biological response modifiers in immune therapyimhaemo.ru/media/documents/hi_16/25_16.pdf ·...
TRANSCRIPT
Biological response modifiers
in immune therapy: What is the role of Polyoxidonium (PO)?
Jean-François Rossi
M Villalba, P Plence, VO Dang-Nghiem, C Alexis
Montpellier FRANCE
N Tupitsyn, FA Shamilov, OA Beznas, IK Vorotnikov
Moscow RUSSIA
Y Gurkovskaya
Moscow RUSSIA
Immune Precision Therapy : The right therapy delivered to the right patient at the right time
2
PROTAGONIST EVALUATION
TARGET (TUMOR)
Mutanome
Metabolic status
Accessibility
MICRO-ENVIRONMENT
Cells
Communication molecules
- Secreted
- Cell surface
Metabolic status
Vascularization
IMMUNE STATUS
Immunome
Immune senescence
Immunogenetic failure
- exhaustion (infections)
- chemotherapy
Targeted therapy impact
BIOLOGICAL CROSSROAD DETERMINATION Microbiota impact
Metabolism
Inflammation
CLINICAL CROSSROADS : THE RIGHT CHOICE Synergism/Antagonism
Prediction
Modelization
Rossi JF et al. Cancer Comm 2019
3
Rossi JF et al. Cancer Comm 2019
Time-windows of opportunity in immune therapy for cancers
4 4 4
Time
PR
CR
MRD Cytokines
Tumor mass
100
Tum
or
mas
s (%
)
Cyt
oki
ne
s co
nce
ntr
atio
n
Chemotherapy
0
100
0 10
“Time-Window” for administration of IEC as:
NK, gdT
“Time-Window” for administration
of CAR-T
Repetitive administration of IEC
for MRD control
Vaccination and blocking immune
checkpoints should be arranged
away from intensive chemotherapy
Immune adjuvants
Rossi JF et al. Cancer Comm 2019
Polyoxydonium (PO): Azoximer Bromide
5
• Copolymer of 1,4-ethylenepiperazine-N-oxide- and
(N-carboxymethylene)-1,4-ethylenepiperazinium bromide
• Water-soluble cationic biodegradable polymer
• Molecular weight: 60-100 kDa
• Synthesized by a partial oxidation of the parent polymer with hydrogen peroxide to introduce
N-oxide groups followed by the quaternization of the nonoxidized 1,4-ethylenepiperazin with
bromoacetic acid
The introduction of N-oxide groups: to minimize toxicity dramatically
The copolymer chains are cleaved to shorter fragments, in vivo, excreted by kidneys
5
x = 0.03 – 0.20, y = 0.60 - 0.80, n ≈ 600
(random polymer)
Immune activity of PO : adjuvant for vaccination
6
• Stimulates inflammatory cytokines : IL6, IL1, TNF-alpha
• Increases activity of PB phagocytes
• Improves bactericidal activity (Staphylococcus Aureus) of leukocytes
• Enhances Ab response to weak AGs and ensures marked immune response to low doses of AG
Enhances immune responses to live brucellosis vaccine Brucella abortus strain 82-PS (penicillin
sensitive) in a guinea pig model
6 6
TARGETS OF
IMMUNE ADJUVANTS
FOR VACCINATION
Clinical usage of PO for adult and children
7
• IMMUNE ADVUVANT FOR VACCINATION
– influenza vaccines Grippol® in which polyoxidonium was mix to antigenic components of the
vaccine-hemagglutinin and neuraminidase (used in more than 50 Million recipients with no toxicity)
– adjuvant for other vaccines (in the pipeline)
• STANDALONE PRODUCT (different dosage)
– unique combination of immune-modulating activity, detoxifying and antioxidant effects
– efficacy and good safety profile when used in complex therapy
– 20+ years of commercial success
• POSTAUTHORIZATION SAFETY STUDY IN SLOVAKIA
Pruzinec P et al. Immunotherapy. 2018 Feb;10(2):131-137)
– 502 subjects were enrolled and 498 (99.2%) subjects completed the study
– 19 (3.8%) subjects experienced a total of 34 adverse events.
