challenges and strategies in designing moa reflective ... · average rp (%) cv (%) sample 1 99 97...
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Challenges and Strategies in Designing
MOA Reflective Bioassays for a Bispecific mAb
Xianzhi Zhou, Ph.D
Analytical Biotechnology Development
Bioassays 2014: Scientific Approaches & Regulatory Strategies
March 24 - 25, 2014
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Introduction
The emerging of multispecific mAbs
– most of the marketed antibodies are monospecific
– complex diseases involve synergistic action of disease-mediating factors
– blockade of different pathological factors for improved therapeutic efficacy
A bi-functional mAb
– anti- ligand1 backbone
– anti-ligand2 scFv attached at the Fc portion
Bioassay design and considerations for each function
2
(G4S)4 VL VH C N
VL VL
VH VH
S-S
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Bioassay Design for Bispecific mAb
Role that Mechanism of Action (MoA) plays in
choosing bioassay format for each function
Strategies for use of cell-based assays and
binding assays at various stages of product
development
– Dual binding ELISA
– For Ligand1 arm:
cell-based assay available
– For Ligand2 arm:
use binding assay at early development stage
develop cell-based assay for later stage
Ligand1
ELISA Plate
His-tag
Ligand2
anti-His-HRP
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Ligand1 Reporter Gene Bioassay
NFκB
Luciferase Reporter Gene
Luminescence
NF κB
IκBα
NF κB
α β
IKK
DD DD
R
T
N
FR
T
N
F
TR
AD
DRIP
TRAF2
MEDI3549
TNF-α
TNF-α
IKK pathway
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Ligand1 Reporter Gene Bioassay Development
Ligand Titration
1 100 10000 1000000
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
TNF-a (pg/mL)R
ela
tive
Lu
min
es
ce
nce
Un
its
Cell Density
5 15 25 37.5 50 75
0
1000
2000
3000
4000
5000
6000
Cells/well (x1000)
S:N
EC75
[Ligand] (ng/mL) Cells/well
Sig
nal
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1 10 100 1000
0
2000000
4000000
6000000
8000000
2 hour
2.5 hour
3 hour
3.5 hour
4 hour
4.5 hour
[MEDI3549] ng/mL
Re
lati
ve
Lu
min
es
ce
nce
Un
its
(R
LU
)
Reporter Gene Bioassay Time Courses
6
Incubation of Cells with mAb/ligand1
mAb Pre-Incubation With Ligand1
0.1 1 10 100 1000 10000
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
0 Minutes
15 Minutes
30 Minutes
45 Minutes
60 Minutes
90 Minutes
MEDI3549 (ng/mL)
Re
lati
ve
Lu
min
es
ce
nce
Un
its
(R
LU
)
[mAb] (ng/mL)
[mAb] (ng/mL)
Sig
nal
Sig
nal
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No Plate Positional Effect
7
row A/H
row B/G
row C/F
row D/E
[mAb] (ng/mL)
RL
U
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Pre-incubate
Drug/Ligand
60±15 min @RT
Add 50 µL mixture
to 50 µL cells
4-4.5 hr @37ºC
Add 100 µL
SteadyGlo
Measure
Luminescence
30-60 min @RT
Specificity, Linearity, Stability Indication
Lu
min
es
ce
nce
L
um
ine
sc
en
ce
[mAb]
Accuracy
103%
Intermediate
Precision
4.8%
0 8 12
Rela
tive
Po
ten
cy
Time
5C
40C
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Bioassay Design for Ligand2 Arm
Ligand2 plays complex role
Unclear signaling pathway
– Ligand2 can act as an agonist or antagonist depending on
cell type and experimental context
– The exact molecular mechanism underlying Ligand2
actions are not known
Stage-based bioassay approach
– Binding assay for early-stage development
– Cell-based assay for late-stage development
9
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Competitive Binding Assay Using SPR
10
Sensor Surface
Receptor
Ligand2 mAb mAb
Ligand2
Sensor Surface
Receptor
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11
Accuracy Expected Potency Level
50% 100% 150% AC
Assay 1 96 101 101 99%
Assay 2 98 100 103 99%
Assay 3 94 100 105 99%
Average 96 100 103 99
CV (%) 2.1 0.6 1.6 0.0
Repeatability
Sample ID Assay 1 Assay 2 Assay 3 Average RP
(%)
CV
(%)
Sample 1 99 97 98 98 1.0
Sample 2 101 100 100 100 0.6
Sample 3 100 102 99 100 1.5
Average RP
(%) 100 100 99
Overall
Accuracy 100%
CV (%) 1.0 2.5 1.0 Overall CV 1.3%
SPR Binding Assay Performance
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Principle of Receptor Phosphorylation Assay
Ligand2 SC region mediate
homo-oligomerization to
tetrameric and higher-order
oligomers
Binding of the multimeric
ligand2 clusters the receptor
Ligand2 binding induces
phosphorylation of the receptor
ELISA to measure
pReceptor
