Anovasia's non-confidential technology presentation (status: May 2012)

1. molecular painting
“just right on the face of it”

2. molecular painting
Technology Presentation
non-confidential, May 2012

3. Executive Summary
• Founded and based in Singapore, Anovasia is a solution provider for the life sciences
industry
• Anovasia has a unique platform technology called Molecular Painting (MP)
– MP is the surface modification of biomembranes
– MP reagents are based on natural products
• The main industrial application areas are:
– Basic and Applied Research
– Diagnostics, Biomarkers
– Vaccine Development
– Targeting and Delivery of Genes, Proteins and/or Drugs
– Cell and Gene Therapy
• Anovasia supplies products from its existing MP range as well as made-to-order,
designer reagents based on client’s individual needs
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4. Technology Overview
• Molecular Painting reagents uniquely allow non-specific biomembrane
surface modification
• All viruses with an envelope posses the potential to be modified by an MP
Reagent
VIRUS
• Other membrane entities such as cells, membrane vesicles, lipsomes and
exosomes can be painted
• Shown for HIV lentivirus and MLV retrovirus (Metzner et al., 2008)
EXOSOME
• Recently shown for wild-type herpes virus and influenza (manuscripts in prep.)
• Recently shown for exosomes (data unpublished)
• Applicable to many other clinically relevant enveloped viruses
LIPOSOME
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5. How does Molecular Painting Work?
- three easy steps
1. Prepare your sample
– example on next slide is a viral gene delivery vector
– other sample types: liposomal drug carrier, exosome, etc.
2. Chose the functionality of your MP reagent
– e.g. fluorescent tag (A)
B C D functional element
– e.g. immune-modulation (B) A
– e.g. ligand-receptor targeting (C) spacer region
– e.g. magnetic particle (D)
biomembrane prong
3. Mix together in a quick 10 min reaction
– see next slide
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6. Viral gene delivery vector modified with two MP reagents
MP reagent 1 - magnetic particle
(purification, targeting, tracking)
virus capsid
virus envelope
proteins
tissue/cell specific
promoter
therapeutic
gene
MP reagent 2 - immune-modulation
(protection, longevity in vivo)
virus shell
(biomembrane)
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7. Key Advantages of the MP Technology
(compared to currently used / state-of-the-art technologies)
• Surface modification is non-specific
– No prior information
– No antibodies required
– All entities can be tagged
– No heavy equipment required
• Existing surface molecules NOT affected
– Viral vectors remain infectious
– Viral life cycle not influenced
• No genetic modification required
– Quick, simple and cheap
• Multiple MP molecules can be attached to the same target
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8. Technology Platform – Wide Fields of Application
• Research
– Tagging
– Isolation and purification
– Imaging
– In vivo tracking
VIRUS • Diagnostics (infectious agents, biomarkers)
– Tagging
– Isolation and purification
– Imaging
• Vaccine Development
– Cell targeting
– In vivo vector protection
EXOSOME • Medical
– Immune evasion of transplant material
• Cell and Gene Therapy
– Cell targeting
– Protection from immune system
LIPOSOME
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9. I. Application Examples
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10. Example: Immune evasion
Imagine a delivery vector in the blood system...
…leading to its destruction.
e.g. viral vector in the blood
…it will be attacked by immunity factors…
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11. Example: Immune evasion and cell targeting
Protected and targeted by MP…
…with a cell targeting MP reagent…
…as well as a immune-protective MP reagent…
e.g. antigen presenting
…it will be protected from immune attack… cell
…allowing increased targeted
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12. Example: Cell Targeting and Delivery
B1 – B4
B1 F
Producer cell R
