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ThioBridge™ - Next
generation conjugation
technology for ADCs
Antony Godwin
Director of Science & Technology
Antibody Drug Conjugates:
Innovation in Design, Development and Manufacture
June 26, 2013
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Presentation Overview
• Company Overview
• Conjugating To Antibodies – The Challenges
• ThioBridge™ Thiol Bridging Conjugation
• Case Studies With mAb And Fab
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Company overview
• Comprehensive portfolio of proprietary technologies
 Pharmacokinetic optimisation of biologics via sitespecific polymer conjugation
 Superior antibody-drug conjugate technologies for
reduced heterogeneity and increased stability
• Business model offers product-specific technology licences
following feasibility studies and collaborative programs
 Multiple partner programs progressing towards clinic
• 45 employees, including 26 PhDs, based in dedicated lab
and office facilities in central London and Coventry
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Enabling Better Biopharmaceuticals
Antibody Drug
Conjugates (ADCs)
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Less heterogeneity
Improved stability
Efficient chemistry
Range of payloads
mAbs, Fabs, scaffolds
Optimisation of halflife & activity
Bispecific & novel
products
 Site-specific
conjugation
 Low reagent use
 Novel polymer for low
viscosity conjugate
 Chemical conjugation to
rapidly produce
• homodimers
• heterodimers
• bispecific products
Leading the way - Approved ADCs
Maleimide linkage not direct to mAb
Kadcyla
(T-DM1, Roche/ImmunoGen)
FDA approval 2013
Amine conjugation
Adcetris
(Seattle Genetics/Millennium)
FDA approval 2011
Average DAR 3.5
Disulfide breaking thiol conjugation
Maleimide linkage to mAb
Based on amine and disulfide breaking conjugation
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Average DAR 4
Payload conjugation to a mAb has challenges:
There is a need to address heterogeneity & stability
Lysine chemistry
• Low efficiency
• Non-specific
• High heterogeneity
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Cysteine chemistry
• Semi-specific
• Bond breaking
• High heterogeneity
• Maleimide instability
Cysteine chemistry
• Re-engineering approach
• Site-specific
• Reduced heterogeneity
• Maleimide instability
ThioBridge™ attaches payload across a native disulfide
bond that is re-bridged
Accessible disulfide bonds:
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mAb
Fab
Bridging conjugation is widely applicable
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Category
Example protein /
peptide
Enzymes
Asparaginase
Amylase
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Cytokines
Interferon α-2a and α-2b
Interferon β
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Hormones
Leptin
Erythropoietin
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Peptides
Tumour imaging ligand
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Blood proteins
Coagulation factors
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Antibody fragments
Anti-CD4 Fab
Anti-TNF α domain
Novel formats
Scaffolds
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Activity
ThioBridge™ S-S bridging reagent
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•
•
•
Combined with payload in single reagent
Compatible with amine and hydroxyl functionalised payloads
Cleavable and non-cleavable linker options
Hydrophilic for improved solubility/reduced aggregation
Thiol bridging linker
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Hydrophilic
spacer
Degradable
or
Nondegradable
linkers
Amenable to payloads
with different MOA’s
ThioBridge™ ADCs are more homogeneous
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More homogeneous
DAR distribution restricted to up to DAR 4 – can adjust distribution of DARs 2, 3 & 4
Avoids highly loaded species
Minimal amounts of non-conjugated antibody
DAR2-4= 94%
DAR2-4= 52%
%
32%
0%
012345678
012345678
ThioBridge™
Maleimide conjugation
to reduced disulfide bonds
Hamblett et al. Clin Cancer Res (2004)
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DAR=0
DAR=1
DAR=2
DAR=3
DAR=4
DAR=5
DAR=6
DAR=7
DAR=8
• Competitive inhibitor
