Biological response modifiers in immune therapyimhaemo.ru/media/documents/HI_16/25_16.pdf · Chemotherapy 0 100 0 10 “Time-Window” for administration of IEC as: NK, gdT “Time-Window”

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


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


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


0.07

90.8 ± 1.6 68.9 ± 1.4 50.4 ± 2.8


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


0.18

88.7 ± 1.5 72.9 ± 0.3 42.3 ± 1.7


0.22

88.1 ± 0.5 74.6 ± 2.1 30.4 ± 4.6


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


% CD83+ cells

26

iDC iDC + PO


10µg (J5-J7)

iDC + PO 100µg

(J5-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


% 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 1µg

(LPS)

mDC (LPS)

iDC + PO 100µg (J0-J7)






CD80

Experiment 2

iDC


mDC + PO 1µg

(LPS)

mDC (LPS)






CD83


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 ?

Documents

Biological response modifiers in immune therapyimhaemo.ru/media/documents/HI_16/25_16.pdf · Chemotherapy 0 100 0 10 “Time-Window” for administration of IEC as: NK, gdT “Time-Window”