1. Ab-XTEN & Ab-XTEN-Drug Conjugates
XTEN
• Long, tunable PK enables up to once-a-month dosing Difficult to conjugate
• Long PK maximizes efficacy and minimizes toxicity Hydrophobic
Low Drug Load
• Clinically validated in 2 products Fc-receptor mis-targeting
ADCC/CDC
• Non-immunogenic
Bivalent
• Biodegradable (safer than PEG) Monospecific
No EPR effect
• 3 publications 4 protein chains
Ab-XTEN
• Genetic fusion to Ab fragments & mimetics Easy, specific conjugation
Highly hydrophilic
• scFv, dAbs, Darpins, Nanobodies, Centyrins, Adnexins, etc.
High Drug Load
• Single protein chain, manufactured in E. coli No FcR mis-targeting
No ADCC/CDC
Mono- or Multi-valent
Ab-XTEN-Drug Mono- or Multi-specific
EPR effect
• Specific conjugation to cytotoxic drugs via free thiols 1 protein chain
• Many available drugs to choose from
• Drug release via linker cleavage or XTEN degradation
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2. AbFr-XTEN: Product Formats
Monovalent AbFr
IgG Drug Conjugate
Target binding
Cytokine Blockers
Receptor Blockers Tumor-Drug Delivery
Antiviral
Bispecific AbFr C1 Binding Multifunctional Fusion
Recycling
ADCC
Tumor-Specific Toxin
Tumor-Specific IL2
Combination Products
Immune activators
• Large number of product opportunities
• Common discovery platform
2
3. XTEN Prevents Aggregation of scFv
Abs (215 nm)
Size Exclusion Chromatography
Monomer aHer2
Dimer
Volume (ml)
XTEN stabilizes the scFv and thus enables clinical use of scFv
3
4. Bispecific scFv-XTEN-scFv
EGFR Her2
SDS/PAGE
160 aEGFR aHer2
110
1.0
Normalized Absorbance
60
Normalized Abs. (OD405 nm)
GFP
0.5
20
Control
10 0.0
0.0001 0.01 1 100 10000
bscFc Concentration (nM)
Versabody Concentration (nM)
• High-affinity, specific binding to EGFR and Her2
• Single protein chain manufactured in E. coli
4
6. AbFr Multimers are Well-Behaved
EGFR Binding
1.6
B SA HRP
Analytical SEC Profiles V
Strept-HRP
V
EGFR-Fc
44 kDa
17 kDa
660 kDa
160 kDa
1.3 kDa
1.2
ELISA Signal
GFP
Anti-GFP
0.8
1 STD
Monomer
0.4
Normalized A215
Dimer
0.75
Tetramer
Hexamer
0
0.5 Control Monomer Dimer Tetramer Hexamer
25
0.25
Plasma Stability
0
10 15 20 Days 0 1 3 7
Retention Volume (ml)
VHH
hexamer
50% pooled cyno plasma in PBS,
37C, Anti-GFP-HRP detection
AbFr Multimers show avidity, no aggregation and good plasma stability
6
7. Chemical Conjugation to scFv
XTEN
medium
short medium long SEC
short
long
44kDa
2 5 2 5 2 5 158kDa
Absorbance 680nm
670kDa
short
med
long
aHer2 aHer2 aHer2 Elution Volume
XTEN allows control of hydrodynamic radius and half-life
7
8. Cell Binding of aHer2-XTEN-AF
Isotype control
Trastuzumab block
XTEN288 XTEN576 XTEN864
Protocol:
SKOV incubated with AF680-labeled scFv-XTEN fusion proteins
Cell binding detected by FACS
XTEN length has little influence on cell binding
8
9. Tumor Uptake of aHer2-XTEN
60
Protocol:
SKOV tumor model
Fluorescence (Fold Increase)
50 3 mice per group
40
Day 0: Inject labeled protein
24hr
30
48hr
Day 1&2: In vivo imaging
20
10
0
Long Medium Short Herceptin
Long-chain XTEN facilitates tumor uptake of aHer2 fragment
9
10. In Vivo Targeting
IgG
aHer2-XTEN
Trastuzumab
A1 A2 A3 A1 A2 A3
Tumor
Inject labeled Heart
protein
3 day Kidney left
distribution
Kidney right
Image organs
Liver 1
Liver 2
Lungs
Protocol:
SKOV3 tumor model, n=3 Spleen
Proteins labeled with AF680
• aHer2-XTEN576 shows specific tumor accumulation
• Trastuzumab shows significant background staining of normal tissues
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