Immunoliposomes for Targeted
Radionuclide Therapy
Outline
• Liposomes
-
George Sgouros, Ph.D.
Russell H. Morgan Dept of Radiology
& Radiological Science
Johns Hopkins University, School of Medicine
Baltimore MD
Overview of structure and function
Enhanced Permeability and Retention (EPR) effect
PEG, size, charge
Doxil, Myocet
• Immunoliposomes
- Composition and structure – different types
- Mode of targeting/contrast w/ untargeted liposomes
• Immunoliposomes in targeted alpha-emitter
therapy
Liposome - structure
Liposome – tumor localization
Enhanced Permeability and Retention (EPR)
Page
A
Liposomes – Composition
B
• Phospholipids chosen
- Surface Charge - interaction with cells, cluster penetration
C
- Membrane fluidity - rigidity
• Size
- Diameter > 250 µm, localize to spleen
• long polymeric chains on surface (PEGylated)
- Evade RES system, increase circulation half-time
Tumor penetration of small unilamellar liposomes – dependence on composition.
Confocal microscopy equatorial slice (100±10 m) images of tumor spheroids following 2hr incubation with SUVs containing (a) DMPC:DC-chol, (b) DMPC:DOPE:DC-chol and (c)
DMPC:DOPE:DOTAP.
- Typically 5% mole of 2000 MW PEG
- Too much (>10%) – micelles, too little - phagocytosed
Kostarelos, et al., Int J Cancer, 2004
60.0
Biodistribution of Liposomes (PEG vs nonPEG) vs In-111
350.0
400 nm
100 nm
1 hr
50.0
300.0
6 hr
1 hr
h
11hour
6 hr
250.0
30.0
200.0
PEG
%ID/g
%ID/g
40.0
150.0
20.0
100.0
10.0
50.0
0.0
0.0
Bloo d
Heart
Lu n g
L iver
S pleen
Kid ney
Sto mach In testin e
M uscle
B on e
Bloo d
Heart
Lu ng
L iver
S pleen
Kid n ey
S to mach In testine
M u scle
Bo ne
1 hr
800
6 hr
Biodistribution of
100, 400 and
650 nm diameter
liposomes in
mice at 1 and 6
h after IV
injection; 6
mice/group
24 hr
ID%/g
600
400
200
At 650 nm, a
very rapid
sequestration to
spleen is shown
resulting in
spleen:liver &
spleen:lung of
~30 as early as
1 hr PI and
lasting to at least
6 hr PI.
In-111
650nm
Non-PEG
1000
0
Blood
Heart
Lung
Liver
Spleen
Kidney
Stomach Intestine
Muscle
Bone
Lymph
Page
h
66
hours
72
h
72
hours
Doxorubicin
Doxil
JAMA. 1991;266:1672-1677
®
Size = 100 nm
Type = large unilamellar vesicles
Hydrogenated soy phosphatidyl
choline (PC), cholesterol, PEG-2000,
distearoyl phosphatidyl ethanolamine
Drug/lipid weight = 0.125
Drug loading: ammonium ion gradient
Sulfate salt precipitate – “striated gel”
Immunoliposome - types
Size = 180 nm
Type = large oligolamellar vesicles
Egg phosphatidyl choline, cholesterol,
Drug/lipid weight = 0.27
Drug loading: hydrogen ion gradient
Citrate fiber bundles
Immunoliposomal - targeting
• No difference in overall tumor concentration
- Gross localization to tumor based on EPR effect
• Significant difference in drug delivery
Type A
Type B
- Increased interaction with target cell population
- Increased drug delivery to target cell population
Type C
= antibody or antibody fragment
= liposome
= PEG chain
Sofou S, Sgouros G. Expert Opin Drug Deliv. 2008
Page
Immunoliposome - targeting
Lipid (tritiated cholesterol)
{
fluorescence
In vivo uptake of liposomes and immunoliposomes in the U87/EGFRvIII tumor xenograft
model
- untargeted
- targeted
- doxorubicin
Mamot, C. et al. Cancer Res 2005;65:11631-11638
Mamot, C. et al. Cancer Res 2005;65:11631-11638
Copyright ©2005 American Association for Cancer Research
Ab targeting – Metastatic dissemination
Therapeutic efficacy of anti-EGFR immunoliposome-doxorubicin in EGFR-overexpressing tumor
models
MDA-MB-468 (5 x 105 sites/cell)
U87 (1-2 x 105 sites/cell)
penetration
Y
penetration
Mamot, C. et al. Cancer Res 2005;65:11631-11638
Copyright ©2005 American Association for Cancer Research
Page
Y
extravasation
Alphas vs Betas
Animal Model: LCV injection
alphas
Survival Curve
SM
- He nucleus (4 amu)
- 80 keV/µm
- 2 to 3 tracks kill cell
- Irrepairable DNA damage
- potent single cell, cluster kill
1.2
Cumulative Survival
T
VB
betas (electrons)
- 0.2 keV/µm
- 103 to 104 tracks to kill cell
- DNA damage is repaired
- cross-fire required
H
1.0
0.8
0.6
0.4
0.2
0.0
0
10
20
30
40
D ays
B3 vertebra lysis 50X
Bright red arterial blood indicates
B4 vertebra
lysis and impinge spinal cord 25X
5
10 NT2 cells, bone, liver metastases,
survival for 3.5 to 4.0 weeks,
90% successful inoculation.
