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Blanka Říhová. Cytotoxicita a imunostimulace - duální protinádorový útok moderních polymerních léčiv. Mikrobiologický ústav AV ČR. OD VÝZKUMU K LÉ Č B Ě. 50 000 nové látky 5 000 testy in vitro 500 testy in vivo 5 klinické testy 1 pou ž ití v klinické praxi. Free drug. Targeted drug. - PowerPoint PPT Presentation
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Cytotoxicita a imunostimulace - duální protinádorový útok moderních
polymerních léčiv
Blanka Říhová
Mikrobiologický ústav AV ČR
50 000 nové látky 5 000 testy in vitro
500 testy in vivo
5 klinické testy
1 použití v klinické praxi
OD VÝZKUMU K LÉČBĚ
Free drug Targeted drug
ACTIVE x PASSIVE
targeting
Active targeting
cell-surface receptors on target tumor cells
Free drug Targeted drug
Target cell Target cell
Plasma membrane
Extracellularmatrix
Endosome
Intracellularfluid
Lysosome
Passive targeting
(accumulation)EPR effect
Effectivelymphaticdrainage
Intact endothelium
a) Normal tissue
b) Tumor tissue
Discontinuousendothelium
Limited lymphatic drainage
Směrované léčivo - pasivní směrování• Složení: polymerní nosič + léčivo• Enhanced Permeation and Retention effect (EPR) Endotel krevních vlásečnic v nádoru je defektní, kapiláry jsou
propustné i pro vysokomolekulární komplexy Ve zdravé tkáni komplex neprojde = cílená terapie Lymfatická drenáž je defektní v nádoru = hromadění léčiva
snížením rychlosti eliminace
N-(2-hydroxypropyl)methacrylamide =
HPMA = P
STRUCTURE OF PRODRUGS
PRODRUGS with ACTIVE TARGETING
PRODRUGS with PASSIVE TARGETING
STRUCTURE OF ANTIBODY-TARGETED HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN
DOX
STRUCTURE OF (PK1) HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN
DOX
STRUCTURE OF PRODRUGS
C CH2OH
O
CH3
CH OH
CH2
NH
C
CCH2
CH3
CH2
CH3
C
C O
X
NH
N
O OH
O OH H O
OH
OCH3
OHNH2.HCl
OMe
x y
O
O
CH3
CH OH
CH2
NH
C
CCH2
CH3
CH2
CH3
C
C O
Gly
Phe
Leu
Gly
NHCH3 OH
O
CH2CH2OHOOH
OOH
OH
OMe
x y
ENZYMATICALLY
ACTIVATED PRODRUGS
pH-SENSITIVE
PRODRUGS
in vivo veritas
Conjugate AConjugate B
Hyperbranched conjugate - type A
NHNH2
NHN=Dox
Dox=NHN
Dox=NHN
NHN=Dox
Dox=NHN
NHN=Dox
X
XNHNH2
X
X-biodegradable spacer
Hyperbranched conjugate – type B
0
20
40
60
80
100
0 10 20 30 40 50 60 70
Hyperbranched A Hyperbranched B Controls
Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugates AHYD or BHYD
(dose 15 mg/kg)
days
su
rviv
ing
mic
e (
%)
0
20
40
60
80
100
0 10 20 30 40 50 60 70
Hyperbranched A Hyperbranched B Controls
Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugates AHYD or BHYD
(dose 5 mg/kg)
days
su
rviv
ing
mic
e (
%)
day size of tumor size of tumor day size of tumor size of tumorconjugate A conjugate B conjugate A conjugate B
8 288 256 11 726 600600 320 600 172726 550 320 600256 500 256 320320 320 550 864405 288 600 500550 288 221 256405 550 550 196
15 600 196 18 550 126600 75 486 32352 108 1080 14196 172 600 14365 787 446 108446 14 1268 787
1186 14 75 075 196 6 0
day size of tumor size of tumor day size of tumor size of tumorconjugate A conjugate B conjugate A conjugate B
22 2025 405 25 3324 13521080 4 3726 01080 4 2304 141080 3240 1960 02025 172 5566 4400256 0 2890 864
1080 0 1437 04 0 14 0
28 6534 1368 31 6613 28905400 256 9216 6006050 0 8438 03468 0 12393 0
