antibody-nanocarrier conjugates for drug targeting and improved cancer therapy n. debotton 1, o....

ANTIBODY-NANOCARRIER

CONJUGATES FOR DRUG TARGETING

AND IMPROVED CANCER THERAPY

ANTIBODY-NANOCARRIER

CONJUGATES FOR DRUG TARGETING

AND IMPROVED CANCER THERAPY

N. Debotton1, O. Harush-Frenkel1, O. Gofrit2 and S. Benita 1

1. Pharmaceutics Department, The School of Pharmacy

2. Urology Department, Hadassah-Hebrew University Medical Center,

N. Debotton1, O. Harush-Frenkel1, O. Gofrit2 and S. Benita 1

1. Pharmaceutics Department, The School of Pharmacy

2. Urology Department, Hadassah-Hebrew University Medical Center,

The Hebrew University Of JerusalemFaculty of Medicine

The Hebrew University Of JerusalemFaculty of Medicine

Drug loaded nanoparticles (NPs)Drug loaded nanoparticles (NPs)

NPs smaller than 500nm can be NPs smaller than 500nm can be administered IV without vascular administered IV without vascular embolization fear embolization fear

Generally made of biocompatible Generally made of biocompatible polyesters, FDA approved for IP/IM polyesters, FDA approved for IP/IM administrationadministration

NPs: long circulation time, high drug NPs: long circulation time, high drug payloads, and long shelf-life as payloads, and long shelf-life as lyophilized powders lyophilized powders

ABRAXANE® - FDA approved NPs formulation

Albumin conjugated to paclitaxel forming the NP (130 nm, 10% w/w drug content)

Drug loaded NPs for cancer therapy

Drug loaded NPs for cancer therapy

mAbs can treat a wide range of diseases

mAbs can treat a wide range of diseases

Major impact in cancerMajor impact in cancer

Solid tumorsSolid tumors

Hematological malignanciesHematological malignancies

8 commercial mAbs are approved 8 commercial mAbs are approved for cancerfor cancer

Cancer cells are known to over-

express antigens

(Prostate Specific Cell Antigen, EGF

receptor, mucin

HER-2 is a HER-2 is a growth factor co-growth factor co-receptor, over-receptor, over-expressed in expressed in

breast, ovarian, breast, ovarian, colon, lung, cervical colon, lung, cervical

and prostate and prostate cancerscancers

Trastuzumab Trastuzumab recombinant recombinant humanized humanized

IgG1 MAb for IgG1 MAb for metastatic metastatic

breast cancerbreast cancer

Cancer cells antigen over-expression

Cancer cells antigen over-expression

Immunonanoparticles (INPs) 1 INP carry up to 20 000 molecules

Immunonanoparticles (INPs) 1 INP carry up to 20 000 molecules

drugdrug

ImmunoNPsImmunoNPs

high drug high drug loading loading capacitycapacity

cell specific cell specific drug deliverydrug delivery

High High affinity affinity densitydensity

More than More than one mAbone mAb

ObjectivesObjectives

Evaluate the cytotoxicity, binding and uptake of Evaluate the cytotoxicity, binding and uptake of immunoNPs to cancerous cell lines immunoNPs to cancerous cell lines

Determine quantitatively the affinity kinetic Determine quantitatively the affinity kinetic parameters of immunoNPs parameters of immunoNPs

Estimate the biodistribution and pk profile of Estimate the biodistribution and pk profile of immunoNPs in miceimmunoNPs in mice

Assess the efficiency of these mAb conjugated Assess the efficiency of these mAb conjugated NPs in a tumor bearing mice modelNPs in a tumor bearing mice model

PEG-PLA

linker

drug

Tween 80®

Acetone

Solutol® HS 15

water

Nanocapsule formation by Nanocapsule formation by interfacial polymer deposition interfacial polymer deposition following solvent displacementfollowing solvent displacement

