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