Download ThioBridge™ conjugate

Drug conjugation to carriers
George Badescu
Next-Generation Drug Conjugates: The Holistic View of Developing
Superior Drug Conjugates
Cambridge, 24 February 2016
Enabling better biopharmaceuticals
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Immunogenicity assessment
Deimmunised proteins & humanised mAbs
Manufacturing cell line development
Antibody creation
to GMP
manufacture
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Manufacturing process development
GMP antibody & protein manufacture
Fill/finish into vials for clinical studies
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Proprietary site-specific linker technology
Half-life extension & ADCs development
Novel toxin payload development
ADC components
 Antibody
 Linker
 Cytotoxic
payload
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ADC creation to
GMP
manufacture
Toxin payloads & linker-payloads for ADCs
Reagent manufacturing & conjugation
Custom chemistry
An integrated solution for creation, selection and manufacture
of next generation ADCs & other biopharmaceuticals
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Enabling better biopharmaceuticals
Proprietary
technologies to
produce product
candidates with
improved properties
Design and
create
ABZENA inside
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Composite Human Antibodies™
Composite Proteins™
ThioBridge™ ADCs
Test and
select
Develop and
manufacture
Services for selection
of product candidates
with greater probability
of successful
development &
commercialisation
Expertise & capabilities to
develop efficient and flexible
manufacturing processes and
produce products for clinical
development
ADC - antibody drug conjugate
5
3
ThioBridge™
Disulfide bridging conjugation
4
ThioBridge™ reagents attach drugs at accessible disulfides
mAb
Fab
5
ThioBridge™ conjugation has been applied to a wide
range of types of disulfide-containing proteins
Category
Protein
Activity
Enzymes
Asparaginase
Amylase

Cytokines
Interferon α-2a and α-2b
Interferon β

Hormones
Leptin
Erythropoietin

Peptides
Octreotide
Tumour imaging ligand

Blood proteins
Coagulation factors (VIIa, VIII & IX)
Antibodies
mAb
Fab
Novel formats
Scaffolds