10571 6912 12152 93752816 2176 4050 30785292 0 10816 0126 0 1099 0
Tumor growth (EL4) after the treatment with hyperbranched conjugates A and B (dose 5mg/kg) ): tumor size in mm3
day size of tumor size of tumor day size of tumor size of tumorconjugate A conjugate B conjugate A conjugate B
35 7497 650 38 10571 09464 0 8750 6613
12166 0 9688 06534 3564 2432 10141470 6200 ex 06050 0 ex 0
10140 0 ex 11300ex ex ex ex
42 5808 0 45 17100 017298 0 7142 0
ex 0 ex 0ex 0 ex 0ex 2601 ex 3179ex 15857 ex exex 10206 ex exex ex ex ex
Tumor growth (EL4) after the treatment with hyperbranched conjugates A and B (dose 5mg/kg) ): tumor size in mm3
day size of tumor size of tumor day size of tumor size of tumorconjugate A conjugate B conjugate A conjugate B
49 10571 0 52 ex 0ex 0 ex 0ex 0 ex 0ex 0 ex 0ex 6613 ex exex ex ex exex ex ex exex ex ex ex
56 ex 0 59 ex 0ex 0 ex 0ex 0 ex 0ex 0 ex 0ex ex ex exex ex ex exex ex ex exex ex ex ex
Tumor growth (EL4) after the treatment with hyperbranched conjugates A and B (dose 5mg/kg) ): tumor size in mm3
GENERAL RULES
Tumor stop to grow in two - three days after the treatment
The first significant shrinkage could be seen in five – six days after the treatment
If there is no response until the day 10, the treatment has to be considered as non-effective
The immune system of the host
Genetic instability/
immune selection
Protection
EquilibriumElimination(Cancer Immunosurveillance)
NK
Re-transplantation
Re-transplantation
8 – 12 days
3 – 8 months
dead mice surviving mice (up to 100%)
strong systemic anti-tumor resistance
lethal dose of cancer cells
polymer-bound drugs
CURED mice
lethal dose of cancer cells! NO TREATMENT!
STRUCTURE OF (PK1) HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN
DOX
Survival of mice – PRIMARY TREATMENT
Dox-HPMAHYD
A B
Survival of mice – RE-TRANSPLANTATION
Dox-HPMAHYD
controls 1x75 mg/kg Dox 1x25 mg/kg
STRUCTURE OF ANTIBODY-TARGETED HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN
DOX
A B
controls Dox-HPMAAM-HuIg Dox-HPMAAM
Survival of mice – PRIMARY TREATMENT
Dox-HPMAAM or Dox-HPMAAM-HuIg
Survival of mice – RE-TRANSPLANTATION
Dox-HPMAAM or Dox-HPMAAM-HuIg
Polymeric drugs based onHPMA
cytotoxic immunomodulation
component
Cytotoxicity and immunostimulation
double attack on cancer cells with polymeric therapeutics
Protection against the second cancer attack
Systemic antitumor resistance initiated by the treatment with polymeric drugs
depends on
Dose
Time
The immune system of the host
Dose
Survival of mice – PRIMARY TREATMENT
Dox-HPMAHYD
A B
Survival of mice – RE-TRANSPLANTATION
Dox-HPMAHYD
controls 1x75 mg/kg Dox 1x25 mg/kg
More aggressive treatment
lower resistance
Too early the immune system is not supplied with a sufficient amount of antigens (cancer cells) for effector-cell activation
Too late the effector mechanisms of the cancer-bearing host’s immune system are already exhausted and unable to be activated, resulting in only very limited cancer resistance
Time
RESISTANCE AGAINST BCL1 LEUKEMIA DEPENDS ON THE INTERVAL BETWEEN TUMOR CELL INOCULATION
AND APPLICATION OF THE ANTIBODY-TARGETED CONJUGATE (DOX-HPMA-B1MAB)
Conjugate survival of survival ofexperimental mice re-transplanted mice
(day) (n = 10) (1 x 104 BCL1 cells i.p.)
