1. XTEN Conjugates – Examples
SH
SH
SH
XTEN Azide
Drugs
· Maytansine
Click
· Auristatin
Peptide + Drug · Doxorubicin
· Paclitaxel
Click
Peptide + Drug
Click
Antibody Mimetics
No disulfides; Link
Antibody Mimetic + Drug via thiol or N-term.
· Darpins
· Nanofitins
Click
· Adnexins
Drug + Drug · Centyrins
· Affibodies
Many others with
disulfides
Antibody + Drug
XTEN gives rapid access to many high-value product formats
1
2. Antibody Mimetic-XTEN-Drug Conjugates
SH
SH
SH
XTEN Azide
XTEN-Drug Precursor Azide
SH + NMal DBCO
Antibody Mimetic
Rapid access to XTEN-Drug conjugates starting from Antibody Mimetics
with free thiol groups or alkynes for Click chemistry
2
3. Peptide-XTEN-Drug Conjugates
Example: LHRH-XTEN-Doxorubicin
SH SH SH
H2N
XTEN 432/864
SH
DBCO Azide
Doxorubicin
SH
LHRH LHRH LHRH
Click
Rapidly create many bifunctional products from purchased parts
Allows rapid optimization of many pharmaceutical properties
3
4. XTEN as a Lead Discovery Platform
Deals
GLP1
Approved XTEN432 Segments for Click Chemistry
Insulin
$530M Glucagon
SH SH SH SH
$537M GIP
Leptin + DBCO
+ Azide
PYY
GHRH
Pramlintide
Prepare “Click-ready” XTEN-peptide segments
Click Click Click Click Click Click Click Click
Bispecific compounds, all 1:1 combinations of 2 peptides
Optimize ratios of the best bispecific compounds
Functional Screens for Diabetes/Obesity – in vitro and in vivo
Rapid optimization of bifunctional drugs in a long half-life context
4
5. Homogeneity by ESI Mass Spec
XTEN PEG
GLP2-XTEN880 G-CSF-PEG20 (Neulasta)
Calc. 84,830 Da
Exp. 84,831 Da
Intensity
82000 84000 86000 88000
Mass (Da) Mass (Da)
Bagal D., et al. (2008) Anal. Chem., 80: 2408-18
1 Species >100 Species
XTEN is homogeneous whereas PEG is highly heterogeneous
5
6. Conjugation Reagents
XTEN versus PEG
Composed of natural amino acids
• Biodegradable PEG is not biodegradable
• No risk of kidney vacuolation PEG builds up in vacuoles
• Made by E. coli fermentation
• CoGs comparable to PEG
Single molecular species PEG is >100 compounds
• Process monitoring by MS Problematic with PEG
• Easy post-reaction purification Problematic with PEG
Greater customization
• Molecular weight Higher, more choice vs PEG
• Number of conjugation sites More flexible than PEG
• Spacing of conjugation sites Reduced viscosity vs PEG
XTEN avoids the key limitations of PEG
6
7. Major Variables of XTEN
XTEN Polymer Length
SH
Type of Conjugation Sites H2N
Cys
SH
H2N
NH2
NH2
SH
SH
SH
Number & Position of Sites H2N
NH2
NH2
SH
SH
SH
SH
H2N H2N
Number of
NH2
NH2
SH
SH
SH
SH
Different Chemistries
H2N H2N
XTEN sequence can be customized to optimize conjugate performance
7
8. XTEN Coupling Chemistries
Payload-N-Mal Payload-Thiol
SH
H2N
N-Mal Sulfo-NHS
N-Mal
N-Mal
SH
Payload-Amine Payload-Azide
H2N H2N
Aldehyde NHS Alkyne NHS
Aldehyde Alkyne
NH2 Azide
Many coupling chemistries exist for conjugation to XTEN
8
9. Peptide Coupling to 1xAmino-XTEN
RP-HPLC ESI-MS
Exp. 79,753 Da
Absorbance
Calc. 79,751 Da
H2N
N-Mal Sulfo-NHS
15.0 20.0 25.0 30.0 35.0
GLP2
SH (39AA) Absorbance Exp. ∆ +3865 Da
Calc. ∆ +3856 Da
Yield: >90%
Exp. 83,836 Da
Calc. 83,826 Da
15.0 20.0 25.0 30.0 35.0
Volume (ml) Mass (Da)
• Coupling to 1xAmino-XTEN is >90% efficient
• Reaction monitoring by HPLC and MS
9
10. Peptide Coupling to 1xThiol-XTEN
RP-HPLC ESI-MS
Exp. 80,685 Da
Calc. 80,685 Da
SH
Absorbance