Barton et al., (2006) Nature Structural Biology Vol. 13:524
Ligand2
Receptor
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13
Day1
Day2
Y R P
Capture Ab: a-R
Detection Ab: a- pR
Seed cells overnight
Treat cells with drug/Ligand2 Mixture
Lyse cells /Transfer lysate to ELISA Plate
Measure pReceptor by ELISA
Phosphorylation Assay Procedure
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Receptor Phosphorylation Upon Stimulation
CCM vs. Serum Free assay media
Reaches maximum at 10 minutes
14
30 min
20 min
15 min
10 min
5 min
0 min
CCM Serum Free Media
[Ligand2] [Ligand2]
Sig
nal
Sig
nal
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Receptor Phosphorylation Assay Optimization
Cells
– Cell Line
– Growth Media optimization
– Assay media: CCM vs. serum free
– Cell number titration
– Passage comparison
Ligand2/mAb
– Pre-incubation time course
– Stimulation Time course
– Ligand2 titration
– Ligand2 lot comparison
– Tetramer vs. rhLigand2
Lysis
– Aspiration vs. direct addition
– Base buffers
– Benzonase concentration
– Lysis Time course
15
Capture Ab
– Coating concentration
– Coating condition
Detection Ab
– Anti-pTYR clone comparison
– Titration
– Detection Time course
– Buffers
Other
– ELISA readout
– Wash buffer conditions
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Cell Titration and Ligand2 Titration
100K
67K
44K
30K
20K
13K
EC80
40K cells/well Sig
nal
[Ligand2]
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Lysis Buffer Optimization
Lysis buffer
Pierce Cell Lysis Buffer
17
1 10 100 1000 10000
0
100000
200000
300000
400000
500000
6000000 min
5 min
10 min
20 min
30 min
40 min
MEDI3617 (ng/mL)
Flu
ore
sce
nce
Aspiration Step Direct Addition
0
2
4
6
8
10
12Pierce RIPA
CST RIPA
Pierce Cell Lysis Buffer
S:N
Lysis time Course
10-20 minutes
[mAb]
Sig
nal
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18
Antibody Optimization: Capture/Detection Ab
Capture Ab: 5 µg/mL
Detection Ab 50 ng/mL
0 1.31 3.28 8.19 20.48 51.2 128 320 800 2000
0
2
4
6
8
10
12
12
4
1.333333333
0.444444444
0.148148148
0.049382716
0
[biotin-anti-hTIE2] ug/mL
Eu-anti-pTYR Titration vs. Biotin-anti-TIE2 Titration
[EU-PY20] ng/ml
S:N
Capture Ab (mg/mL)
Detction Ab (ng/mL)
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19
Phosphorylation Assay Development
Low throughput
– 6 well plate
Variable
19 19
Research Assay
High throughput
– 96 well plate
Variable
Early-stage
1E-3 0.01 0.1 1 10 100 0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
Late-Stage
Increased sensitivity
Improved assay
performance
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Enzyme Fragment Complementation Assay
20
β-gal
Fragment1 β-gal
β-gal substrate
Signal
Receptor
Ligand2
SH2
β-gal
Fragment2
SH2
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21
Expected
Potency
Measured
Potency
50% 54%
75% 77%
100% 102%
125% 128%
150% 148%
DiscoveRx Assay Performance
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Effects of mAb Oxidation on Two Functions
22
The Impact of Met- and Trp-oxidation on mAb Activity
Samples %Met-Ox
(M261)
%Met-Ox
(M436)
%Trp-Ox
(W94)
% RP L1
Bioassay
%RP L2
Bioassay
%RP L2
SPR
Ctrl 6.1 2.8 0.0 98 108 96
Photo-Exposed 45.4 35.2 28.4 45 86 77
Ctrl+0.3% H2O2 100.0 100.0 1.6 109 118 106
Ctrl+0.3% H2O2
(w/photo-exposure) 92.2 89.5 30.1 41 88 75
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Excipient Screening for Photo-Protection
23
Sample Description %RP L1
Bioassay
%RP L2
Bioassay
%RP L2
SPR
W94
Oxidation
Dark Control 98 108 96 0
Photo-Exposed 45 86 77 28.4
Photo-Exposed/10 mM A 54 77 79 26.8
Photo-Exposed/10 mM B 40 79 75 29.6
Photo-Exposed/10 mM C 84 90 111 5.8
Photo-Exposed/10 mM D 50 83 97 41
Photo-Exposed/10 mM E 85 99 115 1.6
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Summary
24
For ligand1:
– cell based assay available and performed well
– used from the beginning of product development
For ligand2
– SPR binding assay used at early stage
– Receptor phosphorylation assay used as characterization assay
– Continuous effort in developing robust cell based assay
Bioassays for each arm of the bispecific antibody:
– Necessary for investigating the two functions
– Reveals the two arms have different photosensitivity
– Correlates with each other
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Acknowledgements
Vicky Bushman
Ashley Mullan
Morgan Wilson
Sarah Ronan
Ken Miller
Julian Relton
Xu-Rong Jiang
Michael Washabaugh
25