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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.

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slide1

Blanka Říhová

Cytotoxicita a imunostimulace - duální protinádorový útok moderníchpolymerních léčiv

Mikrobiologický ústav AV ČR

slide2

OD VÝZKUMU K LÉČBĚ

50 000nové látky

5 000testy invitro

500testy in vivo

5klinické testy

1použití v klinické praxi

slide4

Free drug

Targeted drug

slide6

Active targeting

cell-surface receptors on target tumor cells

slide7

Targeted drug

Free drug

Target cell

Target cell

slide8

Extracellular

matrix

Plasma membrane

Intracellular

fluid

Endosome

Lysosome

slide9

Passive targeting

(accumulation)

EPR effect

slide10

a) Normal tissue

b) Tumor tissue

Intact endothelium

Effective

lymphatic

drainage

Discontinuous

endothelium

Limited lymphatic drainage

sm rovan l ivo pasivn sm rov n
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

structure of prodrugs
STRUCTURE OF PRODRUGS

PRODRUGS with ACTIVE TARGETING

PRODRUGS with PASSIVE TARGETING

structure of prodrugs1
STRUCTURE OF PRODRUGS

ENZYMATICALLY

ACTIVATED PRODRUGS

pH-SENSITIVE

PRODRUGS

slide18

ConjugateA

Conjugate B

slide19

NHN=Dox

Dox=NHN

NH

NH2

X

X

NH

NH2

X

NHN=Dox

NHN=Dox

Dox=NHN

Dox=NHN

X-biodegradablespacer

Hyperbranched conjugate - type A

slide21

Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugates AHYD or BHYD

(dose 15 mg/kg)

100

80

60

surviving mice (%)

40

20

0

0

10

20

30

40

50

60

70

days

Controls

Hyperbranched B

Hyperbranched A

slide22

Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugates AHYD or BHYD

(dose 5 mg/kg)

100

80

60

surviving mice (%)

40

20

0

0

10

20

30

40

50

60

70

days

Controls

Hyperbranched B

Hyperbranched A

slide23

Tumor growth (EL4) afterthetreatmentwithhyperbranchedconjugates A and B (dose 5mg/kg): tumor size in mm3

daysize of tumor size of tumordaysize of tumor size of tumor

conjugateAconjugateBconjugateAconjugateB

8288 256 11 726 600

600 320 600 172

726 550 320 600

256 500 256 320

320 320 550 864

405 288 600 500

550 288 221 256

405550550196

15600 196 18 550 126

600 75 486 32

352 108 1080 14

196 172 600 14

365 787 446 108

446 14 1268 787

118614750

7519660

slide24

Tumor growth (EL4) after the treatment with hyperbranched conjugates A and B (dose 5mg/kg) ): tumor size in mm3

daysize of tumor size of tumordaysize of tumor size of tumor

conjugateAconjugateBconjugateAconjugateB

222025 405 25 3324 1352

1080 4 3726 0

1080 4 2304 14

1080 3240 1960 0

2025 172 5566 4400

256 0 2890 864

1080 0 1437 0

40140

286534 1368 31 6613 2890

5400 256 9216 600

6050 0 8438 0

3468 0 12393 0

10571 6912 12152 9375

2816 2176 4050 3078

5292 0 10816 0

126 0 1099 0

slide25

Tumor growth (EL4) after the treatment with hyperbranched conjugates A and B (dose 5mg/kg) ): tumor size in mm3

daysize of tumor size of tumordaysize of tumor size of tumor

conjugateAconjugateBconjugateAconjugateB

357497 650 38 10571 0

9464 0 8750 6613

12166 0 9688 0

6534 3564 2432 1014

1470 6200 ex 0

6050 0 ex 0

10140 0 ex 11300

ex ex ex ex

425808 0 45 17100 0

17298 0 7142 0

ex 0 ex 0

ex 0 ex 0

ex 2601 ex 3179

ex 15857 ex ex

ex 10206 ex ex

ex ex ex ex

slide26

Tumor growth (EL4) after the treatment with hyperbranched conjugates A and B (dose 5mg/kg) ): tumor size in mm3

daysize of tumor size of tumordaysize of tumor size of tumor

conjugateAconjugateBconjugateAconjugate B

4910571 0 52 ex 0

ex 0 ex 0

ex 0 ex 0

ex 0 ex 0

ex 6613 ex ex

ex ex ex ex

ex ex ex ex

ex ex ex ex

56ex 0 59 ex 0

ex 0 ex 0

ex 0 ex 0

ex 0 ex 0

ex ex ex ex

ex ex ex ex

ex ex ex ex

ex ex ex ex

slide27

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

slide29

Equilibrium

Elimination

(Cancer Immunosurveillance)

gd

NK

Genetic instability/

immune selection

Protection

slide31

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!

