Available via license: CC BY 4.0
Content may be subject to copyright.
Photodynamic therapy (PDT) is an experimental approach for
cancer treatment in which the local or systemic delivery of a
photosensitizer (PS) is followed by tissue illumination with light
of an appropriate wavelength (usually red light delivered by a
laser) (Hasan and Parrish, 1996; Dougherty et al, 1998).
Conventional PS may have some selective accumulation in
tumours (Henderson and Dougherty, 1992; Hamblin and Newman,
1994), but increased tumour targeting may be obtained by the use
of macromolecular carriers which form complexes or covalent
conjugates with PS (Hasan, 1992). The targeting capability of
these carriers may rely on physical properties of the carrier (lipo-
somes and microspheres (Speiser, 1991)), molecular properties
(molecular weight and charge (Kornguth et al, 1989)) or the
specific recognition of molecules associated with tumours (mono-
clonal antibodies (mAbs), lipoproteins and growth factor receptor
ligands). They have been used to deliver cytotoxic drugs (Johnson
et al, 1995), radioisotopes (Buchsbaum et al, 1993a), protein
toxins (Houston, 1993) and PS to tumours, but the latter has the
distinct advantage of not being toxic until illuminated with acti-
vating light, thus reducing toxicity due to non-specific uptake of
the conjugate which can be a problem with conjugates formed
from toxins and radioisotopes. Conjugates between mAbs and PS
have shown promise both in vitro (Pogrebniak et al, 1993;
Vrouenraets et al, 1999) and in experimental animal models of
cancer (Mew et al, 1983; Goff et al, 1996), but have received only
minimal clinical testing (Schmidt et al, 1992). The charge borne by
the immunoconjugate may markedly influence the pharmaco-
kinetics and biodistribution (Slinkin et al, 1993), and manipulation
of the overall charge may increase the therapeutic ratio.
Colorectal cancer is in need of novel and effective therapies,
and certain aspects of it may be appropriate for treatment by PDT.
The ability to selectively target colorectal cancer cells may have
applications to the tumour bed after surgical resection of the
primary tumour (Barr et al, 1990), to disseminated intraperitoneal
carcinomatosis (Veenhuizen et al, 1996), and to the liver metas-
tases (Van Hillegersberg et al, 1992a) which are a frequent cause
of death (Benotti and Steele, 1992). Because of the sensitive
tissues in the peritoneal cavity (Veenhuizen et al, 1997), and the
high accumulation of conventional PS in normal liver (Van
Hillegersberg et al, 1992b), it is attractive to explore the use of
photoimmunoconjugates (PICs) constructed from mAbs targeted
against colorectal cancer antigens, to increase the selectivity of the
PS for tumour over normal tissue. The epithelial membrane
antigen (a homophilic cell–cell adhesion molecule) which is
recognized by several mAbs (including 17.1A) is overexpressed
on many cancers of the gastrointestinal tract (Litvinov et al, 1994).
The murine monoclonal IgG 17.1A has been used clinically to
treat human colorectal cancer both in an unconjugated form to
induce antibody-dependent cellular cytotoxicity (Riethmuller et al,
1994) and as radioimmunoconjugates to target radioisotopes to
residual tumour (Buchsbaum et al, 1993b). As part of our on-going
effort to optimize intraperitoneal photoimmunotherapy (PIT)
(Goff et al, 1991, 1994, 1996) we have recently described the
preparation of charged PICs between the F(ab′)2fragment of anti-
ovarian cancer Mab OC125 and the PS chlorine6 (ce6) (Hamblin et
al, 1996), and have investigated their biodistribution in vivo
(Duska et al, 1997). Toward the long-term goal of applying PIT to
intraperitoneal and hepatic metastases of colorectal cancer, we
report here our initial studies on the preparation of charged PICs
Targeted photodestruction of human colon cancer cells
using charged 17.1A chlorin
e6
immunoconjugates
M Del Governatore1,2, MR Hamblin1, EE Piccinini2, G Ugolini2and T Hasan1
1Wellman Laboratories of Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA;
2Department of Chirurgia III, Ospedale Policlinico S. Orsola, Via Massarenti 9, Bologna 40100 Italy
Summary The goal of this study was to develop a strategy for the selective destruction of colorectal cancer cells. Towards this end,
photoimmunoconjugates were prepared between the anti-colon cancer monoclonal antibody 17.1A and the photosensitizer (PS) chlorin
e6
(c
e6
). Polylysine linkers bearing several c
e6
molecules were covalently attached in a site-specific manner to partially reduced IgG molecules,
which allowed photoimmunoconjugates to bear either cationic or anionic charges. The conjugates retained immunoreactivity as shown by
enzyme-linked immunosorbent assays and by competition studies with native antibody. The overall charge on the photoimmunoconjugate
was an important determinant of PS delivery. The cationic photoimmunoconjugate delivered 4 times more c
e6
to the cells than the anionic
photoimmunoconjugate, and both 17.1A conjugates showed, in comparison to non-specific rabbit IgG conjugates, selectivity for antigen-
positive target cells. Illumination with only 3 J cm–2 of 666 nm light reduced the number of colony forming cells by more than 90% for the
cationic 17.1A conjugate and by 73% for the anionic 17.1A conjugate after incubation with 1 µMc
e6
equivalent of the respective conjugates.
