What is a Spiegelmer?

Therapeutisch wirksame Spiegelmere
Bionnale
6. Mai 2008
Berlin
Dr. Axel Vater, Senior Scientist, Corporate Development
NOXXON Discovers and Develops NonNon-Immunogenic
SpiegelmerSpiegelmer-Based Therapeutics
that can Block Strong ProteinProtein-ProteinProtein-Interactions.
Interactions.
locations:
locations:
Berlin, Germany
Halle a.d. Saale, Germany
Cambridge, MA, USA (NOXXON Inc.)
employees:
employees: 36
2
1
Investors
TVM Capital
Sofinnova Partners
DEWB
Edmond de Rothschild Investment Partners
IBG Beteiligungsgesellschaft SachsenSachsen-Anhalt
Pfizer
Seventure Partners
The Dow Chemical Company
VC Fonds Berlin
Chairman, Board of Directors:
Directors: Dr. Walter Wenninger
What is a Spiegelmer?
Spiegelmers are mirrormirror-image oligonucleotides that
bind and efficiently inhibit target molecules in a
manner conceptionally similar to antibodies.
antibodies.
They are tailortailor-made therapeutic products which
combine the benefits of small molecule drugs and
biopharmaceuticals.
biopharmaceuticals.
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2
Dominant IP Position
> 200 issued patents and
> 100 patent applications
exclusive license for all SELEX processes
in discovery, production and use of Spiegelmers
worldwide exclusivity for mirroring aptamers
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Spiegelmer
Technology
3
Spiegelmer Technology
Combines
Evolutionary Screening Techniques
Selection of RNA structures from libraries of 1015 different compounds
With
Chiral (Mirror) Principles
Use of mirror-image nucleotides that renders RNA fully biostable
7
Combinatorial Oligonucleotide Libraries
A, G, C, U
41 = 4
4
2
Pool of randomized RNA or DNA
= 16
4 3 = 64
4 4 = 256
4 5 = 1,024
4 6 = 4,096
4 7 = 16,384
1015 different sequences
1015 different shapes
Selection of Aptamers
4 25 = 1,125,899,906,842,624
5'-(N)25-3', e.g. 5'-CGCCAGUAAGUAAGCCUCUGUUAAG-3'
8
4
natural target
mirror-image target
SPIEGELMER TECHNOLOGY
selection
RNA library
(1015 compounds)
amplification
Spiegelmer (L-RNA)
binding to the natural target
aptamer (D-RNA)
binding to the mirror-image target
9
Chirality of RNA
Nucleobase
4'
3'
D-RNA
1'
2'
Nucleobase
biologically
unstable!
10
5
Chirality of RNA in the mirror
100 %
biostable!
4'
3'
D-RNA
1'
2'
4'
1'
2'
3'
L-RNA
(Spiegelmer)
biologically
unstable!
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Spiegelmer
Properties
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Spiegelmers are Specific Inhibitors
H3C-(CH2)6-C=O
O
S F L S P
E HQ R V Q QR
G S
K
E
S
K K
P P A
K
L Q P
R
NOX-B11 has the same requirements for binding
to ghrelin as the ghrelin receptor GHS-R!
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Spiegelmers are Biostable
an aptamer, composed of unmodified all-D-nucleotides, and a Spiegelmer,
composed of all-L-nucleotides, were incubated in human serum at 37 °C
and analyzed after the indicated time points; explorative approach
all-D-RNA
all-L-RNA
(aptamer)
L
0 12 24 36 48 60
[seconds]
(Spiegelmer)
0
12
24 36 48 60
[hours]
100
80
60
40
20
14
7
Spiegelmers are Physicochemically Stable
storage of NOX-E36 (1 mM) at 37 °C in water or HGS b uffer for three months;
HGS: isotonic, histidine glycine sucrose buffered parenteral injection formulation;
analysis by ion exchange chromatography
3 months, 37 °C
in water
Control
Absorption [260 nm]
0.4
3 months, 37 °C
in HGS buffer
0.4
0.4
12.4
12.4
12.4
0.3
0.3
0.3
0.2
0.2
0.2
0.1
0.1
0.1
100 %
100 %
100 %
0.0
0.0
0
5
10
15
20
25
0.0
0
5
10
15
20
25
0
Time [min]
Time [min]
5
10
15
20
25
Time [min]
NOX-E36 displays high physicochemical stability in solution
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Spiegelmers are Physicochemically Stable
Inhibition of chemotaxis [% of control]
storage of NOX-E36 (1 mM) at 37 °C in water or HGS b uffer for three months;
HGS: isotonic, histidine glycine sucrose buffered parenteral injection formulation;
functional analysis using a (cellular) chemotaxis assay
100
data after 3 months storage
90
80
70
60
50
40
30
20
10
0
control
Water, 37 °C
HGS, 37 °C
NOX-E36 is fully functional after long-term storage in solution
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8
Spiegelmers are Non-Immunogenic
Serum Dilution [1000-fold]
Compound:
Model:
Readout:
Spiegelmer NOX-B11(3), 1 mg/kg s.c.
