1

Regulation of anti-viral B cell responses

Use of virus-like particles for therapeutic vaccination

against addiction and other chronic diseasesQb-based Vaccines

2

The Production Process The

Expression Technology

•

132 aa

coat

protein

of phage

Qβ

recombinantly expressed

in E. coli (i.e. no replicating

phage

RNA, infectious

phage

particles

etc. around)

• Expression host: E. coli RB791 (K-12 derivative)

• Expression: inducible

(Ptac

)

• Optimized

S/D sequence

• Two

stop

codons

in tandem

(no read-through)

• Plasmid-encoded

resistance: kanR

•

Spontaneous

assembly

of Qbeta particles

within

the

E. coli cytoplasmic space

3

The

Production

Process The

Upstream

Process

• Fedbatch

process

• Semi-defined

medium

• 50 and 800 liter

stirred

tank reactor

• Final cell

density: OD 200

• Induction: lactose

• Final CWW: 190 g/l

• Yield: 12 g/l correctly

folded

and assembled

Qb particles

• Process

time: 18 h preculture

+ 21.5 h main

culture

4

Fermentation –

Development

and Scale-Up

The Production Process

42

fermentations

for

preclinical

and clinical

API at 2.4 L scale

2 fermentations

at 800L scale

8

fermentations

for

scale

up and clinical

API at 50 L scale

5

The

Production

Process Purification

of Qb-Particles

Cell

disruption Clarification

Centrifugation/

TFFHigh pressure

homogenization

at 700±100 bar

Capturing

Anion ex-

change

Endotoxin

removal

Hydroxy-

apatite

Polishing

Size

exclusion

Scale: Cytos 8 g Qbeta/ Lonza 500 g Qbeta Final purity: >98%

6

The Production Process API Manufacturing Process –

Scales & Yields

Step Pilot Scale Phase IIb/III Scale

Fermentation(Fermentation volume Biomass 190

g/L)50 L 9.5 kg

Purification(Biomass Qbeta

15 g/kg)1125 g 17 g

Coupling(Qbeta Ag-Qb(API)) 6 g 5 g

Number

of Doses

(per fermentation, 100 µg)

start dimension product yield

1.1 Mio

doses 18 Mio

doses

100 g 80 g

800 L 150 kg

34 kg 500 g

CMO

CMO

Cytos

7

The Production Process Overview

WCB

Bacterial

Cell

Pellet

API

Fermentation

Purification

Coupling

Formulation

Precursors

Purified

Qbeta

Antigen

Biotechnology

Chemistry

GMP ProcessIMP

8

Antigen Coupling

The Production Process

•

All reaction

steps

(derivatization, coupling), buffer

exchanges, and purification

(removal of reaction

products, etc.) performed

in closed

system

(TFF).

Carrier(Qbeta)

Linker(SMPH)

Antigen(peptide)

NN

O

NO

O

O

O O

OCys

Lys

9

Flexibility of the Approach Vast number of pre-clinical Vaccine have been generated

• 1 Hapten-based Vaccine

• >150 Peptide Based Vaccines

• > 20

Protein Based Vaccines

• Epitopes coupled to Qb range from 100 D to 250 kD

CYT002-NicQbA Novel Vaccine for Nicotine Addiction

11

The Burden of Disease

Consequences of the Smoking Epidemic

Smoking is responsible for:

•

90% of all lung cancer cases

•

80% of all heart attacks before 50 years of age

•

70% of all cases of COPD

•

30% of all cases of cancer and cancer deaths

•

30% of all ischemic heart diseases and strokes

Smoking costs on average 10 years of your life

12

Rationale for a Vaccine Product

Antibodies reduce the amount and rate of nicotine entering the brain:• interrupt reinforcing effect (reward)• prevent relapse in case of single slips after a successful quit

attempt

Postulated Mechanism of Action

Blood-brain barrier

13

Antibody Titers in Humans•

Strong Response in all Groups

0

1000

2000

3000

4000

5000

6000

0 7 14 21 28 35 42 49 56 63Days

IgG

titer

(OD

50)

50 μg CYT002-NicQb

100 μg CYT002-NicQb

100 μg CYT002-NicQb +Alum in PBST

100 μg CYT002-NicQb +Alum in NaAcT

EJI 35:2031-2040.