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Lower tolerability
Accelerated clearance
High instability
High hydrophobicity
Instability of maleimide conjugates:
Deconjugation reactions
antibody conjugate
albumin in serum (A)
albumin conjugate
Maleimide instability can lead to conjugation of drug to albumin
e.g., Senter et al
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ThioBridge™ conjugates are more stable in serum than
maleimide
• Alexa Fluor 488 labelled trastuzumab
• ThioBridge™ & Maleimide conjugates incubated in rat serum at 37 °C for 48 h
• SEC analysis
Relative Fluorescence
ThioBridge™ conjugate
Maleimide conjugate
Significant crossconjugation to albumin
T=0h
T=0h
mAb unstable
Aggregates
T = 48 h
4
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Time (min)
Alexa Fluor 488
T = 48 h
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10
12
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mAb Albumin
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18
20
4
6
Time (min)
8
10
12
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mAb Albumin
Significant instability seen with maleimide conjugate but not
with ThioBridge™ conjugate
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Fragments
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ThioBridge™ is more stable than maleimide in the presence
of human serum albumin
• Same payload (Drug) conjugated to trastuzumab using ThioBridge™ and maleimide
• Both conjugates were incubated in artificial serum (PBS + 20 mg/mL HSA) at 37 °C
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2
3.8
1.9
3.6
1.8
3.4
1.7
3.2
Maleimide ADC (DAR = 4)
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2.8
ThioBridge™ ADC (DAR = 4)
2.6
Average DAR
Average DAR
• HIC was used to determine changes in the average DAR
1.6
1.4
1.2
2.2
1.1
2
1
50
100
ThioBridge™ ADC (DAR = 2)
1.3
2.4
0
Maleimide ADC (DAR = 2)
1.5
0
Time [hours]
Superior stability in artificial serum for ThioBridgeTM
compared with maleimide
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50
Time [hours]
100
ThioBridge™ ADC shows excellent and antigen-selective
cell-killing in vitro
Antigen-positive II Antigen-positive
(BT-474)
II (BT-474)
Antigen-positive I (SK-BR-3)
SK-BR-3 100
50
0
-2
-1
% viability
% viability
Antigen-positive
cell lines
% viability
100
BT-474
100
50
0
0
1
2
3 -2 4 -1
[ADC] (nM)-log scale
Antigen-negative
I (A549)
ThioBridge ADC (DAR=2.8)
100
% viability
% viability
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MCF-7
50
0
Free drug
Free drug
Trastzumab
Trastzum
ThioBridg
ThioBridge ADC (DAR=2.8)
50
0
-1
0
1
2
3
[ADC] (nM)-log scale
4
5
-2
-1
ThioBridge™ ADCs are c.10,000-fold more potent in antigen +ve
cells than in antigen –ve cells
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Trastzuma
ThioBridge ADC (DAR=2.8)
ThioBridg
0
1-2
2-1
30
41
52
3
4
5 0
[ADC] (nM)-log scale
[ADC]
(nM)-logIIscale
Antigen
negative
(MCF-7)
100
-2
Free drug
50
A549
Antigen-negative
cell lines
Free drug
drug
Free
Trastzumab
Trastuzumab
Free drug
Trastzumab
0
1
2
3
[ADC] (nM)-log scale
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ThioBridge™ ADCs are stable in serum
• ThioBridge™ ADC was incubated at 37°C in human serum for 72 h prior to measuring the potency
in antigen-positive (BT474) cells
[ADC](nM) – Log scale
ThioBridge™ ADC retains in vitro potency after incubation in human serum
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ThioBridge™ ADC highly effective in BT474 xenograft model
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Body weight
Tumour volume
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1600
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Body weight (g)
Tumour volume (mm3)
1400
1200
1000
800
600
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Vehicle
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mAb
drug
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Thiobridge ADC (30 mg/kg)
ThioBridge ADC (20 mg/kg)
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ThioBridge ADC (10 mg/kg)
400
10
1
200
1
5
9
13
17
21
25
29
33
37
41
13
17
21
25
29
33
37
41
45
45
Time (days)
Dosing
Schedule
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Time (days)
0
vehicle / mAb /drug (i.v.)