Rabbit (3 groups, n = 5)
Antigen-specific IgG; ELISA
Spiegelmer
(ELISA, Spiegelmer)
250
Spiegelmer + Adjuvant
200
(ELISA, Spiegelmer)
150
Spiegelmer + mBSA
+ Adjuvant
100
(ELISA, Spiegelmer)
Control + mBSA
+ Adjuvant
50
(ELISA, mBSA)
0
0
7
14
28
35
65
95
Time [day]
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Spiegelmers Do Not Trigger TLR-8
human peripheral blood mononuclear cells (PBMC) were treated with 1 µg/ml
Resiquimod (R-848) or D- and L-RNA oligonucleotides (10 µg/ml) and complexed with
DOTAP for 24 h. TNF was determined by ELISA. (thio = phosphorothioate)
thio D-RNA 40
thio D-RNA 41
thio D-RNA 42
liposomes
Resiquimod
thio L-RNA 40
thio L-RNA 41
thio L-RNA 42
L-RNA 40
L-RNA 41
L-RNA 42
0
0.5
1
1.5
2
2.5
3
TNF α [ng/ml]
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9
Spiegelmers Do Not Trigger TLR-3
NFκB in nuclear extracts [∆A450/mg]
human umbilical vein endothelial cells (HUVEC) expressing TLR3, were stimulated
with Poly(I:C), Resiquimod (R848), or Spiegelmers (25 µg/ml) for 2.5 h; cells were
harvested and nuclear extracts were prepared to determine NFkB activation (ELISA).
160
140
120
100
80
60
40
20
0
PBS
Poly (I:C)
R848
NOX-E36 NOX-A12 contr. SPM
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Characteristics of Spiegelmers
Stability
Affinity
Tolerability
Extremly high, proven in vitro and in vivo
High, Kd in low nM range or better
Good, proven in in vivo models
Toxicity
No critical issue so far
Immunogenicity
No critical issue so far
Pharmacokinetics
Efficacy
Intracellular Delivery
Modifications offer a target focused half life
in vivo, ranging from 1 hour to >> 1 day
Proven in various in vivo models for
different Spiegelmers
With specific formulation methods
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Anti-Inflammatory
Spiegelmer:
MCP-1-Inhibitor NOX-E36
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Pathophysiology of MCP-1
MCP-1 (Monocyte Chemoattractant Protein-1) is an early mediator for the
recruitment of activated macrophages to sites of inflammation.
Rheumatoid arthritis (RA)
Renal disease
Systemic lupus erythematosus (SLE)
Multiple sclerosis (MS)
Atherosclerosis / Restenosis
Obesity
Asthma
Cancer
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NOX-E36, Secondary Structure
20
30
10
40
EG
aP
KD
40
3’
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KD Determination NOX-E36-5'PEG
Surface Plasmon Resonance Experiments (Biacore); NHS/EDC
immobilized hu CCL2/ MCP-1, 37 °C 10 µl/min; no mas s transport limitation
NOX-E36-5`PEG
ka [1/Ms]
kd [1/s]
KA [1/M]
KD [M]
1.38-2.20 x105
1.93 x10-4
0.75-1.14x109
0.88-1.34 x10-9
1000
KD ≈ 0.88 – 1.34 nM
R.U.