14

Antibody Amounts and Affinities•

In the Range for Effective Therapy

Antibody concentration: 59 nM = 8.9 mg/l

Best-fit valuesBMAXKD3933731.04 95% Confidence IntervalsBMAXKD34595 to 4408018.92 to 43.17

Volunteer 135, d42

0 50 100 150 2000

10000

20000

30000

40000

Nicotine free (nM)

cpm

boun

d

KD

= 31 nM

Dilution 1:50 3-fold dilution series 1:109350

v1v2

v1v2

day 42

day 0

EJI 35:2031-2040.

15

Antibody

Safety

TQD*

n = 229 100 μg CYT002-NicQb + Alum

Placebo (PBS) + Alumn = 112

0 1 2 3 4 5 6Month 9 12

Follow-up

Point Prevalence

Designing the Phase 2 Study

Continuous Abstinence

Randomized, Double blind, Placebo- controlled, Multi-center

Study (n = 341)

Counselling

*TQD: Target quit date

16

In a Phase 2 Study

Reducing Smoking

% S

mok

ers

PlaceboActive

Time (months)

0102030405060708090

100

0 1 2 3 4 5 6

* *

*p<0.05

PlosOne, in press

17

100% Responders After the First Immunization

Rapid Onset of Action

Months

0 1 2 3 4 5 6

1000000

100000

10000

1000

100

10

1

PlosOne, in press

18

Abstinence is Driven by Antibody Titers*

Rationale for Positive Clinical Outcome%

Sm

oker

s

Months

0

20

40

60

80

100

0 1 2 3 4 5 6

placebo

lowmedium

high

p < 0.05(mt. 2-6)

(* per protocol)

PlosOne, in press

19

Continuous Abstinence is Achievable

CYT002-NicQbhigh respondermedium responderlow responder

Placebo

57% 32% 32%

31%

30 / 5317 / 53 17 / 53

25 / 80

p = 0.014

p = 0.004

20

25

30

35

40

45

50

55

60

High Medium Low Placebo

% N

on-s

mok

er

With CYT002-NicQb Treatment*

Non-smoker (%) Persons (* per protocol)

PlosOne, in press

20

Distribution of Antibody Titers …and Model for Three-fold Increase

low

medium

high% subjects: 33%

33%

33%

Histogram

of logAUC

Bin Center

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

13.0

13.5

14.0

14.5

0

10

20

30

num

bers

ubje

cts

low

medium

high8%

14%

79%

Histogram

Titer

x 3

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

13.0

13.5

14.0

14.5

15.0

15.5

16.0

0

10

20

30

PlosOne, in press

21

0

2500

5000

7500

10000

300 µg NicQb(n=10)

100 µg NicQb(n=159, phase II)

100 µg NicQb High(n=53, phase II)

Ant

i-nic

otin

e G

MT

Effect

of dose: 300ug NicQb+Alum i.m.N

N

O

O

O

CYT002-NicQb -

Dose Optimization -

Titers

p = 0.0011

Anti-nicotine

GMT at week

8 after

i.m. dosing

at weeks

0 and 4

Factor 4.2 increase

PlosOne, in press

22

High Antibody Levels are Reached in a few weeks

If You`re in a RushA

nti-n

icot

ine

IgG

resp

onse

0

5000

10000

15000

20000

25000

0 1 2 3 4 5 6 7 8 9 10 11 12 13

weeks

ELIS

A ti

ter

100 μg, weekly

regimen

100 μg, biweekly

regimen

100μg, monthly

regimen

(Ph. II)

Factor

10.2 increasein antibody

titers

p<0.0001

CYT006-AngQbPhase IIa

Results

24

ACE inhibitors

(Renin

inhibitors)

AIIR blocker

Modulate the Action of Angiotensin

IIHypertension Vaccine

25

HypertensionRemaining Problems

Sources: NHS, (2004); AJH 17: 347-335 (2004)

•

Compliance: An estimated 50-80% do not take all of the prescribed medication

•

Morning pressor

surge: Pharmacokinetic profile of current inhibitors of the RAS may limit efficacy in early morning hours

Vaccine

targeting

angiotensin

II may

address

these

two

issues, due

to long-lived

antibody

responses

26

Vaccine Design

CYT006-AngQb

CYT006-AngQbQbetavirus-like

particle

CGGDRVYIHPF

30 nm

Angiotensin

II

27

Study Outline (2)Two Dose Levels vs. Placebo

N=24 100μg AngQb

placeboN=12

0 1 2 3 4

safety

follow

up

12 months

N=24 300μg AngQb

placeboN=12safety

follow

up24h ambulatoryblood

pressuremeasurement

Injection

The Lancet, 371 (2008) 821-827

The Lancet, 371 (2008) 821-827

28

Antibody Responses

Results (4)