Fessi et al., 1989Fessi et al., 1989

ImmunoNPs preparationImmunoNPs preparation

TEM photographs of PLA100000 NPsTEM photographs of PLA100000 NPs

synthesis validated by MS, IR and NMR

Linker synthesis: OMCCALinker synthesis: OMCCA

N

O

O

O N

O

OO

N

O

O

NH

O

NH2

NHS esterNHS ester stearylaminestearylamine

Maleimide groupMaleimide group

SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylateSMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate

chloroform (40°C)chloroform (40°C)

OMCCA octadecyl-4-(maleimidomethyl) cyclohexane-carboxilic amideOMCCA octadecyl-4-(maleimidomethyl) cyclohexane-carboxilic amide

Conjugation of OMCCA and MAbs


MAb is covalently bound to NPs via thio-ether bond



N

O

HN

O

R

O

NPNPNPNPN

O

HN

O

R

O

S

antibodyantibody

antibodyantibody

NPNPNPNP

Paclitaxel palmitate (pcpl) synthesisA lipophilic derivative of paclitaxel A lipophilic derivative of paclitaxel

(Taxol®) was synthesized: (Taxol®) was synthesized: paclitaxel palmitatepaclitaxel palmitate

Product was validated using Product was validated using HPLC, LCMS, H-NMR HPLC, LCMS, H-NMR

Pcpl potency was equivalent to Pcpl potency was equivalent to paclitaxel in paclitaxel in in vitroin vitro//in vivoin vivo studies studies

Stable NPs were prepared with Stable NPs were prepared with pcplpcpl

Drug encapsulation Drug encapsulation

DrugDrug Initial drug Initial drug content (%)content (%)

Observed drug Observed drug content (%)content (%)

Loading Loading efficiency (%)efficiency (%)

pcplpcpl 13.613.6 8.68.6 6262

Drug content and loading efficiency in NPs

pcpl loaded NPs visualized by light microscopy. No crystals formation

TEM photographs of pcpl loaded NPs

Number of sulfhydryl groups on MAb molecule, before and after Number of sulfhydryl groups on MAb molecule, before and after incubation with Traut’s reagent, as determined by Ellman’s reagentincubation with Traut’s reagent, as determined by Ellman’s reagent

Molar ratio: Traut and Molar ratio: Traut and MAb 1:30MAb 1:30 Native AMB8LKNative AMB8LK Traut modified Traut modified

AMB8LKAMB8LK

SH groups per MAbSH groups per MAb 11.6311.63 30.0330.03

AMB8LK MAbAMB8LK MAb

H-ferritin (acidic) ↑ in patients with colon, testicular, breast, H-ferritin (acidic) ↑ in patients with colon, testicular, breast, pancreas cancerspancreas cancers

AMB8LK (MAT BIOPHARMA) – murine IgG1 recognized H-ferritin AMB8LK (MAT BIOPHARMA) – murine IgG1 recognized H-ferritin with high affinity (KD 2.4x10-9 M)with high affinity (KD 2.4x10-9 M)

[90Y]-anti H-ferritin (Ferritarg P®, MAT BIOPHARMA) is under [90Y]-anti H-ferritin (Ferritarg P®, MAT BIOPHARMA) is under clinical evaluation in patients with refractory Hodgkin’s disease clinical evaluation in patients with refractory Hodgkin’s disease

NP size, nm

Zeta pot.,mV

MAb conc.,mg/ml

MAbs/NP

Obs. pcpl c., mg/ml

% drug cont., w/w

NPs Drug loading

115 -21±1 0.77 82 2.8 9.6 70%

AMB8LK immunoNPs analyzed using Brownian motion as seen by NanoSight

Pcpl loaded AMB8LK immunoNPs characterization

Pcpl loaded AMB8LK immunoNPs characterization

A B

DC

of coumarin-6 NPs and immunoNPs (100ng/mg)

to CAPAN-1 cells, at 4 and 37°C over 1 h

incubation, respectively

Binding

Uptake

NP INP

Binding and uptake of AMB8LK immunoNPs to CAPAN-1 cells

Binding and uptake of AMB8LK immunoNPs to CAPAN-1 cells

Surface Plasmon Resonance (BIAcore)

