complement c5 inhibition blocks the cytokine storm and ... · lp cp ap c3b c3 c3a c3 i convertase...
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1Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK;2Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK;3Department of Pediatrics, Oklahoma University Health Sciences Center, Oklahoma City, OK; 4Ra Pharmaceuticals, Cambridge, MA.
Complement C5 Inhibition Blocks the Cytokine Storm and Consumptive Coagulopathy By Decreasing Lipopolysaccharide
(LPS) Release in E. coli Sepsis
Ravi S. Keshari1, Robert Silasi-Mansat1, Narcis I. Popescu2, Marybeth Langer2, Hala Chaaban3, Cristina Lupu1, Mark K. Coggeshall2, Steven DeMarco4 and Florea Lupu1
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outer membrane
lipopolysaccharide (LPS)
periplasmicspace
cytoplasmic membrane
GRAM-NEGATIVE
peptidoglycan
E. coli
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LP
CP
AP
C3b
C3
C3a
C3 convertaseE.
co
li
LPS release
bacteriolysis
CD14-TLR4
NFk-B signaling
Inflammation Microvascularthrombosis/DIC
opsonization
phagocytosis
C5
C5a
C5b
C5 convertase
C5b9TCC
cytolytic pore
C6-9lysis of host’s cells/tissues
C5 inhibitorC3 inhibitor
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Experimental Approaches
• In vitro: C5 inhibition in whole blood models of Gram negative sepsis (live E. coli or LPS)
• In vivo: C5 inhibition in a baboon model of E. coli sepsis
RA101295 C5 inhibitor: macrocyclic peptide; binds C5 with nanomolaraffinity, and blocks C5 cleavage into C5a and C5b
(Poster #939, Saturday, December 5, 2015, 5:30 PM-7:30 PM.
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Con
trol
RA10
1295
-9
E. c
oli
RA10
1295
-9+ E
. coli
LPS
RA10
1295
-9+
LPS
5000
10000
15000
20000
C5
a (
ng
/ml)
*****
## ##
In vitro: C5 inhibitor (RA101295) reduced the C5b-9
and C5a formation in whole blood sepsis model
Contr
ol
RA
101295-9
E. coli
RA
101295-9
+ E
. coli
LP
S
RA
101295-9
+ L
PS
0
1 0
2 0
3 0
4 0
C5
b-9
(C
AU
/ml)
***
# # #
HEK-TLR4 reporter cell assay for LPS
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E. c
oli
RA10
1295
-9+
E. c
oli
Com
pstat
in+E
. coli
E. c
oli
RA10
1295
-9+
E. c
oli
Com
pstat
in+E
. coli
60
80
100
120
% O
xid
ative
bu
rst
***
******
Neutrophils Monocytes
***
In vitro: C5 inhibitor reduced the oxidative burst
but not bacterial phagocytosis in whole blood
sepsis model
DHR123 oxidation test
% P
ha
go
cyto
sis
E. c
oli
RA10
1295
+ E. c
oli
Com
pstat
in+E
. coli
E. c
oli
RA10
1295
+ E. c
oli
Com
pstat
in+E
. coli
0
50
100
150
Neutrophils Monocytes
NS***
NS***
In vitro phagocytosis test (Phagotest®)
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T-1hr T0 T+2hrE. coli T+8hr
T+24hr T+36hr
In vivo: C5 inhibition in a baboon model
of E. coli sepsis
T+168hr
Euthanasia
C5 inhibitor: RA101295; 10 mg/kg SC
• Vital signs/physiological parameters were recorded• Blood biomarkers were measured
Tissue analysis
Experimental plan
full C5 inhibition
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C5 inhibitor blocked C5b9 formation without affecting C3b
generation thus allowing bacterial clearance by
phagocytosis
E. coli staining
E. c
oli
E. c
oli
+Ra
C5
-I
PMN
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0 2 4 6 8 24 48 72 96 120 144 1680
200
400
600
800
1000
1200
Time (Hrs)
LP
S (
ng
/ml)
LD100
LD100+ RA101295-9
LPS Lipid A staining on blood smears (2hrs postchallenge)
E. c
oli
E. c
oli
+Ra
C5
-I
0
500
1000
1500
Lip
id A
(In
ten
sity, A
U)
Con
trol
E. coli
E. coli+RA C
5-I
C5 inhibitor treatment decreased bacteriolysis and explosive LPS release
LPS in plasma (HEK-TLR4 reporter cell assay)
Con
trol
E. coli
E. c
oli+
RA10
1295
400
800
1200
Lip
id A
(M
FI A
U)
**** ****
LPS (lipid A) staining in the lung
E. coli E. coli +RA101295
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C5 inhibitor reduced sepsis-induced cytokine storm
and neutrophils activation and release
0 2 4 6 8 10 12 14 16 18 20 22 24 1680
50000
100000
150000
Time (hrs)
IL-6
(p
g/m
l)
LD100
LD100+ RA101295-9
0 2 4 6 8 10 12 14 16 18 20 22 24 1680
4000
8000100000
200000
300000
400000
Time (hrs)
TN
Fa
(p
g/m
l)
LD100
LD100+ RA101295-9
Time (hrs)
IL-8
(p
g/m
l)
0 2 4 6 8 10 12 14 16 18 20 22 240
50000
100000
150000
200000
250000
168
LD100
LD100+ RA101295-9
0 2 4 6 8 10 12 14 16 18 20 22 24 1680
20004000
100000
200000
300000
400000
500000
Time (hrs)
IL-1
RA
(p
g/m
l)
LD100
LD100+ RA101295-9
Time (hrs)
G-C
SF
(p
g/m
l)
0 2 4 6 8 10 12 14 16 18 20 22 240
50000
100000
150000
168
LD100
LD100+ RA101295-9
Time (hrs)
MC
P-1
(p
g/m
l)
0 2 4 6 8 10 12 14 16 18 20 22 240
10000
20000
30000
40000
168
LD100
LD100+ RA101295-9
0 2 4 6 80
50
100
150
200
Time (Hrs)
MP
O (
mU
/ml)
LD100
LD100 +RA101295
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C5 inhibitor protects against consumptive
coagulopathy
0 2 4 6 80
200
400
600
800
1000
Time (Hrs)
PA
I-1
(n
g/m
l)LD100
LD100 +RA101295
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CP
AP
LPC5
C5a
C3b
C3
C3a
C5b
C3 convertase
C5 convertase
opsonization
phagocytosis
E. c
oli
C5b9TCC
lysis pore
bacteriolysis
LPS release
CD14-TLR4
NFk-B signaling
Inflammation Microvascularthrombosis/DIC
C6-9
0 50 100 150 2000
50
100
Time (h)
Pe
rce
nt surv
iva
l
LD100 E. coli (n=36)
LD100 E. coli+RA101295 (n=5)
Significant live saving effect
RA101295
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OMRF: R. Silasi-Mansat, R. Keshari, N.I. Popescu, C. Lupu
Ra Pharma: S. DeMarco, A. Ricardo
OUHSC: H. Chaaban
Funding: NIH NGMS R01; NIH NGMS RC1