TB = trabecular bone; VB = vertebral bone; BV = blood vessel; SC = spinal cord;
successful inoculation
SM = skeletal muscle; T = tumor cells H = hematopoietic cells
MR image slices: 0.8 mm thick,
diffusion-weighted, showing
multiple tumor foci, emanating
from marrow spaces in both
femurs and vertebral bodies.
Slices 2 - 4, broken contours
depicts tumor emanating from
left hind limb. Slice 3 region of
enhancement has a horizontal
diameter of 3 mm. Slices 4 & 5
rectangles: tumor emanating
from the marrow region of the
right hind limb. Slice 6: two foci
of high intensity –tumor
originating in the marrow space
of vertebral bodies. Slice 7:
disease emanating from
marrow within a vertebral body.
Mouse survived the imaging
sessions and was sacrificed
soon after completion of
imaging.
Left: Image of excised liver, 4-wks after LCV injection. Right: Section of the liver
containing a metastatic tumor mass. At the periphery of the mass is a thick rim of viable
tumor cells that infiltrate and compress adjacent hepatic parenchyma. At the center, a
large area of tumor necrosis with remnants of congested vasculture and lesser amounts
of hemorrhage and inflammatory cells.
Page
111In-Ab
α-Radioimmunotherapy
targeting of metastases
and
radiation
particles
*
1h PI
24 2h PI
6 h PI
484h PI
particles
Dimension /
range (µm)
35-100
LET in tissue
(keV/µm)
80-150
0.1-3.0
# of DNA hits
required to
inactivate a
cell
1-5
100-1,000
3,000-8,000
tumor
Specific activity of antibody
Therapy
short
penetration
and short half
life
Alpha-Immunoliposome construction
1 Bi-213 / ~1900 antibody
wide
penetration
and extended
damage
Liposome Immunoreactive fraction
lindmo assay
55˚C extrusion, 100nm membrane
Liposomes (~100nm)
DMPE + Cholesterol + DSPE-PEG
pH 8-9
300
Liposomes-DTPA
DSPE-PEG-maleimide + thio-Ab
Liposomes -DTPA + DSPE-PEG-Ab
Immunoliposomes-DTPA + Bi-213
overnight
55 ˚C, 1h
pH 4
Total/bound
total/specific
activity
Liposomes + DTPA
350
DSPE-PEG-Ab
Immunoliposomes-DTPA
Immunoliposomes-Bi-213
250
200
Liposome, no Ab
Liposome, with Ab
150
100
50
Specific activity of 0.1 Bi-213/liposome
Ab
Bi-213
0
0.00E+00 2.00E-06 4.00E-06 6.00E-06 8.00E-06 1.00E-05 1.20E-05
>90% stability after 4h in 10% serum
1/cell#
1/intercept
Immunoliposome 12.82051%
Liposome 4.56621%
Page
Metastatic BCa Targeted alpha-radiotherapy
In-vitro Cell Kill Assay
ANTIBODY
LIPOSOME
100
% survival
Liposome-CHX-DTPA-Bi-213(7.16.4)
Liposome-CHX-DTPA-Bi-213
Antibody, 7.16.4-Bi-213
D0 values:
Liposome, 14 µCi/ml
Immunoliposome, 8 µCi/ml
Antibody, 3.3 µCi/ml
10
Median Survival Time
Control
29.5 days (n=4)
Cold Lipo
1
0
20
40
Lipo-Bi-213
60
µCi/mL
28 days (n=5)
39 days (P < 0.002) (n=5)
Immunolipo-Bi-213(7.16.4)
47.5 days (P < 0.006 ) (n=4)
Antibody: 120µCi of Bi-213-7.16.4
Acknowledgments
Hong Song
Andy Prideaux
Yah-el Har-el
Mohana Lingappa
Rob Hobbs
Caroline Esaias
Karineh Shahverdi
Kostas Kostarelos
Stavroula Sofou
Todd Reilly
Elizabeth Jaffee
Zaver Bhujwalla
Barjor Gimi
VP Chacko
Martin Pomper
Benjamin Tsui
James Fox
Yuchuan Wang
NIH:
Erik Brady
Martin Brechbiel
Diane Milenic
Institute for Transuranium Elements (ITU),
Karlsruhe, Germany:
Christos Apostolides
Alfred Morgenstern
NIH
DOD
David Huso
Kathy Gabrielson
Page
41 days