750
500
250
baseline
0
0
200
400
600
800
Time [s]
24
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NOX-E36 Chemokine Cross Reactivity
Surface Plasmon Resonance (SPR) Experiments (Biacore)
CC
Differentiates
binding to CCL2
by factor of
CXC
Differentiates
binding to CCL2
by factor of
CXC/ XC/ CX3C
Differentiates
binding to CCL2
by factor of
CCL1 /I-309
---
CXCL1/ GROa
---
CXCL12a/ SDF-1a
---
CCL2/ MCP-1
1*
CXCL2/ GROb
---
CXCL12b/ SDF-1b
---
CCL3/ MIP-1a
5.84
CXCL3/ GROg
---
CCL4/ MIP-1b
> 1000-1500/
unspecific
CXCL4/ PF-4
---
> 10.000/
unspecific
XCL1/ Lymphoactin
---
---
CX3CL1/Fraktalkine
---
CCL5/ RANTES
---
CXCL5/ ENA-78
CCL7/ MCP-3
---
CXCL6/ GCP-2
CCL8/ MCP-2
5.6 – 8.9
CXCL7/ NAP-2
CCL11/ Eotaxin
5.6 – 8.9
CXCL8/ IL-8
CCL13/ MCP-4
---
CXCL9/ MIG
CCL14/ HCC-1
---
CXCL10/ IP-10
---
CXCL11/ I-TAC
> 10.000/
unspecific
10.000/
unspecific
2-3
--280/
unspecific
•dissociation constant KD for NOX-E36 ~0.89 nM
CCL6/ CCL9/ CCL10/ CCL12 not expressed in humans
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NOX-E36 Species Reactivity
Surface Plasmon Resonance (SPR) Experiments (Biacore)
CCL2/ MCP-1
Differentiates
binding to human
CCL2 by factor of
KD [nM]
Human
1
0.89
Monkey
1.01
0.90
Canine
1.35
1.20
Porcine
0.92
0.82
Rabbit
---
---
Mouse
---
---
Rat
---
---
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13
Inhibition of MCP-1 in Cell Culture
chemotaxis assay (human monocytic cell line THP-1);
final MCP-1 concentration 0.5 nM; 3 h cell migration at 37°C + resazurin
% of control @ 0.5 nM huMCP-1
100
80
IC50 ≈ 500 pM
60
40
20
0
0.01
0.1
1
10
100
NOX-E36 [nM]
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Efficacy Study in 'Lupus Mice'
Model:
MRL / Faslpr mice (female), spontaneously developing immune
complex nephritis; animals start to develop first nephritis
symptoms at an age of ≈ 12 weeks.
Treatment:
three s.c. administrations per week (10 mg/kg) for ten weeks
(with murine-specific NOX-E36)
treatment for 10 weeks
birth
first
symptoms
start
treatment
end
treatment
0
12
14
24
age of mice [weeks]
28
14
NOX-E36 Reduces Glomerular Destruction
Renal Histology of NOX-E36 vs. vehicle treated MRL/Faslpr Mice
untreated (vehicle)
treated (NOX-E36)
dilated tubuli
protein casts
scarred
glomeruli
periodic acid Schiff stained paraffin sections
destruction of glomerular architecture
- loss of endothelial cells in glomerular capillaries
- fibroblast proliferation; macrophage infiltration
tubulointerstitial infiltration of inflammatory cells
prevented by NOX-E36 treatment
(0.9 µmol/kg s.c. for 10 weeks 3 times per week)
periglomerular accumulation of inflammatory cells
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O. Kulkarni et al. (2007) J. Am. Soc. Nephrol., Vol. 18, 2350-2358.