1 100% 0%
17 100% 30%
11 100% 20%
15 60% 0%
Control (BPS) 0% 0%
„vaccination window – optimal time frame “
treatment given too early or too late after transplantation of cancer cells induces
only a limited resistance
The effect of Dox-HPMAAM/DOX-HPMAHYD mixture (1:2) on the growth of EL4 T cell lymphoma:
conventional mice
days
% o
f su
rviv
al
Survival of mice
0
20
40
60
80
100
0 10 20 30 40 50 60
DOX-HPMAAM/Dox-HPMAHYD mixture (1:2) controls
0
20
40
60
80
100
0 10 20 30 40 50 60
The effect of Dox-HPMAAM/DOX-HPMAHYD mixture (1:2) on the growth of EL4 T cell lymphoma:
nu/nu mice
days
DOX-HPMAAM/Dox-HPMAHYD mixture (1:2) controls
Survival of mice
% o
f su
rviv
al
10 20 30 40 50 60 80700
20
40
60
80
100
0
Hyperbranched B/10mgDox Controls
Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugate BHYD or linear conjugate
Dox-HPMAAM - conventional mice
days
su
rviv
ing
mic
e (
%)
Hyperbranched B/15mgLinear/10 mgLinear/15 mg
10 20 30 40 50 60 700
20
40
60
80
100
0
Hyperbranched B/10mgDox Controls
Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugate BHYD or linear conjugate
Dox-HPMAAM - nu/nu mice
days
su
rviv
ing
mic
e (
%)
Hyperbranched B/15mgLinear/10 mgLinear/15 mg
Conventional mice
full resistance
Immunocompromized (nu/nu) mice
no resistance
Anti-cancer drug testing
conventional immunocompromisedmice mice
Patient
Immune system is immune system is exhaustednot exhausted (nu/nu mice model)
efficient treatment limited efficacy of the treatmentboth components of the only cytotoxic
polymeric drugs are component of the polymericinvolved drug is involved
Acute x chroniccancer
Acute chronic
mice were injected once with a lethal dose of cancer cells
(1x105 EL4 mouse T cell lymphoma)
8 – 10 days a palpable tumor without palpable tumor
6th injection of a low dose of cancer
cells
In 10 – 14 days a palpable tumor
mice were injected 5 times with a low dose of cancer cells(1x104 EL4 mouse T cell
lymphoma)
10 20 30 40 50 60 80700
20
40
60
80
100
0
10 mg Dox-HPMAHYD - acute cancercontrols – acute cancer
Survival of mice suffering from acute or chronic cancer (mouse EL4 T cell lymphoma)
Dox-HPMAHYD
Non-cleavable dendrimer
days
% o
f s
urv
ivin
g m
ice
10 mg Dox-HPMAHYD - chronic cancercontrols – chronic cancer
Immunonormalization
Nine tumor markers
2-microglobulin-fetoprotein (AFP)CA 72-4CA 125CA 15-3CA 19-9CEAFerritinNeuron-specific enolase (NSE)
TAA – No. 6
cytokeratin
cyto
kera
tin
Appli- day E.G. J.K. K.R. K.H. cation
Ist 0 24 23 9 113 45 21 19 33
IInd 0 22 10 10 113 27 25 16 12
IIIrd 0 23 10 8 133 42 12 6 18
healthy donor24 24
NK % of K562 killing
ACTIVITY OF NK CELLS INHUMAN PATIENTS
Polymer drugs based on HPMA
HPMA polymer
drug (doxorubicin)bound through spacer
„Immunogeniccancer cell death“And release oftumor antigenes
Tumor cells sensitive orresistant tothe treatment withpolymeric drugs
Induction of anti-tumor immunity (activation of dendritic cells,natural killer cells, T and B lymphocytes)
AcknowledgementInstitute of Macromolecular Chemistry AS CR, v.v.i.
Karel Ulbrich
Tomáš EtrychMartin Hrubý
Petr ChytilHana Kostková
Daniela PlocováRobert Pola
Jiří StrohalmMartin Studenovský
Vladimír Šubr
AcknowledgementInstitute of Microbiology, AS CR, v.v.i.
