Site-specific conjugation of novel fluorophores and tags to accelerate immunotherapy discovery and development C. Langsdorf, R. Aggeler, B. Agnew, K. Chambers, A. Chen, N. Dolman, Y.-Z. Hu, S. Leonard, K. Oakleaf, M. Wickett, Y. Wu, W. Zhou; Thermo Fisher Scientific, Eugene, OR, USA
The basic cellular mechanisms of internalization and trafficking are important to many areas of cell biology, and especially to the proper function of therapeutic antibodies. Those antibodies intended for use as antibody drug conjugates should specifically bind to target cells and rapidly internalize into acidic compartments. Conversely, antibodies intended to kill cells via direct cell death, complement cascade, or effector cell killing should remain bound to the external surface of target cells as long as possible. However, the ability to study these internalization processes has historically been limited by the lack of tools to directly monitor the internalization and subsequent acidification of extracellular material.
Defined conjugation with pHrodo Red sDIBO for antibody internalization Merge
5 n M p H r o d o iF L R e d S T P H e r c e p t in T im e C o u r s e 1000
M D A -M B - 2 3 1 ( H E R 2 -)
30
0
•Degree of labeling varies between antibody and fluorophore
•Degree of labeling varies for each antibody and fluorophore
•Labeling of binding site can affect immunoreactivity
•Disulfide reduction can affect antibody structural integrity
9
pH
pHrodo Green iFL STP 6.734
A. pH response profile of Herceptin conjugates of amine-reactive pHrodo Red iFL STP and pHrodo Green iFL STP. B. These conjugates will be minimally fluorescent at neutral pH
1 0 0 n M p H r o d o R e d H e r c e p t in
Amine- and thiol-reactive dyes are commonly used to label antibodies. However, the lack of specificity of these bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs.
Defined Conjugation with the SiteClick™ Antibody Labeling System
outside of cells but become brightly fluorescence upon internalization.
Herceptin-pHrodo™ Red iFL STP conjugate traffics to lysosomes
Herceptin conjugates of amine-reactive
6000
p H r o d o R e d s D IB O 4000
2000
0
s ll e
pHrodo™ Red 2.0 - Herceptin NucBlue™ Live ReadyProbes™ Reagent CellMask™ Deep Red Plasma Membrane Stain
+
e
r2
H
e
SiteClick labeling system, then conjugated
Trastuzumab
A.
pH sensitive: pHrodo Red sDIBO
MMAE
Alexa Fluor 647
p H r o d o R e d S T P R it u x a n
were treated with this fluorescently-labeled
F a b lo t 1
antibody-drug conjugate for one hour. B.
F a b lo t 2
Membrane-bound and internalized antibody
SiteClick-conjugated Antibodies Specifically Bind to Cells (Flow Cytometry) C e ll B in d in g o f A le x a F lu o r 6 4 7 s D IB O c o n ju g a t e s
50000
0
G azyva
A le x a F lu o r
SiteClick-conjugated Antibodies Specifically Bind to Cells (High Content Analysis)
S K -B R -3 (H E R 2 + )
S K -B R -3 (H E R 2 + ) 80
500
0
G azyva
H e r c e p t in
S K B R 3 (H e r2 + )
G azyva
H e r c e p t in
M D A - M B - 2 3 1 ( H e r 2 -)
% A p o p to tic C e lls
60
40
20
0
.0
.1 0
5 2
3 .0
0
0
6
6 0
0
.0
0
0 .0 0
0 .0
1
0
0 0
0 0 0 0
4
1
5 2
6 0 .0
.0
0
l
3
.1
0
0
.0 0
0 0
.0
0
5 2
3 6
6 1 0 .0 0
.0 0
0 0
.0
0
0
0
0
4
1
5 2 0 0
.0
0
0
0
o
0
n
0
0
0
tr
6
o
l
3
0
0
1000
M D A -M B -2 3 1 (H E R 2 -)
200
.0
1500
400
0
M e a n A v e r a g e M e m b r a n e In t e n s it y
Herceptin and Gazyva were labeled with pHrodo iFL Red STP . SKBR-3 (Her2+) or MDAMB-231 (Her2-) cells were treated with indicated antibody concentrations, 24 hours at 37ºC. Cells were washed and labeled with Hoechst 33342 15 minutes, then imaged with CellInsight™ CX5 High Content Screening (HCS) Platform.