– Only one (0.1%) subject experienced eight adverse drug reactions (ADRs): restlessness, fatigue,
feeling hot (n = 2), pyrexia (n = 3) and asthenia. There were no renal ADRs or serious ADRs
– VERY GOOD SAFETY PROFILE
7 7
8
In vivo effect of PO on immune cells in 20 patients :
patients with pre- and post-surgery analysis
• 20 patients with breast cancer
• Received 12mg IM Day 1,2,3,5 and 7 (surgery day 8)
• Clinical characteristics
– Median age: 53.5 years (32-78)
– T1-3 N1-3 M0 - 20 patients
o before and after PO administration
Stage n %
I 1 1.8
IIA 5 66.1
IIB 7 12.5
IIIA 7 19.6
1 patient with pathomorphosis degree 4
Disappearance of tumor cells
Before PO
After PO
IHC with anticytokeratin
x200
x50
EXPLORE PO ACTIVITY IN VITRO : T and NK cells
and DC maturation
9
• Specific aim
– to complete data concerning PO activity on immune cells (DC, T, NK cells).
9 9
PROTOCOL FOR IMMUNE EVALUATION
Buffy coat from one donor (blood bank center)
CD3 selection (StemCell)
T-cells (CD3+) NK-cells( CD3- CD56+)
IN TRIPLICATE
Day 0 CD3/CD28 activation (Dynabeads) Numeration CD3-/CD56+
Day 2-4 Examine culture IL2/IL15 irradiation PLH
Day 5 Count/Plate in 96well <5% 7AAD-/CD19+ NK>90%
Cell viability Cytokines
in CD4/CD8 subpopulations
FCS: CD4,8,25,FoxP3,127,IFN-g,IL17
Later (21 days) FCS : CD25,158a/b,56,71 ,45RA,16,45RO,621,NKG2D,57
IN TRIPLICATE
FCS: BD-LSR Fortessa (Becton Dickinson) and all data were
analysed using FlowJo software (Tree Star, Ashland, OR, USA)
2 experiments
EFFECT OF PO on T-lymphocytes
10
PO increases T cell proliferation. Purified T cells from 3 independent healthy donors were
activated by anti-CD3/CD28 costimulation in triplicate.
The indicated concentrations of PO were added at Day 0.
The number of cells was quantified. The average of the
control, non treated cell, values was standardized to 1 to
decrease interdonors variability. Graphs represent means
± SEM; * p0.05, ** p0.01, *** p0.001 ANOVA test
compare to non treated cells.
Donor 1
Donor 2
Donor 3
11 11
Non activated Activated
Eff. Mem.
Naive
Eff. Mem.
Naive
CD4+
Eff. Mem.
Naive
Non activated Activated
Eff. Mem.
Naive
CD8+
Efficient T cell activation after CD3/CD28 costimulation. Purified T cells were activated by anti-CD3/CD28 costimulation for 5 days.
CD127 and and CD25 expression were analyzed by FACs.
EFFECT OF PO on T-lymphocytes
12 12 12
Donor 1
Donor 2
Donor 3
EFFECT OF PO
on T-lymphocytes
PO does not affect activation
of CD4+ cells. T cells were activated and the % CD25+, CD127+ and
CD45RAdim cells (left) and the MFI of the positive
population or CD45RAdim (right) were analyzed by
FACs in the CD4+ compartment.
13 13 13
*
Donor 1
Donor 2
Donor 3
EFFECT OF PO
on T-lymphocytes
PO does not affect activation
of CD8+ cells. T cells were activated and the % CD25+, CD127+ and
CD45RAdim cells (left) and the MFI of the positive
population or negative (CD45RA) (right) were
analyzed by FAC in the CD8+ compartment.
14 14 14
CD8+
Donor 1
Donor 2
Donor 3
CD4+
EFFECT OF PO
on T-lymphocytes
PO does not affect interferon-g
expression. T cells were activated for 5 days and then treated
for 4 h with PMA (50ng/ml)/Ionomycin (1µg/ml).
The % of IFNg+ cells (left) and the MFI of the
positive population were analyzed by FAC in the
CD4+ (up) or CD8+ (down) compartments.
PO (> 1μg/mL) decreases the number of T regs
15
T-Reg. T-Reg. T-Reg. T-Reg. T-Reg.
