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
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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
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sc
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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
0
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
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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
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)
EC50 =1.3x10-8 g/ml
Fold Induction = 8 fold
CD28 Agonist Bioassay CD28 Blocking Bioassay
Bio
lum
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nce (
RL
U)
Log10[anti-CD28 agonist Ab], g/ml Log10[abatacept], M
ICOS Blocking Bioassay
- 9 - 8 - 7 - 6 - 5 - 4
0
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
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LU
)
- 7 - 6 - 5 - 4 - 3
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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
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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: [email protected] Research Use Only. Not for Use in Diagnostic Procedures.