GLP2 15.0 20.0 25.0 30.0 35.0
N-Mal (39AA)
Exp. ∆ +4146 Da
Absorbance
Calc. ∆ +4145 Da
Yield: >95%
Exp. 84,831 Da
Calc. 84,830 Da
15.0 20.0 25.0 30.0 35.0
Volume (ml) Mass (Da)
• Coupling to 1xThiol-XTEN is >95% efficient
• Reaction monitoring by HPLC and MS
10
11. Tri-valent Coupling to 3xThiol-XTEN
RP-HPLC ESI-MS
SH Exp. 82,833 Da
SH
SH
Calc. 82,836 Da
Absorbance
GLP2
(39AA)
15.0 20.0 25.0 30.0 35.0 40.0
N-Mal
Exp. ∆ +12,444 Da
Absorbance
Yield: 67% Calc. ∆ +12,434 Da
Exp. 95,277 Da
Calc. 95,270 Da
15.0 20.0 25.0 30.0 35.0 40.0
Volume (ml) Mass (Da)
• Trivalent coupling to 3xThiol-XTEN is 67% efficient
• Attractive for viscosity reduction, dose reduction, multivalent targets
• Other valencies available on request
11
12. Peptide Coupling to 1xDBCO-XTEN
RP-HPLC ESI-MS
Exp. 81,115 Da
Calc. 81,113 Da
Absorbance
DBCO
Click Azide 18 20 22 24 26 28
Exp. 81,407 Da Exp. ∆ +292 Da
Calc. 81,404 Da Calc. ∆ +291 Da
Absorbance
Yield: >99%
18 20 22 24 26 28
Volume (ml) Mass (Da)
• Click coupling using DBCO-Azide is >99% efficient
• Reaction monitoring by HPLC and MS
12
13. Tri-valent Coupling to 3xDBCO-XTEN
RP-HPLC ESI-MS
Exp. 84,123 Da
Calc. 84,120 Da
Absorbance
DBCO DBCO DBCO
20 22 24 26 28 30 32
Click Azide Exp. 84,995 Da Exp. ∆ +872 Da
Calc. 84,994 Da Calc. ∆ +874 Da
Absorbance
Yield: >95%
20 22 24 26 28 30 32
Volume (ml) Mass (Da)
• Click coupling of 3 peptides to 3xDBCO-XTEN is >95% efficient
• Attractive for viscosity reduction, dose reduction, multivalent targets
• Reaction monitoring by HPLC and MS
13
14. Rat PK of Conjugate XTEN
Dose: 2 mg/kg SC; 3 rats per group
10000
Plasma Concentration (ng/ml)
Recombinant GLP2-XTEN
T1/2= 36.1 hr
1000
Conjugate GLP2-XTEN
T1/2= 37.4 hr
100
0 1 2 3 4 5 6 7 8
Days
Recombinant and Conjugate GLP2-XTEN have similar PK profiles
14
15. In vitro Plasma Stability of Conjugate XTEN
Human plasma (37C)
160
Concentration (% of Day 0)
140 Recombinant GLP2-XTEN
120
100
80
Conjugate GLP2-XTEN
60
40
20
0
0 1 2 3 4 5 6 7 8 9 10
Days
• Half-life of thiol-maleimide linker >240hr
• Linker stability does not limit in vivo drug exposure
15
16. LHRH-XTEN-Doxorubicin
Internalization
Proteolysis of XTEN
Linker Cleavage (Optional)
Endosome
Cytosol
16 Toxin can be released by cleavage of the linker or the XTEN
17. Cost of Goods Prediction
for Large Scale Production of XTEN
Fermentation 15,000 L fermenter
8 g/L titer
Purification Standard commercial resins
45% overall process yield
Formulation Frozen Liquid
Capacity ~50 kg per fermentation
~35 day cycle time
Cost <$150/g
(versus GMP grade PEG ~$200/g)
Amunix expects to make GMP-grade XTEN at lower cost than PEG
when produced at large scale
17
18. Comparison of XTEN versus PEG
Property XTEN PEG
Large Hydrodynamic Radius Yes Yes
In vivo Half-Life Very Long Very Long
Immunogenicity Extremely Low Extremely Low
Manufacturing Process Recombinant Chemical
Conjugation Chemistries Many Many
Solubility Very High Very High
Reagents for PK Assays Available Available
Biodegradable = Safety Benefit Yes No
Single Molecular Species Yes No
Peptide Mapping of Payload/Polymer Junction Yes No
Chemical Structure Confirmation by MS Yes No
RP-HPLC Peak Resolution High Low
Capture by Ion Exchange Yes No
Number & Position of Functional Groups Fully Flexible Few Choices
Patent Life Extension Yes Limited
18 XTEN offers multiple advantages over PEG
