Bio

lum

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(R

LU

)

Log[anti-HVEM Ab], g/ml

IC50 =3.7x10-8 g/ml

Activation with LIGHT LigandInhibition of LIGHT signaling

with anti-HVEM blocking Ab

7. HVEM/LIGHT Bioassay

- 3 - 2 - 1 0

0

4 . 0 1 04

8 . 0 1 04

1 . 2 1 05

1 . 6 1 05

L o g 1 0 [ a n t i - C D 2 8 a g o n i s t A b ] , g / m l

Bio

lum

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sc

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(R

LU

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EC50 =1.3x10-8 g/ml

Fold Induction = 8 fold

CD28 Agonist Bioassay CD28 Blocking Bioassay

Bio

lum

inesce

nce (

RL

U)

Log10[anti-CD28 agonist Ab], g/ml Log10[abatacept], M

ICOS Blocking Bioassay

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

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5 . 0 1 05

1 . 0 1 06

1 . 5 1 06

l o g [ a n t i - I C O S a g o n i s t A b ] , g / m L

Bio

lu

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(N

an

o-L

uc

R

LU

)

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1 1 07

2 1 07

3 1 07

4 1 07

L o g [ a n t i - I C O S b l o c k i n g A b ] , g / m L

Bio

lu

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(N

an

o-lu

c R

LU

)

a A P C / C H O ( I C O S L n e g a t i v e )

I C O S L a A P C / C H O

EC50 =2.6x10-7 g/ml

Fold Induction = 5.3 fold

ICOS Agonist Bioassay

ICOS Agonist Bioassay ICOS Blocking Bioassay

CD28 Agonist Bioassay CD28 Blocking Bioassay

April 2018 Corresponding author: Jey.Cheng@promega.comFor Research Use Only. Not for Use in Diagnostic Procedures.