Homogeneous ADC created from an anti-HER2 antibody
fragment (Fab) using ThioBridge™-MMAE
ThioBridge™ Fab drug
conjugate
SDS-PAGE analysis of
reaction mixture
ThioBridge
Drug
conjugate
 Site-specific conjugation
 Conversion nearly 100%
 Low reagent consumption
 Homogeneous product (DAR 1)
 Simple purification and characterisation
ThioBridge™ anti-HER2 Fab-MMAE conjugate is bridged at
the disulfide
100%
100%
100%
100%
Complete re-bridging of the reduced disulfide was
observed for this conjugation reaction so structural
integrity of the Fab was maintained
Using ThioBridge™ can improve homogeneity
100
Crude reaction mixture
Av DAR=3.95
Clinical grade product
Av DAR=3.8
• Competitive inhibitor
80
%
60
40
• Lower tolerability
20
• Accelerated clearance
• High instability
0
• High hydrophobicity
0%
012345678 012345678
ThioBridge™
Brentuximab-vc-PAB-MMAE
Maleimide conjugation
to reduced disulfide bonds
ADCETRIS®
9
DAR 4 Conjugation optimisation using Design of
Experiments (DoE)
•
Screening for factors influencing yield and determine factor interactions
•
Successfully achieved conversion to greater than 90% DAR 4 with simple optimisations in
reagent concentrations and pH
•
A powerful approach for optimisation of conjugation
> 90% conversion
to DAR 4
HIC-UV of unpurified reaction:
mAU
WVL:280 nm
intensity
94 % DAR 4
Extrapolated range
Experimental range
Example of a surface
model: response surface
plot (2 factors) for %
DAR 4 achieved
5
3
4.0
5.0
6.0
7.0
8.0
9.0
10.0
minutes
11.0
12.0
13.0
mi
14.0 n 15.0
ThioBridge™ conjugates were stable in different sera
Method: Alexa Fluor 488 conjugated to trastuzumab using ThioBridge™ or maleimide
chemistry with average of 2 disulfides reduced. Conjugates incubated in sera from different
species at 37 ⁰C for 96 h; analysed using SEC
50 LU
ThioBridge™
conjugate: 0 h
30
10
0
PBS
5.0
7.0
9.0
11.0
13.0
100 LU
15.0
17.0
19.0
min
ThioBridge™
conjugate: 96 h
80
50 LU
Maleimide
conjugate: 0 h
30
10
0
PBS
5.0
7.0
9.0
11.0
13.0
100 LU
15.0
17.0
19.0
min
Maleimide
conjugate: 96 h
80
60
human
60
human
40
mouse
40
mouse
20
rat
20
rat
monkey
0
5.0
7.0
9.0
11.0
13.0
15.0
17.0
19.0
min
monkey
0
5.0
7.0
9.0
11.0
13.0
15.0
17.0
19.0
min
ThioBridge™ conjugates were more stable than
maleimide conjugates in human and animal sera
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ThioBridge™ conjugates were more stable in vivo
Distribution of Alexa fluor 488 ADC species in blood pool 48 h after i.v. administration to SCID
mice based on analysis of serum by SEC with fluorescence detection
100
Peak area (%)
80
ThioBridge TM
Maleimide
60
40
20
0
Intact ADC
Cross-conjugated &
dissociated species
Aggregates
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Different ThioBridge™ brentuximab ADCs had similar in
vitro potency in Karpas 299 cells
ThioBridge™ brentuximab ADCs (DAR 4) were prepared with four different reagents with the same
linker-payload.
MMAE
Adcetris
ThioBridge™ ADC 1
ThioBridge™ ADC 2
ThioBridge™ ADC 3
ThioBridge™ ADC 4
Cell Viability [%]
100
50
0
10 0
10 1
10 2
10 3
10 4
Conc (pM)
The four different ThioBridge™ brentuximab ADCs with
MMAE and a cleavable linker had similar potencies in vitro in
Karpas 299 cells
We thank Dr Karpas of Cambridge University for supply of the Karpas 299 cell line
ThioBridge™ brentuximab ADCs with different linker
structures have different efficacy profiles in vivo
ThioBridge™
ThioBridge™
ThioBridge™
ThioBridge™
ADC 1
ADC 2
ADC 3
ADC 4
Adcetris™
• Karpas-299 xenograft model at 0.5 and 1 mg/kg (single dose, i.v. administration)
• The flexibility of the ThioBridge™ technology allows quick screening of reagent configurations
and selection of optimal ADC
ThioBridge™ brentuximab ADCs with different linker structures
have different efficacy profiles in vivo
ThioBridge™
ThioBridge™
ADC 1ADC 1
ThioBridge
ADC 2ADC 2
ThioBridge
TM
3
Tumour volume (mm )
3
Tumour volume (mm )
2000
1000
0
2000
1000
10
20
30
40
50
20
30
40
3
1000
40
Days post treatment
Adcetris
Control ADC
Vehicle
50
•
Two ThioBridge™ brentuximab ADCs (DAR 4)
with structurally different linkers were tested
in Karpas-299 xenograft model at 0.4 mg/kg
(Q4Dx4, i.v. administration)
•
Different ThioBridge™ reagents generated
ADCs with different in vivo efficacy profiles
2000
1000
0
0
30
20
50
Vehicle
0.4 mg/kg, Q4Dx4
Dosing
3000
Tumour volume (mm )
3
10
Adcetris
Adcetris™
2000
20
ThioBridge ADC 1
Days post treatment
Vehicle
0.4 mg/kg, Q4Dx4
Dosing
10
40
Days
0
Control ADC
0
60
0 10 20 30 40 50 60 70 80
Days post treatment
3000
ThioBridge ADC 2
80
0
0
0
Tumour volume (mm )
100
Vehicle
0.4 mg/kg, Q4Dx4
Dosing
3000
Percent survival
Vehicle
0.4 mg/kg, Q4Dx4
Dosing
3000
Kaplan-Meier Plot
TM
0
10
20
30
40
Days post treatment
50
In Vivo Imaging of ThioBridge™ anti-PSMA Minibody Variants