0 1 10 100 500 PO
(µg/ml)
0
20
40
60
80
100
0 1 10 100 500
%
CD
4+
/Fo
xP
3+
/C
D1
27
+
PO (µg/ml)
% CD4+/FoxP3+/CD127+
• in vitro chronic PO treatment lacked toxic effect
• increased T cell proliferation
• without negatively affecting any of the activation
markers tested.
16 16 16
EFFECT OF PO on T-lymphocytes:
Summary
EFFECT OF PO on NK cells
17
d0 d2 d5 d8 d11 d14 d17 d19 d21
iPLH
PO
FACS analysis
CD3+-depleted PBMC cells were
incubated at different PO
concentrations and cell number
analyzed at day 7, 14 and 21.
unstim PO 1
PO 1
0
PO 1
00
PO 5
00
PSO 0
.5%
PSO 5
%
0
20
40
60
80
100
% viablility
pe
rce
nta
ge
unstim
PSO 0.5%
PSO 5%
PO 1
PO 10
PO 100
PO 500
unstimPO
1
PO 1
0
PO 1
00
PO 5
00
PSO 0
.5%
PSO 5
%
0
1
2
3
4
1*E
6 c
ell
cell count
Pharmacological activity
Experiment 1
Experiment 2
cell count % viablility
cell count
Control
PO 1 μg/mL
PO 10 μg/mL
PO 100 μg/mL
PO 500 μg/mL
PO (1 to 10 μg/mL) induces expression of CD25, CD69 and CD71
on NK cells, that is correlated to high anti-tumor effect
18 18 18
Krzywinska et al , 2015 #7034; 2016 #7114
CD25 CD71
PO affects NK cell cytotoxicity, dependent of the E:T ratio
19
• Cytotoxicity increase was found in a lower E:T (1:3) ratio
19 19
no targ
et
[1:1
]
[1:3
]0
1
2
3
4
5
Degranulation
control
1
10
100
500
Cytotoxicity and degranulation
[1:1
]
[1:3
]0
10
20
30
40
50
cytotoxicity assay
% ta
rge
t ly
sis
control
1
10
100
500
target: B cell lymphoma
(patient 2)
Control
PO 1 μg/mL
PO 10 μg/mL
PO 100 μg/mL
PO 500 μg/mL
PO did not induce spontaneous degranulation.
However, PO increased degranulation against target cells.
Effect of PO on NK cells : summary
• EXPANSION : no effect on 21 days of culture except at 500 μg/mL
• DEVELOPMENT: no effect (CD62L and CD 57)
• ACTIVATION: no effect (CD64 and CD45RO/RA)
• MATURATION: PO increases CD25, CD71 and KIR expression
• ACTIVITY: no modification of cytotoxicity and degranulation that are preserved
20
Dendritic Cells (DC) maturation
21
Mannose
receptor
v
5
Apoptotic cell
RFc
Soluble Ag
Microorganism or
mannosylated antigen
CCR1
CCR5 CCR7
CD86
CMH I
MO CD14+
Immature DC Ag capture
and processing
Mature DC
Ag presentation
GM-CSF + IL-4 or IL13
5-7 days
TNF- or CD40L+Poly-IC
and/ or agonistic
anti-CD40 Zhou et al
Hybridoma 1999,18:471
or PGE2
2 days
CD83
MHC restricted peptides
CTL Activation in vitro
In vivo injection
Proteins, lysates, RNA,
apoptotic cells
CD86
CD80
CMH II
apheresis
PBMC
Tarte K, Fiol G, Rossi JF, Klein B Leukemia 2000,14:2182
Several concentrations of PO (1 μg/ml, 10μg/ml and 100 μg/ml) were added from Day 0-7.
Day 5, DC maturation was induced adding various concentration of PO (1 μg/ml, 10 μg/ml
and 100 μg/ml) or LPS (50ng/ml) as positive control.