ThioBridgeTM ADC 10 mg/kg (i.v.)
ThioBridgeTM ADC 20 mg/kg (i.v.)
ThioBridgeTM ADC 30 mg/kg (i.v.)
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 Complete responses at highest ADC doses
 Sustained for at least 60 days
 Significantly better efficacy in ADC groups
 Well-tolerated at all doses
ThioBridge™ is ideally suited for conjugation of antibody
fragments (Fab)
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Advantages of using ThioBridge™ to create ADCs using Fabs
ThioBridge™ Fab-DC
SDS-PAGE analysis of
reaction mixture
ThioBridge
Drug
conjugate
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Site-specific conjugation
Conversion nearly 100%
Low reagent consumption
Single reaction product (DAR = 1.0)
Easy purification and characterisation
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ThioBridge™ Fab drug conjugate
100%
100%
100%
100%
Complete bridging of ThioBridge™ Fab drug conjugate and excellent purity
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ThioBridge™ Fab and mAb ADCs
internalise into antigen-positive cells
• A fluorescent mock payload (FITC) was conjugated to trastuzumab Fab and mAb using ThioBridge™
• Internalisation into antigen-positive cells (SKBR3) was investigated by confocal microscopy
ThioBridge™ mAb conjugate
ThioBridge™ Fab conjugate
ThioBridge™ Fab and mAb conjugates internalise efficiently into antigen-positive cells
: ADCs obtained using a bis-alkylating reagent internalise into antigen-positive cells. (a)
conjugate. (b) Fab-FITC conjugate.
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ThioBridge™ conjugation of Fabs does not
impair antigen binding
• ELISA in reversed orientation (Fab / FabDC immobilised on the plate)
• Detection with:
• biotin-HER2 / streptavidin-HRP
• 6His-HER2 / aHis-mAb-HRP
ThioBridge™ Fab-DC retains high affinity
binding to antigen
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ThioBridge™ Fab drug conjugate shows potent and
selective cell-killing in vitro
Antigen-positive cell line
Antigen-negative
Antigen-negative cell
cell line
line
ThioBridge™ Fab-DC
Drug
Drug
[Fab-drug conjugate] (nM) – log scale
% viability
Fab
ThioBridge™ Fab-DC
% viability
Fab
[Fab-drug conjugate] (nM) – log scale
In antibody positive cells, the activity of ThioBridge™ Fab-DC matches free drug
No effect of ThioBridge™ Fab-DC observed in antigen negative cells
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ThioBridge™ Fab drug conjugate highly
effective in BT474 xenograft model
Vehicle
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Vehicle
mAb (20 mg/kg)
Drug (same dose as in Fab-DC)
Fab-DC (20 mg/kg)
Dosing schedule
• q2d vehicle, free drug & ThioBridge™ Fab-DC
• q4d mAb
mAb
drug
Fab-DC
Potential CMC advantages of using ThioBridge™ to create ADCs
• Controlled DAR distribution (no greater than DAR 4)
• Easier characterisation
• Low reagent stoichiometry (near 1:1 stoichiometry)
• Improved aggregation profile
• Excellent stability
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CONFIDENTIAL
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Conclusions:
• Improved homogeneity
 No re-engineering of mAb
 Site directed to disulfide
bonds
• Improved stability
 Re-bridged disulfide bonds
 Preserved structural integrity
• PEG spacers
 Reduced aggregation
ThioBridge™
 Improved reagent solubility
IP & partners
Granted Patents
Commercial Partners
Half-life extension
Antibody-drug conjugates
 Claims cover reagents, conjugation
process and conjugate products
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 Feasibility studies and collaborations
ongoing with non-disclosed partners
Thank you for your attention
Antony Godwin
Director of Science & Technology
[email protected]