Surface Plasmon Resonance (BIAcore)

The excitation of surface plasmon by light is denoted as The excitation of surface plasmon by light is denoted as surface plasmon resonance (SPR)surface plasmon resonance (SPR)

SPR technology enables detection of biomolecular SPR technology enables detection of biomolecular events and provides quantitative information on:events and provides quantitative information on:

Specificity – how specific is the binding between 2 Specificity – how specific is the binding between 2 molecules?molecules?

Concentration – how much of a molecule is present and Concentration – how much of a molecule is present and active?active?

Kinetics – what is the rate of association/dissociation?Kinetics – what is the rate of association/dissociation?

Affinity – how strong is the binding?Affinity – how strong is the binding?

The sensogram

Surface Plasmon ResonanceSurface Plasmon Resonance

Kon (1/Ms)Kon (1/Ms) Koff (1/s)Koff (1/s) KD (M)KD (M) Chi2Chi2

AMB8LKAMB8LK 3.45x1043.45x104 1.53x10-41.53x10-4 6.67x10-96.67x10-9 0.4250.425

AMB8LK AMB8LK immunoNPsimmunoNPs 3.3x1043.3x104 1.27x10-41.27x10-4 8.16x10-98.16x10-9 0.3510.351

Rate and affinity constants obtained from the Rate and affinity constants obtained from the interaction between matrix bound AMB8LK interaction between matrix bound AMB8LK

MAb or AMB8LK INPs and H-ferritin solution MAb or AMB8LK INPs and H-ferritin solution

BIAcore studiesBIAcore studies

Incubation without trastuzumab

incubation with native trastuzumab

incubation with Traut modified trastuzumab

Number of sulfhydryl groups on MAb molecule, determined by Ellman’s reagent

Traut : MAb molar ratioNative

trastuzumabModified

trastuzumab

SH groups per MAb at 1:30 1.4 31.5

SH groups per MAb at 1:50 1.3 48.5

Trastuzumab thiolationTrastuzumab thiolation

Thiolated trastuzumab binding to SK-BR-3 cells at 4°C over 1 h incubation, visualized by FITC anti-human IgG

NP size nm

Zeta pot. ,mV

MAb c.,mg/ml

MAbs/NP

pcpl conc., mg/ml

Drug cont., % w/w

NPs Drug loading

110 -20 0.675 63 2.5 8.6 65%

TEM photographs of trastuzumab INPs (23

MAbs/NP) following incubation over 1h with

anti-human IgG Ab conjugated to 12nm

gold particles

Pcpl loaded trastuzumab immunoNPs characterization

Pcpl loaded trastuzumab immunoNPs characterization

Debotton et al., J. Cont. Debotton et al., J. Cont. Rel,127, 219-230, 2008.Rel,127, 219-230, 2008.

Uptake of coumarin-6 pcpl NPs and INPs to PC-3 cell at 37°C over 3h

The fluorescence intensities of coumarin-6 (100ng/mg) in both formulations was equivalent

Debotton et al., J. Cont. Rel.,127, Debotton et al., J. Cont. Rel.,127, 219-230, 2008.219-230, 2008.

NPsNPs INPsINPs

Uptake of trastuzumab pcpl loaded immunoNPs to PC-3 cells


Debotton et al., J. Cont. Debotton et al., J. Cont. Rel.,127, 219-230, 2008.Rel.,127, 219-230, 2008.