2000
Herceptin and Gazyva were azidemodified and labeled with Alexa Fluor 488 sDIBO. SKBR-3 (Her2+) or MDA-MB-231 (Her2-) cells were treated with indicated antibody concentrations, 24 hours at 37ºC. Cells from triplicate samples were analyzed on the CellInsight™ CX5 High Content Screening (HCS) Platform. Conjugated Herceptin specifically binds to Her2+ cells. All negative controls provide minimal signal.
C
C e ll B in d in g o f A le x a F lu o r 4 8 8 s D IB O C o n ju g a t e s
In te n s ity (R F U )
600
SKBR-3 + Gazyva SKBR-3 (Her2+) + Herceptin MDA-MB-231 (Her2-) + Gazyva MDA-MB-231 (Her2-) + Herceptin
pHrodo Fabs - High Affinity for all IgG Subclasses
100
800
M D A -M B -2 3 1 (H E R 2 -)
(Her2+)
F a b o n ly
A p o p t o s is I n d u c t io n ( C e l lE v e n t ™ C a s p a s e - 3 / 7 G r e e n )
6 4 7 In t e r n a li z a t i o n
0
Specific Internalization of pHrodo iFL Red STP conjugates
5
J u rk a t (C D 2 0 -)
o
R a m o s (C D 2 0 + )
Herceptin-pHrodo iFL Red STP Conjugate Specifically Internalizes
H e r c e p t in
Specific Internalization and Cell Killing Response of Trastuzumab Alexa Fluor 647/MMAE Conjugates
0
G azyva
F a b lo t 5
1.5 µg aliquots of Rituximab were labeled with pHrodo Red anti-human IgG Fab conjugates. CD20+ Ramos cells were loaded with 30 nM Rituximab labeled with prelabeled pHrodo Fabs or with pHrodo iFL Red STP for 16 hours at 37ºC. pHrodo Red Fab – Rituximab complexes are brightly fluorescent when internalized into CD20+ cells and require the presence of antibody to internalize into cells.
0
H e r c e p t in
F a b lo t 4
R itu x im a b + F a b
0
e H
100000
F a b lo t 3 10
0
tr
k n la
p e rc
a G
B
ti
v zy
a x u it R
n
a
n
k la
H
e
B
ti p e rc
a G
n
n
a v zy
a x u it
Rituxan, Gazyva, and Herceptin were labeled with pHrodo iFL Red STP or pHrodo iFL Red STP . CD20+ Ramos cells treated with antibody conjugates, 16 hours at 37ºC. Cells from triplicate samples were analyzed on the Attune™ NxT Flow Cytometer with AutoSampler. Internalization of positive controls (Rituxan & Gazyva) gives bright signal. Negative control (Herceptin) is not internalized - signal is similar to unlabeled cells.
150000
Herceptin and Gazyva were azidemodified and labeled with Alexa Fluor 647 sDIBO. CD20+ Ramos and CD20- Jurkat cells were treated with antibody conjugates, 16 hours at 37ºC. Cells from triplicate samples were analyzed on the Attune™ NxT Flow Cytometer with AutoSampler. Conjugated Gazyva specifically binds to CD20+ Ramos cells. All negative controls provide minimal signal.
Alexa Fluor 647 MMAE trastuzumab NucBlue Live
Alexa Fluor 647 MMAE trastuzumab NucBlue Live
n
5000
200000
C. MDA-MB-231 (Her2 )
+ B. SK-BR-3 (Her2 )
o
10000
0
n
0
was observed in MDA-MB-231 cells.