IN TRIPLICATE LPS
PO improves DC maturation (experiment 1)
22
Conditions Addition PO D0-D7
Nb cells (106/mL)
Viability (%)
CD1a+ (%) CD14+ (%)
iDC control 1.6 ±
0.07
93.5 ± 0.9 57.9 ± 1.1 68.8 ±1.9
iDC PO 1μg/mL 1.6 ±
0.19
90.1 ± 0.9 68.0 ± 2.2 51.2 ± 5.5
iDC PO 10μg/mL 1.6 ±
0.07
90.8 ± 1.6 68.9 ± 1.4 50.4 ± 2.8
iDC PO 100μg/mL 1.7 ±
0.02
89.7 ± 1.1 57.4 ± 1.1 56.8 ± 6.2 mDC control 1.4 ±
0.09
89.1 ± 0.1 60.0 ± 0.6 14.5 ± 2.4
mDC PO1μg/mL 1.5 ±
0.16 87.7 ± 1.7 58.2 ± 3.7 13.1 ± 5.1
mDC PO10μg/mL 1.4 ±
0.15 86.6 ± 1.1 50.4 ± 7.4 11.4 ± 1.3
mDC 100μg/mL 1.7 ±
0.07 87.7 ± 5.1 47.0 ± 1.2 10.0 ± 1.2
iDC PO 1μg/mL 1.4 ±
0.18
88.7 ± 1.5 72.9 ± 0.3 42.3 ± 1.7
iDC PO 10μg/mL 1.4 ±
0.22
88.1 ± 0.5 74.6 ± 2.1 30.4 ± 4.6
iDC PO 100μg/mL 1.3 ±
0.04
87.8 ± 1.1 67.2 ± 4.9 38.3 ± 0.1
Ad
ditio
n P
O
at D
6
Expansion of PO-treated DC
23
Addition of PO at Day 0
PO (µg/ml)
mean of Nbr of cell (*106 /ml) T test
iDC 0 1.80 ± 0.07
mDC
1.87 ± 0.08
iDC 1 1.84 ± 0.03
mDC
2.03 ± 0.16
iDC 10 1.68 ± 0.05
mDC
2.05 ± 0.13
iDC 100 1.85 ± 0.14
mDC
2.08 ± 0.12
Addition of PO at day 5
iDC 1 1.29 ± 0.11 0.005
iDC 10 1.34 ± 0.03 0.003
iDC 100 1.25 ± 0.04 0.0003
o Good generation of monocyte-derived DC in the presence of PO
o Addition of PO at day 5 reduces the numbers of iDCs
Viability of the cells
24
Addition of PO at Day 0
PO (µg/ml) % of viable cells T test
iDC 0 90.04 ± 0.83
mDC
89.16 ± 0.56
iDC 1 91.47 ± 0.58
mDC
90.00 ± 0.75
iDC 10 89.80 ± 1.46
mDC
89.67 ± 1.26
iDC 100 90.27 ± 0.90
mDC
89.73 ± 0.44
Addition of PO at day 5
iDC 1 88.67 ± 0.48
iDC 10 88.43 ± 0.78
iDC 100 86.33 ± 1.08 0.014
o Good viability of the PO-treated DC
o Small decrease of viability at 100 µg/ml
Phenotypic analyses of PO-treated DC at Day 7:
% HLA-DR+ CD80high cells
25
iDC
iDC
+ P
O 1
µg
(J5-J
7)
iDC
+ P
O 1
0µg
(J5-J
7)
iDC
+ P
O 1
00µg
(J5-J
7)
iDC
+ P
O 1
µg
(J0-J
7)
iDC
+ P
O 1
0µg
(J0-J
7)
iDC
+ P
O 1
00µg
(J0-J
7)
mD
C (
LP
S)
mD
C+ P
O 1
µg
+ L
PS
mD
C+ P
O 1
0µg
+ L
PS
mD
C+ P
O 1
00µg
+ L
PS
0
2 0
4 0
6 0
8 0
1 0 0
Ce
llu
les
HL
A-D
R+
CD
80
hig
h (
%)
* * * *
p = 0 ,1 2 6 2
p = 0 ,0 8 1 7
p = 0 ,0 8 1 7mDC (LPS) mDC + PO
1µg (LPS)
mDC + PO
10µg (LPS)
mDC + PO
100µg (LPS)
HLA-
DR
CD
80
iDC iDC + PO
1µg (J5-J7) iDC + PO
10µg (J5-J7)
iDC + PO 100µg
(J5-J7)
iDC + PO
1µg (J0-J7)
iDC + PO
10µg (J0-J7)
iDC + PO 100µg
(J0-J7)
HLA-
DR
CD
80
Phenotypic analyses of PO-treated DC at Day 7:
% CD83+ cells
26
iDC iDC + PO
1µg (J5-J7) iDC + PO
10µg (J5-J7)
iDC + PO 100µg
(J5-J7)
iDC + PO
1µg (J0-J7) iDC + PO
10µg (J0-J7)
iDC + PO 100µg
(J0-J7)
HLA-DR
CD
83
mDC (LPS) mDC + PO
1µg (LPS)
mDC + PO
10µg (LPS)
mDC + PO
100µg (LPS)
HLA-
DR
CD
83
iDC
iDC
+ P
O 1
µg
(J
5-J
7)
iDC
+ P
O 1
0µ
g (
J5
-J7
)
iDC
+ P
O 1
00
µg
(J
5-J
7)
iDC
+ P
O 1
µg
(J
0-J
7)
iDC
+ P
O 1
0µ
g (
J0
-J7
)
iDC
+ P
O 1
00
µg
(J
0-J
7)
mD
C (
LP
S)
mD
C+
PO
1µ
g +
LP
S
mD
C+
PO
10
µg
+ L
PS
mD
C+
PO
10
0µ
g +
LP
S
0
5 0
1 0 0
1 5 0
Ce
llu
les
CD
83
(%
)
p = 0 ,1 0 5 5
* *
*
p = 0 ,0 8 0 2
p = 0 ,0 8 7 9
* * * *
One way ANOVA
Tukey’s multiple comparaisons test
Phenotypic analyses of PO-treated DC at Day 7:
% HLA-DR+ CD40high cells
27
iDC iDC + PO
1µg (J5-J7)
iDC + PO
10µg (J5-J7)
iDC + PO 100µg
(J5-J7)
iDC + PO
1µg (J0-J7)
iDC + PO
10µg (J0-J7)
iDC + PO 100µg
(J0-J7)
HLA-
DR
CD
86
mDC (LPS) mDC + PO
1µg (LPS)
mDC + PO
10µg (LPS)
mDC + PO
100µg (LPS)
HLA-
DR
CD
86
iDC
iDC
+ P
O 1
µg
(J5-J
7)
iDC
+ P
O 1
0µg
(J5-J
7)
iDC
+ P
O 1
00µg
(J5-J
7)
iDC
+ P
O 1
µg
(J0-J
7)
iDC
+ P
O 1
0µg
(J0-J
7)
iDC
+ P
O 1
00µg
(J0-J
7)
mD
C (
LP
S)
mD
C+ P
O 1
µg
+ L
PS
mD
C+ P
O 1
0µg
+ L
PS
mD
C+ P
O 1
00µg
+ L
PS
0
5 0
1 0 0
1 5 0
Ce
llu
les
HL
A-D
R+
CD
86
hig
h (
%)
*
* * * *
p = 0 ,0 9 2 7
*
One way ANOVA
Tukey’s multiple comparaisons test
PO improves maturation process of DC
MFI of CD80 and CD86 expression on live cells
28
iDC
mDC + PO 100µg (LPS)
mDC + PO 10µg (LPS)
mDC + PO 1µg
(LPS)
mDC (LPS)
iDC + PO 100µg (J0-J7)
iDC + PO 10µg (J0-J7)
iDC + PO 1µg (J0-J7)
iDC + PO 100µg (J5-J7)
iDC + PO 10µg (J5-J7)
iDC + PO 1µg (J5-J7)
CD80
Experiment 2
iDC
mDC + PO 100µg (LPS)
mDC + PO 1µg
(LPS)
mDC (LPS)
iDC + PO 100µg (J0-J7)
iDC + PO 10µg (J0-J7)
iDC + PO 1µg (J0-J7)
iDC + PO 100µg (J5-J7)
iDC + PO 1µg (J5-J7)
CD83
mDC + PO 10µg (LPS)
Proliferation of allogenic T-cells stimulated with PO-treated DC
29
iDC
1 10 100 PO (µg/ml)
0
mDC
Ratio 1/3
Ratio 1/6
Ratio 1/12
Ratio 1/24
o Good immunogenicity of PO-treated DC
o on allogeneic T cells
At day 7, viable DC were counted using
the MUSE counter and plated with
allogeneic CFSE-labeled T cells to
investigate DCs immunogenicity. A total of
1.5 x105 responding allogeneic PBMC per
well were cultured in 96 round-bottom
microplates with various amount of DC
(ratio of DC/effector T cells from 1/10 to
1/900).