Cells were treated with 3[H]-pcpl Cells were treated with 3[H]-pcpl solution, 3[H]-pcpl loaded NPs solution, 3[H]-pcpl loaded NPs and 3[H]-pcpl loaded trastuzumab and 3[H]-pcpl loaded trastuzumab INPs over 3 h.INPs over 3 h.

The equivalent amount of pcpl per The equivalent amount of pcpl per well was 5.2µg. Results are well was 5.2µg. Results are normalized against total protein in normalized against total protein in cells. Values are mean±SD. N=5cells. Values are mean±SD. N=5

Efficacy of pcpl loaded trastuzumab immunoNPs on PC-3 cells

Efficacy of pcpl loaded trastuzumab immunoNPs on PC-3 cells

Percentage of PC-3 cell survival at 37°C over 3 h with trastuzumab immunoNPs, pcpl NPs and pcpl solution (10µM). N=6

SC5b-9 levels in two SC5b-9 levels in two sera obtained sera obtained following incubation following incubation with different with different formulations. N=3 formulations. N=3

Pharmacokinetic and biodistribution study in mice


4 mice/group (Balb/C 8 week old healthy male)4 mice/group (Balb/C 8 week old healthy male)

IV administration of 0.225 µCi of [3H]-pcpl IV administration of 0.225 µCi of [3H]-pcpl equivalent to a total dose of 7.5mg/kg of pcpl:equivalent to a total dose of 7.5mg/kg of pcpl:

[3H]-pcpl solution (Cremophor EL : Ethanol) [3H]-pcpl solution (Cremophor EL : Ethanol)

[3H]-pcpl loaded NPs[3H]-pcpl loaded NPs

[3H]-pcpl loaded trastuzumab INPs[3H]-pcpl loaded trastuzumab INPs

Time points: 5min, 1, 2, 6, 24 and 48 hTime points: 5min, 1, 2, 6, 24 and 48 h

Biodistribution and pharmacokinetics Biodistribution and pharmacokinetics

paclitaxel-palmitate concentration in blood with time

iv injection, N=4

Debotton et al., J. Cont. Rel.,127, Debotton et al., J. Cont. Rel.,127, 219-230, 2008.219-230, 2008.

FormulationFormulation Cmax, Cmax, µg/mlµg/ml

Terminal half Terminal half life, hlife, h

AUC,AUC,µg/ml X hµg/ml X h

MRT , MRT , hh

pcpl solutionpcpl solution 9.79.7 8.38.3 84.584.5 9.19.1

pcpl NPspcpl NPs 51.151.1 14.614.6 132.3132.3 12.212.2

pcpl immunoNPspcpl immunoNPs 45.245.2 2020 137.5137.5 15.315.3



Pharmacokinetics parameters in mice of [3H]-pcpl Pharmacokinetics parameters in mice of [3H]-pcpl following IV administration of 7.5mg/kg pcpl solution, pcpl following IV administration of 7.5mg/kg pcpl solution, pcpl NPs and pcpl immunoNPsNPs and pcpl immunoNPs

t ½ of trastuzumab was 75.1 h (Beige athymic female t ½ of trastuzumab was 75.1 h (Beige athymic female mice), Mandler et al, 2004 mice), Mandler et al, 2004