*range

from

10 patient

samples

0

4000

8000

12000

0 4 8 12 16 20 24 28 32 36 40 44 48

weeks

ELIS

A ti

ter

affinity* = 1-5 nMt1/2

= 123 days

300 μg AngQb

100 μg AngQb

Placebo

p<0.01

The Lancet, 371 (2008) 821-827

29

Mean Change of ABP: 300 μg vs. Placebo

Results (3)

Day-time

Blood Pressure

-9.0 / -4.0 mm Hg p=0.015 / p=0.064

at 8am -25 / -13 mm Hg p<0.0001 / p=0.0035

The Lancet, 371 (2008) 821-827

30

Results (6)

placebo

(SBP/DBP), n=12300μg AngQb

(SBP/DBP), n=21

708090

100110120130140150160170

09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 00 01 02 03 04 05 06 07 08

Time (24-hour)

Am

bula

tory

bloo

dpr

essu

re(m

mH

g)

run-

in

The Lancet, 371 (2008) 821-827

31

Conclusions

• Treatment with AngQb

was safe and very well tolerated

•

Treatment with a 300μg dose of AngQb

led to a statistically

significant reduction of the day-time ambulatory blood pressure which was in the range of classical drugs

•

A pronounced blood pressure reduction was observed in early morning hours, when most cardiovascular events occur

•

Next

studies

are

in preparation

where

state-of-the-art

formulations

and treatment

regimens

will be

applied

to explore

the

full

potential of this

promising

vaccine

candidate

Of the Study

32

Study

02 vs. Study

01

3002412

3004623

0 4 12

0 4 102 6

10 nM 1 nMweek

week? nM

33

0

10'000

20'000

30'000

40'000

0 2 4 6 8 10 12 14 16 18

weeks

ELIS

A ti

ter

Study 02: 300 ug AngQb responder

Study 02: non-responder

Study 01: 100 ug AngQb

Study 01: 300 ug AngQb

Study

02 vs. Study

01Antibody titers

34

Results From Second StudyChange of Daytime

ABPM vs. Placebo

-14

-12

-10

-8

-6

-4

-2

0

2week

14 week

4 week

8 week

12 week

14

Study

01 Study

02

Del

ta m

mH

g

systolic diastolic

35

The Components

CYT003-QbG10

Virus-like Particle Qbeta

5‘

GGG GGG

GGG

GGA CGA TCG TCG

GGG GGG

GGG

3‘

Oligonucleotide

G10

CpG-loaded VLPs

36

The Manufacturing Process (1) Purification of Qbeta

Particles

Cell disruption/

Clarification

Capturing Purification Qb

PrecipitationPolyethyleneimine

(NH4

)2

SO4

Gel filtrationSepharose

CL4B

37

Qb

Structure Virus-Like Particle

Built up from 90 dimers, located on the

edges of 20 hexagons and 12 pentagons

“abgestumpfter

Ikosaeder”

Qβ

coat protein monomers

Qβ

coat protein dimer

38

The Manufacturing Process (2) Purification of Qbeta

Dimers

Disassembly Qb

DimersPurification

DTTMgCl2

CIXSP-Sepharose

FFGel filtration

Sepharose

CL4B

39

The Manufacturing Process (3) Packaging/Reassembly

Qb

Dimers

G10

1. Reassembly

2. Oxidation (H2

O2

)

TFF

Purification

TFF

QbG10

40

61.0x10

71.0x10

81.0x10

91.0x10

101.0x10

10 20 30 40

Mol

ar M

ass

(g/m

ol)

Time (min)

Molar Mass vs. Time 5507 Cytos IS0

VLP IntegritySubunits Reassemble Correctly into the VLP

Diameter (Qbeta VLP) = 30 nm

Electron

MicroscopyAF4-MALS (Asymmetric

Flow

-

Field

Flow

Fractionation-

Multi Angle Light Scattering)

Size-exclusion

HPLCRel. peak

area:Release: 99.0%2.5 years, -80°C

98.8%6 months, +5°C

96.1%

Dynamic

Light Scattering:

