Recombinant Antibody NanoLuc Fusions for Immunoassays Poster

Recombinant Antibody-NanoLuc Fusions for Immunoassays
Nidhi Nath, Rod Flemming, Marjeta Urh
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
Email: [email protected]
4. Recombinant Antibody-NanoLuc Binding
Activity
•
ELISA Format
Recombinant Antibody NanoLuc
Anti Human HRP
Anti Human HRP
Antibody-NanoLuc
9
9
C e tu x im a b
C e tu x im a b -N L u c
0
0
0
0
.0
0
0
.0
C e tu x im a b C o n c . ( g /m l)
0
0
0
l)
0
1
0
0
0
C o n c . ( g / m
T ra s tu z u m a b
0
1
%
0
0
1
.1
0
1
.0
0
0
0
.0
0
0
.0
0
.0
0
1
1
1
0
0
0
0
0
1
1
.1
0
0
.0
.0
0
1
1
1
0
0
0
0
0
0
1
NanoLuc
1
0
0
1
T ra s tu z u m a b -N L u c
50
0
8
1
1 0
1
5 .0
.1
1 0
T ra s tu z u m a b
8
1 0 0 % s e ru m
0
5 .0
9
1
1 0 9
1 0
5 0 % s e ru m
100
.0
1 .0
1 .0
1 0 % s e ru m
0
1 0
N o s e ru m
1
1 0
1 .5
150
0
1 .5
.0
Renilla
1 0 9
1
NanoLuc is an extremely bright and small (19kDa)
bioluminescent protein
2 .0
R LU
R L U
NanoLuc is cloned with antibody heavy chain, expressed in CHO
cells and purified
.0
CHO cells
Firefly
•
EGFR or HER2
EGFR or HER2
0
Recombinant
antibody-NanoLuc plasmid
Cetuximab/Trastuzumab
with NLuc fusion
Cetuximab/Trastuzumab
without NLuc fusion
.0
Express
•
Pharmacokinetics of antibody drugs is a critical parameter
that determines the dosing and efficacy of the drug.
Two step ELISAs are commonly used to determine the
concentration of drug in the serum.
We optimized a single step immunoassay using AntibodyNanoLuc conjugate
1
Transfect
5. One Step Pharmacokinetics Experiment
o f M a x im u m S ig n a l
1. Recombinant Antibody-NanoLuc Fusion
L o g [T r a s tu z u m a b ( g /m l)]
550 aa/44 Å3
312 aa/32 Å3
171 aa/14 Å3
In a ELISA assay, binding affinity of NanoLuc fused antibody was
similar to that of native antibody
Site-specific recombinant antibody drugs fused to NanoLuc
offers a single step competition ELISA protocol for
quantitating drugs during pharmacokinetics experiment
Attributes of Recombinant AntibodyNanoLuc
5. Binding Activity of Antibody-NanoLuc
using Competition Assay
• NanoLuc is site-specifically fused to the Cterminus of the heavy chain of the antibodies
Cetuximab-NanoLuc
• Unlike chemical labeling, recombinant method
results in homogeneous labeling with NanoLuc to
antibody ratio = 2
8. One Step Assay for Detection of Anti
Drug Antibody (ADA)
NanoLuc fused
Antibody drug
Administration of antibody drugs
results in the presence of antidrug antibodies (ADAs) which
may impact efficacy, pharmacokinetics and may result in
adverse reaction
Panitumumab
or
Cetuximab
• NLuc is attached away from antigen binding site
resulting in no impact on antibody-antigen binding
EGFR
ADA
Antigen
Cetuximab or Panitumumab were titrated into 0.1µg/ml Cetuximab-Nanoluc
before binding to EGFR plate.