% [3H]-pcpl from initial dose/g tissue % [3H]-pcpl from initial dose/g tissue

Organ Organ Formulation Formulation 5min 5min 1h 1h 2h2h 6h6h 24h24h 48h48h

HeartHeartpcpl sol.pcpl sol.pcpl NPspcpl NPs

pcpl immunoNPspcpl immunoNPs

3.06±1.83.06±1.84.71±1.24.71±1.26.83±2.16.83±2.1

5.45±1.335.45±1.338.03±0.38.03±0.35.86±0.25.86±0.2

1.45±0.41.45±0.42.39±0.82.39±0.83.34±0.43.34±0.4

0.99±0.30.99±0.32.12±0.52.12±0.52.79±0.82.79±0.8

0.59±0.20.59±0.22.56±0.62.56±0.62.36±0.52.36±0.5

0.62±0.40.62±0.41.22±0.31.22±0.31.62±0.71.62±0.7

liverliverpcpl sol.pcpl sol.pcpl NPspcpl NPs


9.21±4.69.21±4.69.09±0.79.09±0.7

11.06±2.911.06±2.9

28.66±0.8728.66±0.87**58.92±1.7**58.92±1.727.61±2.827.61±2.8

6.39±16.39±1*43.49±2.4*43.49±2.428.87±5.828.87±5.8

8.04±2.28.04±2.2*61.21±9.8*61.21±9.837.42±4.737.42±4.7

3.97±0.33.97±0.3*51.45±7*51.45±737.54±1.237.54±1.2

2.02±0.52.02±0.5*32.71±4.1*32.71±4.1

24.11±424.11±4

KidneyKidneypcpl sol.pcpl sol.pcpl NPspcpl NPs


7.32±3.57.32±3.55.51±1.25.51±1.28.25±2.38.25±2.3

9.39±2.49.39±2.44.43±0.54.43±0.55.83±1.25.83±1.2

2.15±0.52.15±0.53.93±0.73.93±0.72.67±0.32.67±0.3

1.64±0.41.64±0.45.13±0.35.13±0.36.79±2.26.79±2.2

1.1±0.31.1±0.35.59±0.75.59±0.76.48±26.48±2

1.06±0.31.06±0.32.64±0.82.64±0.85.01±3.85.01±3.8

Spleen Spleen pcpl sol.pcpl sol.pcpl NPspcpl NPs


3.06±23.06±26.78±0.86.78±0.86.23±0.66.23±0.6

2.27±0.22.27±0.2*17.68±1.8*17.68±1.827.08±5.327.08±5.3

2.57±0.72.57±0.721.14±4.221.14±4.225.11±7.625.11±7.6

2.86±0.62.86±0.6**32.35±3**32.35±321.42±3.521.42±3.5

1.87±0.31.87±0.3*41.15±2.4*41.15±2.429.67±4.429.67±4.4

1.41±0.51.41±0.5*32.48±3.7*32.48±3.722.5±1.122.5±1.1

* P value of unpaired t test with Welch correction was at least 0.05* P value of unpaired t test with Welch correction was at least 0.05** P value of unpaired t test with Welch correction was at least 0.005** P value of unpaired t test with Welch correction was at least 0.005 Debotton et al., J. Cont. Rel.,127, 219-230, 2008.Debotton et al., J. Cont. Rel.,127, 219-230, 2008.





Pharmacokinetic behavior of immunoNPs was Pharmacokinetic behavior of immunoNPs was similar to that of NPs similar to that of NPs

MAb lost its intrinsic molecular pharmacokinetic MAb lost its intrinsic molecular pharmacokinetic properties properties

MAb will mainly function as a targeting moiety MAb will mainly function as a targeting moiety rather than a therapeutic moietyrather than a therapeutic moiety

More pronounced stealth character of INPs over More pronounced stealth character of INPs over NPs owing to the presence of the MAb NPs owing to the presence of the MAb

Routh and Leibovich, 2005Routh and Leibovich, 2005

Orthotopic prostate cancer model in mice


Second leading cause of cancer-related death for Second leading cause of cancer-related death for man in the USman in the US

All patients become refractory to androgen ablation: All patients become refractory to androgen ablation: hormone refractory prostate cancer (HRPC)hormone refractory prostate cancer (HRPC)

10-20% will develop metastases 10-20% will develop metastases

Management of metastatic prostate cancer: Management of metastatic prostate cancer: hormonal, radiotherapy, chemotherapy is usually hormonal, radiotherapy, chemotherapy is usually palliative palliative

