AltermuneKary B. Mullis

The α-gal epitope

• α-gal is NOT produced in humans, chimps, orangutan or gt -/- mouse

• A strong anti-α-gal response is already ubiquitous in all humans

The anti-Gal response

0.1uMIgG2

Altermune Linker

alpha-gal epitopeAptamer willbind to M2e

The Altermune Linker

Spiegelmer StrategyEulberg and Klussmann

ChemBioChem 2003

Altermune linker attaches to pathogen

Altermune linker attracts alpha-Gal antibodies

M2target of amantadine

M2e MSLLTEVET: The Achilles Heel of

influenzais bicistronic

A human to human influenza virion detected

by Altermune

natural killer cell attacks infected cell

Total DNA Linker

Today

Model system with Ab to Phenylarsonateand Haemophilus influenza bacteria in rats.

gt-/- mice

Building linker to hemagglutinin peptide from H3N2

Made M2e peptides

0

25

50

75

100%

Pro

tec

ted

200 100 30 10 3

Fab-Ars Dose

Protection from Hib Bacteremia by Fab-Ars Linker

% Protected: percentage of rats in each treatmentgroup that had sterile blood cultures, i.e. <20 HibCFU/ml.

Protection of Neonatal Rats From Hib Bacteremia by Treatment with Anti-Arsonate Antibodiesand [Fab Anti-HibPS-Arsonate] Linker* Group Anti-Ars Fab41-Ars Hib CFU/ml of Blood† (mg) (g) 1 - - >1 x106, 5.0 x 105

2 0.1 - 5.6 x 105, 5 x 105, 2.7 x 105, 1.5 x 105, 1.0 x 105, 6.9 x 104

3 - 200 4.0 x 105, 7.0 x 104, 6.0 x 104

4 - 100 >1 x 106, 2.5 x 105, 1.7 x 105, 1.0 x 105

5 - 30 1.0 x 105, 1.0 x 105, 4.0 x 104, 2.0 x 104

6 - 10 1.6 x 105, 9.0 x 104, 6.0 x 104, 1.0 x 104

7 - 3 >1 x 106, 2.6 x 105, 9.0 x 104, 3.0 x 104

8 - 1 >1 x 106, >1 x106, 6.4 x 105, 1.7 x 105

9 - 0.3 >1 x 106, >1 x 106, 7.0 x 104, 4.0 x 104

10 - 0.1 >1 x 106, 4.1 x 105, 1.8 x 105, 1.5 x 105

11 0.1 200 <20, <20, <20, <20, <20, <20, <20, <20, <20, <20, <20

12 0.1 100 1.4 x 103, <20, <20, <20, <20, <20, <20, <20, <20, <20, <20

13 0.1 30 1.2 x 105, 1.2 x 104, 2.5 x 103, 2.2 x 103, 2.2 x 103, <20, <20, <20, <20, <20, <20,

14 0.1 10 1.1 x 105, 2.8 x 104, 2.7 x 104, 1.3 x 104, 9.8 x 103, 6.2 x 103, 4.9 x 103, 2.4 x 103, 2.4 x 103, <20, <20

15 0.1 3 >1 x 106, 6.4 x 105, 1.8 x 105, 1.1 x 105, 5.1 x 104, 2.5 x 104, 1.7 x 104, 4.6 x 103, 4.6 x 103, 4.3 x 103, 2.3 x 103

16 0.1 1 >1 x 106, 2.0 x 105, 1.9 x 105, 1.3 x 105, 2.8 x 104, 2.5 x 104, 2.4 x 104, 8.7 x 103, 7.5 x 103, 1.9 x 103

17 0.1 0.3 >1 x 106, 6.2 x 105, 5.4 x 105, 4.5 x 105, 3.0 x 105, 7.5 x 104, 6.3 x 104, 5.2 x 104, 2.1 x 104, 1.3 x 104, 1.6 x 103

18 0.1 0.1 >1 x 106, 4.0 x 105, 3.2 x 105, 2.1 x 105, 2.1 x 105, 2.0 x 105, 2.0 x 105, 1.4 x 105, 1.1 x 105, 1.0 x 105, 1.4 x 104

O

O

O

OHOH

HOOH OH

OH

OO

OH

HO

NHAc

OH O NH

O

POO

OHO

AATTGAATAAGCTGGTATGTTGGTCACATAACGCCAACGCCAAAACTCTGAGTCGGGAAATCACTCCCAATTA

OO

N3 +

CuSO4, sodium ascorbate

10

O

OO

HO HO

OHHOHO

HO

O O

HO

OH

AcHN

HOO

O

POO

OHO

AATTGAATAAGCTGGTATGTTGGTCACATAACGCCAACGCCAAAACTCTGAGTCGGGAAATCACTCCCAATTA

OO

N

NN

13

14

Aptamer binds to 161 aa peptide from H3N2 hemagglutinin

Ruth Arnon, JBC 279, 2004

Appendix A Reduction of infectivity in vitro of influenza virus on chicken cells by Altermune linker (apt-gal).

1.00E+07

1.10E+08

2.10E+08

3.10E+08

4.10E+08

5.10E+08

6.10E+08

7.10E+08

8.10E+08

TC

ID50

Mean virus titers 4.63E+08 1.27E+08 9.67E+07 6.13E+07 5.73E+07

cntrl 1uM apt 0.2 uM apt 1 uM apt-gal 0.2 uM apt-gal

Appendix B

Degradation of Lethal Factor Aptamer Cocktail or Protective Antigen Cocktail, Unmodified (UN) versus Modified (PEG or

Phosphorothioate) Under Varying Conditions

CTRL 5 min. 15 min. 30 min.

UN PEG UN PEG UN PEG UN PEG

Nuclease

CTRL 1h 2h 4h 6h

UN PEG UN PEG UN PEG UN PEG UN PEG

70% FBS 70% FBS

CTRL 2h 4h 6h 24h

UN Th UN Th UN Th UN Th UN Th

UN Th UN Th UN Th UN Th UN Th

CTRL 1h 4h 6h 24h

Nuclease

UN=UnmodifiedPEG=PEG-modifiedTh=Phosphorothioate modified

Survival Curve of A/J Mice Immunized with Human Serum, Challenged

with BAS and Treated with α-gal PAA-12 Aptamer and Doxycycline

•A/J mice were immunized intraperitoneally with either 1X PBS or 1% human serum 1X per week for 5 weeks.

•Mice were challenged intranasally with either 1X PBS or 1X106 BAS (Sterne strain).

•Mice were treated with either PBS (with or without human serum) or PAA-12 aptamer drug ( 75 ug /dose with human serum) at 2 hours following challenge and every 24 hours thereafter for 10 days and either PBS or doxycycline (1.5 mg/kg – mouse dose) at 12 or 24 hours following challenge and for every 24 hours thereafter for 14 days:

•Survival was monitored daily for 28 days.

The Future:Priming the immune system

for specific diversion by Altermune

A set of immunogens is chosen for:

•Convenient synthesis

•Safety

•The distinct spectrum of immune responses provoked

Various immunogens with their resultant immune responses

M13 Filamentous Phage

Collaborators• Dr. Alex Lucas, Childrens’ Hospital of Oakland

Research Institute, Oakland, CA

• Dr. Rick Lyons, University of New Mexico, Albuquerque

• Dr. Ron Cook, Biosearch, Novato, CA

• Dr. Carol Cardona, UC Davis, CA

• Dr. Jeeva Vivikananda, Dr. Jonathan Kiel, Brooks Air Force Base, San Antonio, TX

supported by DARPA