Technology: any membrane
particle can be quickly and
B1 Binding protein easily surface modified for
F Fusogenic protein targeting and delivery in vivo
R Receptor Target cell
Application examples:
a) Antigen presenting cells can be targeted for more effective vaccination
b) Cancer cells can be targeted. The virus can be carrying a suicide gene
c) B2 for example could be a fluorescent tag for delivery to a biomarker.
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13. II. Technology Details
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14. The Key to MP – the GPI Anchor
o Glycosylphosphatidylinositol
anchor
o Post-translational modification
o Conserved backbone structure
o External face of cell membrane
o GPI signalling sequence
o Recombinant anchoring
o Releasing enzymes
o Hypermobility
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15. Engineering Methods for Viral Vectors
Metzner C et al., Virology 2008
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16. Detecting Virus Surface Modification
Metzner C et al., Virology 2008
Viral Particles
Purification Analysis
CD59his
Painting
RV c
CD59 kD
170
130
MLV CA 95
- + - + - + 72
CD59his 5%
ME PC VP 56
43
34
LV CD59 26
17
HIV p24
100 %
10 %
Before
After
CD59his 10% - + - + - + Marker
ME PC VP
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17. Nanoparticles for Linking to MP Reagents
Ni
Iron, phospholipid micelle
Ni-NTA coated MNPs
Nickel based MNP Type 1 Nickel based MNP Type 2
Transmission electron microscopy 50 nm 5-100 nm
5-10 nm
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18. Binding and Purification by Nanoparticles
varying conditions
M
70 kD GFP-GPIhis
55 kD
40 kD
35 kD
25 kD
CD59his M M
15 kD
10 kD
a-CD59
Type 2 MNP
a-GFP
Fe/Ni-NTA MNP
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19. Recent Publications
• Metzner, C., Mostegl, M.M., Günzburg, W.H., Salmons, B. and Dangerfield, J.A. (2008) Association of
glycosylphosphatidylinositol-anchored protein with retroviral particles. FASEB Journal 22(8): 2734-2739.
• Metzner, C., Salmons, B., Günzburg, W.H. and Dangerfield, J.A. (2008) Rafts, anchors and viruses – A role for
glycosylphosphatidylinositol-anchored proteins in the modification of viruses and viral vectors. Virology 382(2): 125-31.
• Dangerfield, J.A. and Metzner, C. Main and co-editor for the E-Book “GPI Membrane Anchors - The Much Needed
link” (2010) Bentham Science Publishers. eISBN: 978-1-60805-123-6.
• Metzner, C., Legler, D.F. and Dangerfield, J.A. (2010) Chapter 6: Surface engineering of biomembranes with GPI-
anchored proteins and its applications, p. 83-97. In J. A. Dangerfield, Metzner C. (ed.), GPI Membrane Anchors - The
Much Needed Link, vol. 1. Bentham Science Publishers. E-Book, eISBN: 978-1-60805-123-6.
• Metzner, C. and Dangerfield, J.A. (2011) Chapter 3: Surface modification of retroviral vectors for gene therapy, p. 41-
72, in K. Xu (ed.), Viral Gene Therapy, vol. 1. InTech Open Access Publishers. E-Book, eISBN: 978-953-307-539-6.
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20. Summary of Proven Features for MP
• Tag and isolate from dilute samples
• Link to nano- and micro-particles
• Deliver genetic material
and/or
• Deliver a functional protein load
• Protect from active serum
• Possible with any protein (tested so far)
• Proteins are always functional (tested so far)
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21. III. Products and Contact Details
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22. Products
please see www.anovasia.com for more details
• Off-the-shelf MP Reagents
Ano-P001: Green-Glow*
Ano-P002: Immuno-Sheath*
Ano-P003: Targo-Tag*
• Made-to-Order MP Reagents
Based on customer requirements
• MP Reagents in Development
AnoR&D4: Immuno-Stim
AnoR&D5: Strepto-Multilink
AnoR&D6: Tomato-Tag
*contains linker(s)
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23. Contact
John A. Dangerfield, PhD
Managing Director
Anovasia Pte Ltd
26 Kandahar Street
Singapore 198888
Reg. No. 201101692G
Tel: +65 9339 4927
Fax: +65 6774 5569
john@anovasia.com
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