C
1000
1 0 n M A n tib o d y
A le x a F lu o r 6 4 7 M e d ia n F lu o r e s c e n c e
2000
M e d ia n F lu o r e s c e n c e S D
3000
R
M e d ia n F lu o r e s c e n c e S D
3 0 n M A n tib o d y
15000
15
SK-BR-3 cells and HER2- MDA-MB-231 cells
was visible in SK-BR-3 cells. C. No signal
3 0 n M A n tib o d y 1 0 n M A n tib o d y
R it u x a n p H r o d o R e d F a b s
F o ld C h a n g e S D
20000
4000
pHrodo Fabs provide a bright signal when bound to internalizing antibody
with sDIBO-Alexa Fluor 647 and sDIBO-
DIY tool kit: sDIBO Biotin sDIBO amine sDIBO amine-reactive
In t e r n a liz a t io n o f p H r o d o ™ G r e e n 2 .0 C o n ju g a t e s
5000
Direct Monitoring of ADC Internalization in Live Cells Using Dual SiteClick Alexa Fluor 647/MMAE Trastuzumab Conjugates
5 µg aliquots of Herceptin were labeled with pHrodo Red anti-human IgG Fab conjugates. HER2+ SK-BR-3 cells were loaded with 1 µg/ml of fluorescently-labeled Trastuzumab and 50 nM LysoTracker Deep Red for 30 minutes at 37ºC. B. pHrodo Red Fab - Trastuzumab complexes are brightly fluorescent when internalized to acidic lysosomes but not on the cell surface.
MMAE (Monomethyl auristatin E). HER2+
Antibody-pHrodo iFL STP Dye Conjugates Specifically Internalize In t e r n a liz a t io n o f p H r o d o ™ R e d 2 .0 C o n ju g a t e s
Trastuzumab was amine-labeled with pHrodo iFL Red STP or with pHrodo Red-sDIBO using the SiteClick labeling system. SKBR-3 (Her2+) or MDA-MB-231 (Her2-) cells were treated with 100 nM Herceptin conjugates, 16 hours at 37ºC. Cells from triplicate samples were analyzed on the CellInsight™ CX5 High Content Screening (HCS) Platform. pHrodo Red labeled Herceptin specifically internalizes into Her2+ cells, regardless of conjugation chemistry.
A. Trastuzumab was azide-activated with the
Standards: Alexa Fluor 488 sDIBO Alexa Fluor 555 sDIBO Alexa Fluor 647 sDIBO
CellLight™ Lysosomes-GFP
H
H
Updated Detection Molecules for SiteClick Antibody Labeling
c
c
c r2 e
SK-BR-3 cells and become brightly IgG antibodies contain two Fc N-glycans that provide an ideal target for conjugation. The SiteClick™ labeling system involves three steps: 1, enzymatic removal of terminal galactose residues on heavy chain glycans; 2, enzymatic addition of galactose-azide (GalNAz); 3, the covalent click conjugation of fluorophore-modified dibenzocyclooctynes to the azide-modified sugars. This system allows efficient site-selective attachment of one or multiple fluorescent dyes, radiometal chelators, or small-molecule drugs to antibodies.
Trastuzumab labeled with pHrodo Red Fabs are fluorescent in acidic organelles pHrodo Red Fab – Trastuzumab complex LysoTracker Deep Red
pHrodo Red iFL STP traffic into HER2+
fluorescent in acidic lysosomes.
5 µg aliquots of Herceptin were labeled with Alexa Fluor 594 anti-human IgG Fab conjugates. HER2+ SK-BR-3 cells were loaded with 1 µg/ml of fluorescently-labeled Trastuzumab and 50 nM LysoTracker Deep Red for 30 minutes at 37ºC. Alexa Fluor 594 Fab - Trastuzumab complexes are visible both on the cell surface and where they have trafficked to lysosomes.