slide33

A

B

Survival of mice –PRIMARY TREATMENT

Dox-HPMAHYD

Survival of mice –RE-TRANSPLANTATION

Dox-HPMAHYD

  • controls1x75 mg/kg Dox 1x25 mg/kg
slide35

A

B

Survival of mice –PRIMARY TREATMENT

Dox-HPMAAM or Dox-HPMAAM-HuIg

Survival of mice – RE-TRANSPLANTATION

Dox-HPMAAM or Dox-HPMAAM-HuIg

  • controls Dox-HPMAAM-HuIg Dox-HPMAAM
slide36

Polymeric drugs based on

HPMA

cytotoxic immunomodulation

component

slide37

Cytotoxicity and immunostimulation

double attack on cancer cells with polymeric therapeutics

slide39

Systemic antitumor resistance initiated by the treatment with polymeric drugs

depends on

Dose

Time

The immune system of the host

slide41

A

B

Survival of mice –PRIMARY TREATMENT

Dox-HPMAHYD

Survival of mice –RE-TRANSPLANTATION

Dox-HPMAHYD

  • controls1x75 mg/kg Dox 1x25 mg/kg
slide43

Too earlythe immune system is not supplied with a sufficient amount of antigens (cancer cells) for effector-cell activation

Too latethe 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

slide45

RESISTANCE AGAINST BCL1 LEUKEMIA DEPENDS ON THE INTERVAL BETWEEN TUMOR CELL INOCULATION AND APPLICATION OF THE ANTIBODY-TARGETED CONJUGATE (DOX-HPMA-B1MAB)

Conjugatesurvival of survival of

experimental micere-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%

slide46

„vaccination window – optimal time frame “

treatment given too early or too late after transplantation of cancer cells induces only a limited resistance

slide47

The effect of Dox-HPMAAM/DOX-HPMAHYD mixture (1:2) on the growth of EL4 T cell lymphoma: conventional mice

Survival of mice

100

80

60

% of survival

40

20

0

0

10

20

30

40

50

60

days

DOX-HPMAAM/Dox-HPMAHYD mixture (1:2)

controls

slide48

The effect of Dox-HPMAAM/DOX-HPMAHYD mixture (1:2) on the growth of EL4 T cell lymphoma:

nu/nu mice

Survival of mice

100

80

60

% of survival

40

20

0

0

10

20

30

40

50

60

days

DOX-HPMAAM/Dox-HPMAHYD mixture (1:2)

controls

slide49

Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugate BHYD or linear conjugate Dox-HPMAAM - conventional mice

100

80

60

surviving mice (%)

40

20

0

10

20

30

40

50

60

80

70

0

days

Hyperbranched B/15mg

Hyperbranched B/10mg

Controls

Dox

Linear/15 mg

Linear/10 mg

slide50

Survival of mice bearing EL4 T cell lymphoma and treated with hyperbranched conjugate BHYD or linear conjugate Dox-HPMAAM - nu/nu mice

100

80

60

surviving mice (%)

40

20

0

0

10

20

30

40

50

60

70

days

Hyperbranched B/15mg

Hyperbranched B/10mg

Controls

Dox

Linear/15 mg

Linear/10 mg

slide51

Conventional mice

full resistance

Immunocompromized (nu/nu) mice

no resistance

slide52

Anti-cancer drug testing

conventionalimmunocompromised

micemice

slide53

Patient

Immune system isimmune system is exhausted

not exhausted(nu/nu mice model)

efficient treatmentlimitedefficacy of thetreatment

both components of theonly cytotoxic

polymericdrugs arecomponentof the polymeric

involved drug is involved

slide54

Acute x chronic

cancer

Acutechronic

mice were injected once witha lethal dose of cancer cells

(1x105 EL4 mouse T cell lymphoma)

mice were injected 5 times with a low dose of cancer cells

(1x104 EL4 mouse T cell lymphoma)

8 – 10 days a palpable tumorwithout palpable tumor

6th injection of a low dose of cancer cells

In 10 – 14 days a palpable tumor

slide55

Survival of mice suffering from acute or chronic cancer (mouse EL4 T cell lymphoma)

Dox-HPMAHYD

Non-cleavable dendrimer

100

80

60

% of surviving mice

40

20

0

10

20

30

40

50

60

80

70

0

days

controls – chronic cancer

controls – acute cancer

10 mg Dox-HPMAHYD - chronic cancer

10 mg Dox-HPMAHYD - acute cancer

slide58

Nine tumor markers

2-microglobulin

-fetoprotein (AFP)