A n ti C e tu x im a b _ Ig G
% o f M a x im u m S ig n a l
C e tu x im a b
7
10
P a n itu m u m a b
a n ti C e tu x Ig G (5 0 % s e ru m )
R L U
100
50
6
10
5
10
4
10
3
a n ti C e tu x -Ig G (1 0 0 % s e ru m )
1
0
0
0
1
0
.0
0
.0
10
•
•
1
0
0
0
1
1
.1
0
1
.0
0
0
.0
0
L o g [a n ti C e tu x im a b Ig G ( g /m l)]
IC50 reached at approximately 0.1µg/ml and 0.2 µg/ml of unlabeled
Cetuximab and Panitumumab, respectively.
Cetuximab-NanoLuc has similar binding affinity as unlabeled Cetuximab
and Panitumumab
6. Antibody-NLuc Conjugates for
Biosimilar Screening
3. Recombinant Antibody-NanoLuc
Conjugates
.0
L o g [C e tu x im a b o r P a n itu m u m a b ( g /m l)]
0
0
0
1
1
0
0
0
.0
1
1
1
0
0
0
0
0
0
.0
.1
0
1
• Proof-of-concept: We made recombinant NanoLuc
fusion of Trastuzumab (Herceptin) and Cetuximab
(Erbitux) and show homogeneous labeling of
antibodies and several applications
150
1
• Lot-to-lot reproducibility prevents expensive and
time consuming revalidation of assays
A concentration series of Trastuzumab spiked into serum was competed
against 0.1 µg/ml of Trastuzumab-Nluc, then added to HER2 plate. After 1hr
incubation plates were washed and NLuc substrate added and plate was read.
• Higher concentration of ADA results in loss of signal
• NanoLuc-Antibody fusion allow a single step ADA detection
assays directly from serum sample
9. Summary
Mouse anti-EGFR
M.wt.
For screening, mouse antibodies to
EGFR may
• compete directly with CetuximabNanoLuc for the same binding site
IgG-NanoLuc Non- reducing SDS-PAGE
(Loss of signal)
• NanoLuc was site-specifically
fused to C-terminus of the heavy • can bind to alternate site on EGFR
(no change in signal)
chain
.0
0
25
20
0
0
1
0
37
0
Heavy chain
1
50
0
1
• NanoLuc is fused to the heavy
chain of the antibodies
Heavy chain-NanoLuc
M S -2 6 9 -P 1 A B X
.1
75
0
Reducing SDS-PAGE
L S -C 8 8 0 0 1
1
100
Light chain
1. Trastuzumab;
3. Cetuximab
2. Trastuzumab-rNanoLuc; 4. Cetuximab-rNanoluc
•
Site specific attachment of NanoLuc reporter away
from the antigen binding site
•
Well defined ratio of two NanoLuc per antibody
•
Small and bright reporter results in sensitive assays
•
Batch to batch reproducibility of reagent
2. Applications
•
Ligand binding assay for Pharmacokinetics
•
Anti drug antibody assays
•
ELISAs for analyte detection
ab 11000 6
50
.0
150
1. Recombinant antibody NanoLuc fusions have
several advantages including
ab 846 5
0
250
100
1
4
0
3
.0
2
0
1
1
M.wt.
150
0
1. Trastuzumab;
3. Trastuzumab-rNanoLuc;
Mouse anti-EGFR antibodies were titrated into 0.1 mg/ml Cetuximab-Nluc,
then added to EGFR plate.
0
50
• Antibodies have 2 NanoLuc per
antibody
• Homogeneous population means
2. Cetuximab
4. Cetuximab-rNanoluc batch-to-batch reproducibility
EGFR
.0
75
0
IgG
1
150
0
250
100
Cetuximab-NLuc
4
0
3
o f M a x im u m S ig n a l
2
%
1
L o g [M o u s e a n ti-E G F R A b ( g /m l)]
Several of the mouse antibodies bound competitively with CetuximabNanoLuc. However, the IC50 for those antibodies was at least 10-fold
higher than the Cetuximab-Nanoluc concentration.
www.promega.com
3. We have also developed novel chemical conjugation
method for oriented and efficient attachment of
NanoLuc to the proteins and antibodies.

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