Hydrodynamic

Radius: 13.5 nm

3.4-3.5 MDa, Qbeta 3.5-3.6 MDa, CYT002-

NicQb

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0-50

0

100

200

300

350

1 - SS3_051011_HPLCsecprot_051007A-01 051007C-01 #5 [modified by ss UV_VIS_12 - DW1_050919_HPLCSECPROT_050916C01_050916B01 #5 [modified by wdbegovic, 4 peaks manually assigned, normamAU

min

21 1 -

6.75

5

2 -

8.62

7

3 -

11.0

57

4 -

13.4

90

5 -

15.2

38

6 -

16.7

93

WVL:260 nm

UV260nm

41

CYT003-QbG10 Analytics/Characterization

Appearance

Slightly opalescent, colourless solution

pH

pH 7.2

Conductivity

15.9 mS/cm

Oligonucleotide

content

20-27 μg/100 μg protein

Peptide map

10 main signals (identified by MS)

SE-HPLC

Relative peak area drug substance 99%

Host cell DNA

< 5 ng

DNA/100 μg protein

Host cell protein

1-5 ng

HCP/100 μg protein

Bacterial endotoxins

≤

2.5 EU/100 μg protein

Total viable aerobic count

0 CFU/ml

42

Study Outline (1) A Placebo-controlled

Phase IIb

Study

•

Randomized, double-blind, placebo- controlled, multicentre, phase

IIb

study

in 35 centers

in six

EU countries

Design:

Treatment:

•

Allergic

rhinoconjunctivitis

due

to sensitization

against

house

dust

mites

Indication:

•

299 patients were randomized into 2 different dose groups (0.5 mg and 1 mg) and placebo

Patients:

•

6 weekly injections with efficacy assessment during week 7 and 8 and follow-up for 12 months

43

Results (1) Combined

Symptom and Medication

Score*

Assessed

Symptoms:

Blocked

nose, runny

nose, sneezing

attacks, itchy

nose, reddened

eyes, itchy

eyes, tearing, (absent, mild, moderate, severe: 0 –

3 points)

Rescue

medication:

Step

1: Local

antihistamines

(Azelastine)

Step

2: Oral antihistamines

(Cetrizine)Step

3: Local

corticosteroids

(Mometason)

(ATSS + ARMS)ACS =

2

ACS: Average

Combined

ScoreATSS: Average

Total Symptom ScoreARMS: Average

Rescue

Medication

Score

* Allergy

(2007) 62, 1023-1028

44

Results (2) Combined

Symptom and Medication

Score

median is

shown PlaceboN=95

0.5 mgN=93

1 mgN=94

ACS 0.515 0.454 0.312

Δ to placebo -12% -39%

p vs. placebo n.s. p=0.035

45

Qb Vaccines in Clinical Development at Cytos

Indications

& Targets

Project Indication Antigen

CYT001-DerQb Technology test der p1 peptide

(20 aa)CYT002-NicQb Smoking cessation nicotineCYT006-AngQb Hypertension angiotensin

II peptide

(8 aa)

CYT007-TNFQb Psoriasis TNFα

peptide

(20 aa)

CYT009-GhrQb Obesity ghrelin peptide

(8 aa)

CYT013-IL1bQb Diabetes IL-1β

protein

(167 aa)

CYT003-QbG10CYT005-AllQbG10

Allergy

/ Asthma - - - -

CYT004-MelQbG10 Melanoma melan

A peptide

(21 aa)

46

Clinical Experience with Qb Vaccines at Cytos

Flow

of Phase I / Phase II Projects

2002 2003 2004 2005 2006 2007PoC

Smoking

Allergy

Asthma

Hypertension

Psoriasis

Obesity

Ph

I

Ph

I Ph

II PoC Dose Regimen

Ph

I

Ph

I Ph

IIa

Ph

I/IIa

Ph

I/IIa

Ph

IIa Ph

IIa

Foreign

antigens

PoC

Melanoma Ph

I/IIa

2004 2005 2006 2007 2008 2009

Diabetes Ph

I/IIa

Ph

II

Ph

IIa

Peptide Self

Antigens Protein Self

Antigens

Ph

IIbPh

II Ph

IIb

der p1

nicotine

- - - -

ang

II

TNFα

ghrelin

melan-A

IL1b

Phase II

±

allergens

47

Clinical Experience of 6 Years Overview

(January

2009)

# of subjects

treated

with Total Active

drug Responder

rate, AbsReversibility

of Ab resp.

_Qb 898 645 100% 100%

_QbG10 422 277

Total 1320 922

No safety

relevant observations

in 19 completed

clinical

trials