Prostate cancer Prostate cancer

Tumor development

following PC-3.38 prostate

inoculation in SCID/Bg mice

Day 41Day 41Day 27Day 27

Day 60Day 60

Day 95Day 95 Day 105Day 105

Day 48Day 48



Prostate cancer over - expresses HER2

receptorHealthy

Cancer

Hematoxylin and Hematoxylin and Eosin stainingEosin staining

Immunostaining for Immunostaining for HER-2 receptorHER-2 receptor



Treatment groups:Treatment groups:

SalineSaline

Paclitaxel-palmitate solution Paclitaxel-palmitate solution

Paclitaxel-palmitate loaded NPs Paclitaxel-palmitate loaded NPs

Tastuzumab pcpl loaded immunoNPsTastuzumab pcpl loaded immunoNPs

10mg/kg pcpl, injected IV, once a week over 3 10mg/kg pcpl, injected IV, once a week over 3 weeks from day 54 following PC-3.38 prostate weeks from day 54 following PC-3.38 prostate inoculationinoculation

Effect of immunoNPs in-vivoEffect of immunoNPs in-vivo

Detection of tumor size in live mice by luciferase assay using CCCD

following 3 consecutive treatment injections of

different pcpl formulations. Mice pictures were taken

at day 95

Saline


Pcpl NPs trastuzumab NPs

pcpl sol.

N=8, values are mean±SE Debotton et al., J. Cont. Rel.,127, 219-230, 2008.Debotton et al., J. Cont. Rel.,127, 219-230, 2008.

1st inj 2nd inj 3rd injinoculation


Paclitaxel palmitate loaded INPs inhibit the tumor growth much more than the pcpl solution

and pcpl NPs (p< 0.05)

Statistical analysis day 95:

pcpl NPsTrastuzumab INPs

Trastuzumab INPs pcpl sol



Possible reasons for incomplete eradication of Possible reasons for incomplete eradication of tumors:tumors:

No effective chemotherapy for prostate cancerNo effective chemotherapy for prostate cancer

Regimen needs to be adjustedRegimen needs to be adjusted

VEGF play a crucial role in the VEGF play a crucial role in the neovascularization and progression of HRPC, neovascularization and progression of HRPC, and therefore, needs to be targetedand therefore, needs to be targeted

Conclusions IConclusions I

Optimal conditions were identified for the Optimal conditions were identified for the manufacturing of immunoNPs comprising manufacturing of immunoNPs comprising significant drug contentssignificant drug contents

ImmunoNPs were shown to bind specifically ImmunoNPs were shown to bind specifically to cells over expressing antigens recognized to cells over expressing antigens recognized by the mAbs by the mAbs

ImmunoNPs were shown to enhance drug ImmunoNPs were shown to enhance drug penetration markedly into cellspenetration markedly into cells

Conclusions IIConclusions II

Drug loaded immunoNPs were Drug loaded immunoNPs were significantly more toxic than NPs and significantly more toxic than NPs and drug solution on cancer cellsdrug solution on cancer cells

The affinity (KD) of the antibody is not The affinity (KD) of the antibody is not affected by the conjugation process affected by the conjugation process

ImmunoNPs did not activated the ImmunoNPs did not activated the complement system in complement system in ex vivoex vivo assay assay

Conclusions IIIConclusions III

Similar pk behavior of drug loaded NPs Similar pk behavior of drug loaded NPs and INPs was observed and INPs was observed

Coupling of the mAb to the NPs alters Coupling of the mAb to the NPs alters the intrinsic pk properties of the mAbthe intrinsic pk properties of the mAb

The INPs elicited a significant anti-The INPs elicited a significant anti-tumor activity as compared to pcpl tumor activity as compared to pcpl solution and NPs.solution and NPs.

antibody-nanocarrier conjugates for drug targeting and improved cancer therapy n. debotton 1, o....

Documents

drug targeting

npsdruginitial drug

mab conjugated nps

high drug payloads

studiesstable nps

lyophilized powders

ww drug contentmabs

loading efficiency