Renal Macrophage Infiltration
untreated (vehicle)
macrophage count (Mac2+ cells)
in and around the glomeruli
tubulointerstitial T cell count (CD3+ cells)
treated (NOX-E36)
prevented by mNOX-E36-PEG treatment
(0.9 µmol/kg s.c. for 10 weeks 3 times per week)
Mac2+: 39 % (13.4 8.2)
CD3+: 62 % (20.3 7.7)
O. Kulkarni et al. (2007) J. Am. Soc. Nephrol., Vol. 18(8), 2350-2358.
30
15
Renal Effects of mNOX-E36 Therapy
mNOX-E36-PEG (10 mg/kg)
Glomerular filtration rate
↑↑
Albuminuria [ratio albumin/creatinin]
↓↓
IgG [glomerular score]
Activity index [score] *
~
↓↓
Chronicity index [score]
↓
Glomerular MΦ [Mac2+ cells/glom] *
↓
Interstitial MΦ [Mac2+ cells/hpf] *
T cells
[CD3+
cells/hpf] *
↓↓
↓↓
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O. Kulkarni et al. (2007) J. Am. Soc. Nephrol., Vol. 18(8), 2350-2358.
NOX-E36 reduces lung damage
Lung Histology of NOX-E36 vs. vehicle treated MRL/Faslpr Mice
untreated (vehicle)
O. Kulkarni et al. (2007) J. Am. Soc. Nephrol., Vol. 18(8), 2350-2358.
treated (mNOX-E36)
32
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NOX-E36 Improves Skin and Eye Symptoms
untreated (vehicle)
treated (NOX-E36)
prevented by NOX-E36 treatment
two-sided enophthalmos
and conjunctivitis
(treatment for 10 weeks 3 times per week)
normal appearance: - eyes
loss of fur (alopecia)
- skin
loss of vibrissae
- vibrissae
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O. Kulkarni et al. (2007) J. Am. Soc. Nephrol., Vol. 18, 2350-2358.
Efficacy Study in Diabetic Mice
Model:
Nephropathy in male, unilaterally nephrectomized obese
diabetic (db/db) mice
Treatment:
unilateral nephrectomy at age 6 weeks
treatment with mNOX-E36-3‘PEG from age 16 – 24 weeks
three s.c. administrations per week (14 mg/kg)
Unilateral
nephrectomy
Treatment with
mNOX-E36-PEG
↓↓↓
↓↓↓↓↓↓
↓↓↓
0
1
2
3
4
5
6
Month of age
34
17
Efficacy Study in Diabetic Mice
Glomerulosclerosis score is improved by mNOX-E36
35
V. Ninichuk et al. (2008) Am. J. Pathol., Vol. 172(3).
Efficacy Study in Diabetic Mice
Functional parameter: Glomerular filtration rate
400
GFR (ml/min)
p = 0.001
300
200
100
0
control mNOX-E36
PEG
SPM
V. Ninichuk et al. (2008) Am. J. Pathol., Vol. 172(3).
36
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NOX-E36 for Inflammatory Kidney Diseases
proprietary Spiegelmer developed by NOXXON
method of action: potent (picomolar) inhibitor of
pro-inflammatory chemokine MCP-1
lead indication: Lupus nephritis, with potential for other
inflammatory diseases
proof of concept for Lupus nephritis and Diabetic nephropathy
in relevant animal models
large market potential in Lupus, a major disease with
unmet medical need (≈ 900,000 patients in US)
expected to enter phase I in late 2008
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NOXXON's Development Pipeline
Drug
Discovery
Drug
Candidate
Preclinical
Research
NOXNOX-E36 (MCP(MCP-1) Lupus Nephritis
NOXNOX-A12 (SDF(SDF-1) Diabetic Retinopathy
Preclinical
Development
Clinical
Development
Phase I Q4 / 2008
Phase I 2009
NOXNOX-B11 (ghrelin
(ghrelin)) Obesity
NOXNOX-C89 (CGRP) Migraine
NOXNOX-F37 (vasopressin
(vasopressin)) Heart Failure
pre-clinical PoC shown,
ready for out-licensing
NOXNOX-A50 (HMGA1b, intracellular)
intracellular) Cancer
NOXNOX-D14 (Complement
(Complement inhibitor)
inhibitor) Inflammation
(undisclosed)
undisclosed) Diabetes
Drug Discovery Partnership Pfizer
Drug Discovery Partnership Roche
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Thank You!
www.noxxon.net
Dr. Axel Vater
Senior Scientist
Corporate Development
Tel.: +49 - (0)30 - 726247 - 100
Fax: +49 - (0)30 - 726247 - 225
[email protected]
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