Maleimide control
ThioBridge™ PEG 6u
ThioBridge™ PEG 36u
• Excellent radiolabeling efficiency and immunoreactivity for all three conjugates
ImaginAb Inc:
Jean Gudas, VP of R&D
Michael Torgov, Ph.D
Tove Olafsen, Ph.D
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ThioBridge™ conjugates of an antibody fragment with
desferrioxamine and 89Zr – images at 24 h
Maleimide control
ThioBridge™ PEG 6u
ThioBridge™ PEG 36u
24 h
Nude mice
• Maleimide conjugation of desferrioxamine on cysteine residues destabilized the protein and
resulted in rapid clearance through the kidneys
• Disulfide bridging through ThioBridge™ stabilized the antibody fragment and reduced
kidney clearance
ImaginAb Inc:
Jean Gudas, VP of R&D
Michael Torgov, Ph.D
Tove Olafsen, Ph.D
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CyPEG™
Stable conjugation of thiols
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CyPEG™
Cysteine thiol conjugation and stabilisation schematic
CyPEG™ Reagent
= Payload/fluorescent dye or other functional molecule
Reduced or cys-engineered
antibody
Efficient Michael addition conjugation with a mono-sulfone reagent
followed by stabilisation (‘locking’) of the formed conjugate with NaBH4
CyPEG™ conjugation to an antibody mimetic thiol was efficient
• CyPEG™ PEGylation of an Affibody* was compared with other thiol-reactive PEG reagents
• CyPEG™ reactivity was comparable to the maleimide reagent
• CyPEG™ was more reactive than vinyl sulfone, acrylate and haloacetamide reagents
PEGylation with CyPEG™ was successfully achieved with a 1:1 ratio of reagent to protein
* Anti-Her2 Affibody® with single free cysteine; a PEG size of 5 kDa was used for all reagents
CyPEG™ Stabilisation with borohydride did not affect mAb
binding affinity by ELISA
•
Borohydride locking step stabilises the conjugate preventing retro-Michael reactions
•
Mild borohydride treatment an established method for stabilising bioconjugates, e.g.,
PEG-GSCF (N-terminal amine conjugation with PEG-aldehyde)
•
Antibody shown to retain full binding efficiency post borohydride treatment
4
A630 (nm)
3
2
1
0
0.001 0.01
0.1
1
10
100 1000 10000
[Compound] (ng/mL)
Native Trastuzumab, n=1
Trastuzumab + Reagent + NaBH4
Trastuzumab + NaBH4
ADC preparation example: CyPEG™ val-cit-PAB-MMAE addition
to partially reduced trastuzumab - DAR profile by HIC
• CyPEG™ trastuzumab ADC DAR profile under 3 h; average DAR (0-8) = 4.0
31.20
26.20
30
% Area at 280 nm
25
17.30
20
15
9.20
7.00
10
1.50
3.70
1.10
5
2.80
0.00
0
pH 7.0
Conjugation reaction pH
DAR 0
DAR 1
DAR 2
DAR 3
DAR 4
DAR 5
DAR 6
DAR 7
Typical profile for mono-cysteine conjugation observed
(e.g., malemide conjugation)
DAR 8
DAR >8
HIC purified DAR 4 CyPEG™ADC analysed by native SEC-MS
Intact mass for
Trastuzumab
148250 Da
Mass ion for Locked
CyPEGTM ADC
158175 Da
Compound
= CyPEGTM
val-cit-PAB-MMAE
TOF MS ES+
Mass by LC/MS (Da)
CyPEG™ trastuzumab ADC DAR 4
158175 Da
trastuzumab
148250 Da
Mass difference (ADC – ADC)
Mass of conjugated reagent
9925 Da
4 x 2481 = 9924 Da
Deconvoluted MS allowed for determination of the intact mass and confirmed full
borohydride stabilisation
CyPEG™ DAR 4 ADC Stability versus equivalent maleimide
based ADC: Incubation in PBS with excess HSA, 7 d, 37 °C
• ADCs purified to DAR 4 using HIC and then incubated in PBS with HSA (20 mg/mL; 50 mol
equivalents to ADC) over 7 d
• HIC used to determine the DAR profile after 7 d
% change in DAR 4 at 7 d
CyPEG™ ADC
0
Maleimide ADC
30
CyPEG™ DAR 4 ADC showed excellent stability in presence of HSA
whereas the maleimide ADC degraded by 3̴ 0% to DAR < 4 variants
HiPEG™
Covalent conjugation at polyhistidine motifs
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HiPEG™: Covalent conjugation at polyhistidine motifs
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•
•
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Utilises terminal or intra-protein polyhistidines as site for covalent conjugation
Conjugation to N or C-terminal histidines reduces interference with protein binding
Stoichiometrically efficient and predictable conjugation process
His-tag available for purification post-conjugation
ADC services & technologies provided by Abzena
Abzena provides one of the broadest range of services to support the creation
and development of ADCs of any company in the industry supported by a wealth
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assessment
ThioBridge™
CyPEG™
Over 20 prepared
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15,000 L GMP
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100g Toxin
50g linker
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Lysine
50g Toxin-linker
10g ADC
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