challenges and strategies in designing moa reflective ... · average rp (%) cv (%) sample 1 99 97...
TRANSCRIPT
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
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
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
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
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
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
No Plate Positional Effect
7
row A/H
row B/G
row C/F
row D/E
[mAb] (ng/mL)
RL
U
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
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
Competitive Binding Assay Using SPR
10
Sensor Surface
Receptor
Ligand2 mAb mAb
Ligand2
Sensor Surface
Receptor
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
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
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
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
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
Cell Titration and Ligand2 Titration
100K
67K
44K
30K
20K
13K
EC80
40K cells/well Sig
nal
[Ligand2]
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
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)
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
Enzyme Fragment Complementation Assay
20
β-gal
Fragment1 β-gal
β-gal substrate
Signal
Receptor
Ligand2
SH2
β-gal
Fragment2
SH2
21
Expected
Potency
Measured
Potency
50% 54%
75% 77%
100% 102%
125% 128%
150% 148%
DiscoveRx Assay Performance
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
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
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
Acknowledgements
Vicky Bushman
Ashley Mullan
Morgan Wilson
Sarah Ronan
Ken Miller
Julian Relton
Xu-Rong Jiang
Michael Washabaugh
25