CA 72-4

CA 125

CA 15-3

CA 19-9

CEA

Ferritin

Neuron-specific enolase (NSE)

slide59

TAA – No. 6

cytokeratin

cytokeratin

slide60

ACTIVITY OF NK CELLS IN

HUMAN PATIENTS

Appli- day E.G. J.K. K.R. K.H.

cation

Ist 0 24 23 9 11

3 45 21 19 33

IInd 0 22 10 10 11

3 27 25 1612

IIIrd 0 23 10 8 13

3 42 12 6 18

healthy donor 24 24

NK % of K562 killing

slide61

Polymer drugs based on HPMA

HPMA polymer

drug (doxorubicin)

bound through spacer

Tumor cells sensitive or

resistant to

the treatment with

polymeric drugs

„Immunogenic

cancer cell death“

And release of

tumor antigenes

Induction of anti-tumor immunity (activation of dendritic cells,

natural killer cells, T and B lymphocytes)

slide62

Acknowledgement

Institute of Macromolecular Chemistry AS CR, v.v.i.

Karel Ulbrich

Tomáš Etrych

Martin Hrubý

Petr Chytil

Hana Kostková

Daniela Plocová

Robert Pola

Jiří Strohalm

Martin Studenovský

Vladimír Šubr

slide63

Acknowledgement

Institute of Microbiology, AS CR, v.v.i.

            • Jaroslav Betka
            • Jan Bouček
  • Ondřej Hovorka
  • Helena Chmelová
  • Markéta Ibrahimová
  • Martina Kabešová
  • Jiřina Kovářová
  • Marek Kovář
  • Lubomír Kovář
  • Veronika Pakanová
  • Milada Šírová
  • Jakub Tomala
  • David Větvička
slide65

NK cell

(LAK cell)

macrophage

(APC)

dendritic cell

CD8 CTL

lymphocyte

CD4 Th

lymphocyte

antibody

Ab-secreting

Plasma cell

slide66

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

120

100

80

% survival

60

40

20

0

0

60

80

40

20

days

SC from mice cured with Dox-HPMA-HuIg

SC from mice suffering from tumor growth

SC from mice cured with doxorubicin

slide67

CD8+ 10:1

resistant donors

control (EL-4 only)

CD8+ 20:1

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

100

80

60

survival (%)

40

20

0

0

10

20

30

40

50

60

days

spleen cells 100:1

slide68

The treatment generates a tumor-specific long-lasting memory, which is based mainly on effector cell of specific immunity, the CD8+ CTLs

slide69

MYELOTOXICITY

Sample CFU-s/spleen

P-AH-DOX45.2±5.3

Doxorubicin8±3.0

Control35±3.7

slide72

Apoptosis

immunogenic non-immunogenic

cell death cell death

slide73

CRT

originaly termed high-affinity calcium-binding protein

slide74

CRT

is a Ca2+ - binding lectin chaperone that is mostly present in the ER lumen

slide75

Phagocytosis,

processing,

maturation,

cross-presentation

Translocation

of CRT to

cell surface

P

elF2a

elF2a

Anthracyclins,

GADD/PP1

inhibitors

PS turnover,

apoptosis

slide76

HMGB1 =

high mobility group box 1

slide77

Engulfed

tumor cell

in phagosome

(a)

(c)

(b)

MHC Class I

TLR4

Immature

DC

Phagocytosis

Antigen

processing and

presentation

MHC Class II

Scavenger

receptors

HMGB1

TLR4

Mature DC

Binding of HMGB1

to TLR4

Tumor

cell

Released HMGB

Cell surface

-CRT

Dying

tumor

cell

Anthracyclines

Anthracyclines

slide78

Expression of calreticulin (CRT) and heat shock protein (HSP) 110 on mouse EL4 T cell lymphomaa

Expression induction local/systemic

sample of anti-cancer immune response

CRT HSP 110

Dox - HPMAHYD + - +

Dox - HPMAAM - + +

free Dox + - +c/-d

slide79

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

slide80

Our preliminary results suggest an expression of Hsp on cancer cells after the treatment with conjugates where the drug is bound through amide bond

slide82

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

slide83

60

50

40

30

% of regression

20

10

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Treatment

METASTATIC LUNG TUMORS

D-P-HuIg

slide84

40

35

30

25

20

x 109 cells/L

15

10

5

0

1

2

3

4

11

12

13

14

15

16

17

18

25

26

27

28

29

30

31

32

33

34

LEUKOCYTES

free drug

D-P-HuIg

slide85

eIF2α

eukaryotic translation initiation factor

slide86

eIF2α

anthracyclins enforce the early phosphorylation of eIF2α, a process normally observed in the ER under conditions of cellular stress

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