Thank you !
macrophage(APC)
dendritic cell NK cell(LAK cell)
CD4 Thlymphocyte
CD8 CTLlymphocyte
Ab-secretingPlasma cell
antibody
Winn's assay
Recipients were naive B/6 mice, injected s.c. with splenocytes (SC) together with 1 x 105 EL- 4 lymphoma cells. SC isolated 24 days after
EL-4 cell transplantation
SC from mice cured with Dox-HPMA-HuIgSC from mice suffering from tumor growthSC from mice cured with doxorubicin
days
% s
urv
ival
0 20 40 60 80
120
100
80
60
40
20
0
The anti-tumour immunity could be transferred to naive mice: Winn´s assay
tumour neutralization in vivo: 1x105 EL-4 cells were transplanted s.c. together with cells of the Dox-PHPMA-HuIg-cured animals to naïve recipients
spleen cells 100:1 resistant donors CD8+ 20:1 CD8+ 10:1 control (EL-4 only)
0
20
40
60
80
100
0 10 20 30 40 50 60
days
surv
ival
(%
)
The treatment generates a tumor-specific long-lasting memory, which is
based mainly on effector cell of specific immunity, the CD8+ CTLs
MYELOTOXICITY
Sample CFU-s/spleen
P-AH-DOX 45.2 ± 5.3
Doxorubicin 8 ± 3.0
Control 35 ± 3.7
Immunogenic cancer cell death
Immunogenic chemotherapy
Apoptosis
immunogenic non-immunogeniccell death cell death
CRT
originaly termed high-affinity calcium-binding protein
CRT
is a Ca2+ - binding lectin chaperone that is mostly present in the ER lumen
Phagocytosis,processing,maturation,
cross-presentation
PS turnover,apoptosis
Translocationof CRT to
cell surface
Anthracyclins,GADD/PP1inhibitors
elF2
elF2
P
HMGB1 =high mobility group box 1
(b)(a) (c)
Tumorcell
Mature DC
Dyingtumor
cell
Engulfedtumor cell
in phagosome
MHC Class IImmature
DC
Phagocytosis
Scavengerreceptors
HMGB1TLR4
Released HMGB
Anthracyclines
Anthracyclines
Cell surface-CRT
TLR4
Antigenprocessing andpresentation
Binding of HMGB1to TLR4
MHC Class II
Expression induction local/systemic sample of anti-cancer immune response
CRT HSP 110
Dox - HPMAHYD + - +
Dox - HPMAAM - + +
free Dox + - +c/-d
Expression of calreticulin (CRT) and heat shock protein (HSP) 110 on mouse EL4 T cell lymphomaa
CRT expression depends on intracellular release of the drug and is detectable only
after the treatment with HPMA-based conjugates containing drug bound
through a hydrazone bond
Our preliminary results suggest an expression of Hsp on cancer cells after the treatment with conjugates where the drug
is bound through amide bond
An idea of „immunogenic cancer cell death“ is valid also for
polymeric nanotherapeutics
Dox-HPMA copolymer, PK1 (FCE 28068)
Paclitaxel-HPMA copolymer (PNU 166945)
Cisplatin-HPMA copolymer (AP 5280)
Dox-HPMA copolymer-gal, PK2 (FCE 28069)
Dox-HPMA copolymer-HuIg
No polymer-related toxicity was observed
60
50
40
30
20
10
01 2 3 4 5 6 7 8 9 10 11 12 13 14
% o
f re
gre
ssio
n
Treatment
METASTATIC LUNG TUMORS
D-P-HuIg
40
35
30
25
20
15
10
5
01 2 3 4 11 12 13 14 15 16 17 18 25 26 27 28 29 30 31 32 33 34
x 10
9 c
ells
/LLEUKOCYTES
free drug D-P-HuIg
eIF2α
eukaryotic translation initiation factor
eIF2α
anthracyclins enforce the early phosphorylation of eIF2α, a process normally
observed in the ER under conditions of cellular stress
Immunomobilization