p H r o d o R e d iF L S T P
-
pHrodo Red iFL STP 6.689
LogEC50
Herceptin labeled with pHrodo Red via amine-reactive or SiteClick chemistry both give bright, specific signals
r2
8
s
7
ll
6
s
•Stable covalent bond to cysteine residues
Alexa Fluor 594 Fab – Trastuzumab complex LysoTracker Deep Red
ll
2
•Stable covalent bond to lysines and terminal amines
Trastuzumab labeled with pHrodo Red Fabs are fluorescent in acidic organelles
e
Thiol Labeling
Fluorescently labeled IgG
c
Amine Labeling
5
internalization for many antibody
Trastuzumab was azide-activated with the SiteClick labeling system, then click conjugated with pHrodo Red-sDIBO. Cells were treated with 10 nM pHrodo Red-labeled Trastuzumab for 16 hours at 37ºC, 5% CO2. Media was replaced with Live Cell Imaging Solution with 1% BSA, NucBlue Live Cell Stain, and 50 nM LysoTracker Deep Red for 30 minutes at 37ºC. Red spots indicate internalization of trastuzumab into acidic intracellular vesicles that are positive for LysoTracker Deep Red. Cells imaged on EVOS™ FL Auto cell imaging system.
e
RFU
4
4
scalable screening of antibody
+
Conventional Antibody Labeling Strategies
p H r o d o R e d M E A N _ R in g S p o tA v g In te n C h 2
p H r o d o ™ G r e e n iF L S T P
6
provide rapid labeling for fast,
r2
20
SKBR-3 (Her2+) or MDA-MB-231 (Her2-) cells were treated with 5 nM pHrodo iFL Red STP Herceptin at 37ºC for indicated time. Cells from triplicate samples were analyzed on the CellInsight™ CX5 High Content Screening (HCS) Platform. Conjugated Herceptin internalizes into Her2+ cells over time. Her2- negative cells provide minimal signal.
p H r o d o ™ R e d iF L S T P
with human or mouse IgG to
e
B.
Prelabeled Fab fragment
H
10
s
0
p H r e s p o n s e o f t r a s t u z u m a b c o n ju g a t e s
pHrodo Green can be complexed
pHrodo Red Trastuzumab
NucBlue Live
0
T im e ( H o u r s )
A.
prelabeled with pHrodo Red or
samples. 200
New pHrodo™ Red and Green pH Sensor Dyes
Fc-Specific Fab fragments
400
ll
Here we demonstrate three approaches to directly visualize internalization of therapeutic antibodies and ADCs in live cells. Two of these techniques employ site-specific antibody labeling to screen for and further characterize internalizing clones. Additionally, we demonstrate synthesis of antibody-drug conjugates via defined conjugation. The internalization of these ADCs into HER2positive breast cancer cells shows a strong correlation with target cell killing. Further applications are enabled by site-specific conjugation of biotin, including fluorescent signal enhancement and cell depletion or enrichment. Finally we functionally characterize bispecific antibodies created via bioorthogonal antibody heterodimerization. We believe the insights provided by these approaches have the potential to accelerate biotherapeutic lead generation and development.
S K B R 3 (H e r2 + )
e
M e a n R in g s p o t a v g in t
800
600
Quick Antibody Labeling for Internalization Screening
LysoTracker Deep Red
Herceptin-pHrodo™ Red iFL STP conjugate signal increases over time
Abstract
A n tib o d y C o n c e n tra tio n ( g /m L )
A n tib o d y C o n c e n tra tio n ( g /m L )
HER2+ SK-BR-3 cells and HER2- MDA-MB-231 cells were treated with Trastuzumab Alexa Fluor 647/MMAE conjugates for 72 hours. Cells were then labeled with CellEvent™ Caspase-
The anti-human Fabs specifically bind to the Fc of Human IgG1, IgG2, IgG3, and IgG4 by ELISA
The anti-mouse Fabs specifically bind to the Fc of Mouse IgG1, IgG2a, IgG2b, and IgG3 by ELISA
3/7 Green detection reagent and analyzed on a Thermo Scientific™ ArrayScan™ VTI HCS reader A. SK-BR-3 cells internalized antibody-drug conjugate, while MDA-MB-231 cells had minimal internalization. B. Apoptosis was observed in SK-BR-3 cells at higher ADC concentrations, while MDA-MB-231 cells had minimal apoptosis.
[email protected]
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