PO Is active
on DC maturation
CONCLUSIONS:
PO is an immune modifier both in vitro and in vivo
30
• PO is a biological response modifier for T, NK and DC with dose response starting at
pharmacological dose (10-100 μg/mL) with no toxicity on immune cells
• NK cells
• PO increases NK cell maturation, decreases proliferation and increases NK cell cytotoxicity
• Probably PO induces NK cell maturation, by stoping proliferation and stimulating effector functions
• T cells
• PO increases T-cell proliferation with no toxicity
• Not affecting activation biomarkers for both CD4 and CD8+ in the absence of targets
• Decreases T-regs
• DC
o No IMPAIR of PO on the differentiation process
o PO impacts the DC maturation process, mainly at 10μg/mL
o PO was shown to increase the expression of co-stimulatory molecules leading to an increase DC
immunogenicity monitored by T cell proliferation.
• In vivo study suggests that PO impacts on CD8+ T-cells in patients with advanced breast cancer
31
Time-windows of opportunity in immune therapy for cancers
31 31 31
Time
PR
CR
MRD Cytokines
Tumor mass
100
Tum
or
mas
s (%
)
Cyt
oki
ne
s co
nce
ntr
atio
n
Chemotherapy
0
100
0 10
“Time-Window” for administration of IEC as:
NK, gdT
“Time-Window” for administration
of CAR-T
Repetitive administration of IEC
for MRD control
Vaccination and blocking immune
checkpoints should be arranged
away from intensive chemotherapy
POLYOXIDONIUM
Personalized vaccination with Neo-Ag:
APAVAC®
32
Apavac® is based on calcium hydroxyapatite (HAC) microparticles capturing TAA (and Neo-Ag) bound to Heat-Shock Proteins
TUMOR BIOPSY HOMOGENATE
MIX WITH APAVAC BEADS
HSP-associated tumor antigens
HSP (heat shock proteins)
TAA (Tumor-Associated Antigens)
10 μM beads
CAPTURE HSP-BOUND
TAA/Neo-Ag
Apavac® HAC beads
INJECT TO PATIENTS
TAA-loaded Dendritic cells
Apavac® technology for 1) Enrichment of TAA (Neo-Ag) 2) Facilitated antigen capture 3) Adjuvant effect
SERUM or
33 33
Company snapshot APAVAC® preclinical validation
Randomized, Placebo-Controlled, Double-Blinded Chemoimmunotherapy Clinical Trial in a Pet Dog Model of Diffuse Large B-cell Lymphoma Laura Marconato, Patrick Frayssinet, Nicole Rouquet, Stefano Comazzi, Vito Ferdinando Leone, Paola Laganga, Federica Rossi, Massimo Vignoli, Lorenzo Pezzoli, Luca Aresu.
Clinical Cancer Research Published 1 February 2014
Clinicaloutcome(TimeToProgressionandLifeSpanShortening)
APAVAC® CONTROL P
n=12 n=7
FirstTTP(mean;d) 332 59 0.0004
FirstTTP(median;d) 304 41 0.0004n=12 n=7
LSS(mean;d) 468 136 0.0001
LSS(median;d) 505 159 0.0018
n=6 n=3SecondTTP(mean;d) 140 35 0.10
SecondTTP(median;d) 127 32 0.0167
RECENT RETROSPECTIVE ANALYSIS
300 petdogs with NHL (148 CTvs152 CT+V)
OS at 1,2,3 years (DLBCL): 30,16,10%vs 55,28,10%
Marconato L et al. J ImmunoTher Cancer accepted
APAVAC® : response
34 34
SUGGESTED STUDIES
Post surgery for metastatic risk
With vaccine
With Cellular Therapy
NK/NK-CAR
Tgd
35
35 35 35 35
Thank you for your attention спасибо за внимание
დიდი მადლობა თქვენი
ყურადღებისთვის
Merci pour votre attention
How do you imagine
a prospective (bio-)clinical study in cancer patients ?