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Cytotoxicita a imunostimulace - duální protinádorový útok moderních polymerních léčiv - PowerPoint PPT Presentation


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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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Blanka Říhová

Cytotoxicita a imunostimulace - duální protinádorový útok moderníchpolymerních léčiv

Mikrobiologický ústav AV ČR


OD VÝZKUMU K LÉČBĚ

50 000nové látky

5 000testy invitro

500testy in vivo

5klinické testy

1použití v klinické praxi


Free drug

Targeted drug


ACTIVE x PASSIVE

targeting


Active targeting

cell-surface receptors on target tumor cells


Targeted drug

Free drug

Target cell

Target cell


Extracellular

matrix

Plasma membrane

Intracellular

fluid

Endosome

Lysosome


Passive targeting

(accumulation)

EPR effect


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


N-(2-hydroxypropyl)methacrylamide = HPMA = P


Structure of prodrugs
STRUCTURE OF PRODRUGS

PRODRUGS with ACTIVE TARGETING

PRODRUGS with PASSIVE TARGETING




Structure of prodrugs1
STRUCTURE OF PRODRUGS DOXORUBICIN

ENZYMATICALLY

ACTIVATED PRODRUGS

pH-SENSITIVE

PRODRUGS


in vivo veritas DOXORUBICIN


Conjugate DOXORUBICINA

Conjugate B


NHN=Dox DOXORUBICIN

Dox=NHN

NH

NH2

X

X

NH

NH2

X

NHN=Dox

NHN=Dox

Dox=NHN

Dox=NHN

X-biodegradablespacer

Hyperbranched conjugate - type A



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


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


Tumor with hyperbranched conjugates Agrowth (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


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


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


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


GENERAL RULES conjugates A and B (dose 5mg/kg) ): tumor size in mm

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 conjugates A and B (dose 5mg/kg) ): tumor size in mm


Equilibrium conjugates A and B (dose 5mg/kg) ): tumor size in mm

Elimination

(Cancer Immunosurveillance)

gd

NK

Genetic instability/

immune selection

Protection


Re-transplantation conjugates A and B (dose 5mg/kg) ): tumor size in mm


Re-transplantation conjugates A and B (dose 5mg/kg) ): tumor size in mm

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 conjugates A and B (dose 5mg/kg) ): tumor size in mm

DOX


A conjugates A and B (dose 5mg/kg) ): tumor size in mm

B

Survival of mice –PRIMARY TREATMENT

Dox-HPMAHYD

Survival of mice –RE-TRANSPLANTATION

Dox-HPMAHYD

  • controls1x75 mg/kg Dox 1x25 mg/kg



A DOXORUBICIN

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


Polymeric drugs based on DOXORUBICIN

HPMA

cytotoxic immunomodulation

component


Cytotoxicity and immunostimulation DOXORUBICIN

double attack on cancer cells with polymeric therapeutics



Systemic antitumor resistance initiated by the treatment with polymeric drugs

depends on

Dose

Time

The immune system of the host


Dose with polymeric drugs


A with polymeric drugs

B

Survival of mice –PRIMARY TREATMENT

Dox-HPMAHYD

Survival of mice –RE-TRANSPLANTATION

Dox-HPMAHYD

  • controls1x75 mg/kg Dox 1x25 mg/kg


More aggressive treatment with polymeric drugs

lower resistance


Too early with polymeric drugsthe 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


Time with polymeric drugs


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%


„vaccination window – optimal time frame “ BETWEEN TUMOR CELL INOCULATION AND APPLICATION OF THE ANTIBODY-TARGETED CONJUGATE (DOX-HPMA-B1MAB)

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


The effect of Dox-HPMA BETWEEN TUMOR CELL INOCULATION AND APPLICATION OF THE ANTIBODY-TARGETED CONJUGATE (DOX-HPMA-B1MAB)AM/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


The effect of Dox-HPMA BETWEEN TUMOR CELL INOCULATION AND APPLICATION OF THE ANTIBODY-TARGETED CONJUGATE (DOX-HPMA-B1MAB)AM/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


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


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


Conventional mice with hyperbranched conjugate B

full resistance

Immunocompromized (nu/nu) mice

no resistance


Anti-cancer drug testing with hyperbranched conjugate B

conventionalimmunocompromised

micemice


Patient with hyperbranched conjugate B

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


Acute x chronic with hyperbranched conjugate B

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


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


Immunonormalization (mouse EL4 T cell lymphoma)


Nine tumor markers (mouse EL4 T cell lymphoma)

2-microglobulin

-fetoprotein (AFP)

CA 72-4

CA 125

CA 15-3

CA 19-9

CEA

Ferritin

Neuron-specific enolase (NSE)


TAA – No. 6 (mouse EL4 T cell lymphoma)

cytokeratin

cytokeratin


ACTIVITY OF NK CELLS IN (mouse EL4 T cell lymphoma)

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


Polymer drugs based on HPMA (mouse EL4 T cell lymphoma)

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)


Acknowledgement (mouse EL4 T cell lymphoma)

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


Acknowledgement (mouse EL4 T cell lymphoma)

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


  • Thank you ! (mouse EL4 T cell lymphoma)


    NK cell (mouse EL4 T cell lymphoma)

    (LAK cell)

    macrophage

    (APC)

    dendritic cell

    CD8 CTL

    lymphocyte

    CD4 Th

    lymphocyte

    antibody

    Ab-secreting

    Plasma cell


    Winn's assay (mouse EL4 T cell lymphoma)

    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


    CD8+ 10:1 (mouse EL4 T cell lymphoma)

    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


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


    MYELOTOXICITY memory, which is based mainly on effector cell of specific immunity, the CD8

    Sample CFU-s/spleen

    P-AH-DOX45.2±5.3

    Doxorubicin8±3.0

    Control35±3.7


    Immunogenic cancer cell death memory, which is based mainly on effector cell of specific immunity, the CD8


    Immunogenic chemotherapy memory, which is based mainly on effector cell of specific immunity, the CD8


    Apoptosis memory, which is based mainly on effector cell of specific immunity, the CD8

    immunogenic non-immunogenic

    cell death cell death


    CRT memory, which is based mainly on effector cell of specific immunity, the CD8

    originaly termed high-affinity calcium-binding protein


    CRT memory, which is based mainly on effector cell of specific immunity, the CD8

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


    Phagocytosis, memory, which is based mainly on effector cell of specific immunity, the CD8

    processing,

    maturation,

    cross-presentation

    Translocation

    of CRT to

    cell surface

    P

    elF2a

    elF2a

    Anthracyclins,

    GADD/PP1

    inhibitors

    PS turnover,

    apoptosis


    HMGB1 = memory, which is based mainly on effector cell of specific immunity, the CD8

    high mobility group box 1


    Engulfed memory, which is based mainly on effector cell of specific immunity, the CD8

    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


    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


    CRT expression (HSP) 110 on mouse EL4 T cell lymphoma 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 (HSP) 110 on mouse EL4 T cell lymphomaexpression of Hsp on cancer cells after the treatment with conjugates where the drug is bound through amide bond



    Dox-HPMA copolymer, PK1 (FCE 28068) for polymeric nanotherapeutics

    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 for polymeric nanotherapeutics

    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


    40 for polymeric nanotherapeutics

    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


    eIF2α for polymeric nanotherapeutics

    eukaryotic translation initiation factor


    eIF2α for polymeric nanotherapeutics

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


    Immunomobilization for polymeric nanotherapeutics


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