By contrast, 1 µM free c
e6
gave only a 35% reduction in colonies. These data suggest photoimmunoconjugates may have applications in
photoimmunotherapy where destruction of colorectal cancer cells is required. © 2000 Cancer Research Campaign
Keywords: photodynamic therapy; photoimmunotherapy; photosensitizer; polylysine; laser
56
British Journal of Cancer
(2000) 82(1), 56–64
© 2000 Cancer Research Campaign
Article no. bjoc.1999.0877
Received 20 April 1999
Revised 13 July 1999
Accepted 16 July 1999
Correspondence to:
T Hasan
17.1A photoimmunoconjugates
57
British Journal of Cancer (2000) 82(1), 56–64
© 2000 Cancer Research Campaign
with the intact mAb 17.1A and their in vitro interactions with a
human colorectal cancer cell line recognized by the mAb (HT29)
and a cell line which showed no binding to the mAb (OVCAR-5).
MATERIALS AND METHODS
Cell line and monoclonal antibody
Two tumour cells lines were employed. HT29 cells derived from a
human colorectal adenocarcinoma were a generous gift from Dr K
Tanabe (Massachusetts General Hospital, Boston, MA, USA).
NIH:OVCAR-5 human ovarian cancer cells were purchased from
Dr T Hamilton (Fox Chase Cancer Center, Philadelphia, PA,
USA). The growth medium for HT29 was DMEM/F12 (50/50
mixture) and that for OVCAR-5 was RPMI-1640. Both media
contained 15 mMHEPES and L-glutamine and were supplemented
with 10% heat-inactivated fetal calf serum (FCS) (Whittaker
Bioproduct, Walkersville, MD, USA), 100 U ml–1 penicillin and
100 µg ml–1 streptomycin, and maintained in an incubator at 37°C
in an atmosphere of 5% carbon dioxide. 17.1A murine mAb was a
kind gift from Centacor (Malvern, PA, USA). Rabbit IgG and
mouse IgG was obtained from Sigma (St Louis, MO, USA).
Conjugation procedure
The procedure has been described in detail elsewhere (Hamblin et
al, 1996). Briefly poly-L-lysine (average MW 25 000) was treated
in DMSO with the N-hydroxysuccinimide ester of ce6 to give pl-
ce6. This was then reacted with pyridyldithiopropionic acid N-
hydroxysuccinimide ester (SPDP) to form the functionalized
derivative pl-ce6-SPDP. This was then split into two parts and one
part was treated with an excess of succinic anhydride to give the
negatively charged functionalized pl-ce6-succ-SPDP. 17.1A mAb
was reduced for 1 h with 5 mMmercaptoethylamine hydro-
chloride, dialysed (1 mMEDTA) and then reacted with either
pl-ce6-SPDP or pl-ce6-succ-SPDP to form the cationic and anionic
PICs respectively. The procedure was repeated with rabbit IgG.
The conjugates were purified by chromatography on Sephadex
G200 columns, and characterized by absorption and fluorescence
spectrophotometry, and polyacrylamide gel electrophoresis. The
structures of the PICs are shown in Figure 1.
Enzyme-linked immunosorbent assay
Cells were grown to 100% confluence in 96-well plates for 24 h
with medium containing FCS, than washed 3 times with PBS and
fixed with 0.25% glutaraldehyde. After 1 h cells were washed with
PBS and plates blocked with PBS containing 5% FCS for 1 h, then
0.1 ml PBS containing the appropriate dilution of PIC or mAb was
added to each well. After 2 h incubation at room temperature in
the dark, wells were washed 3 times with PBS containing 0.05%
Tween 20 and had added 0.1 ml of horseradish peroxidase conju-
gated-F(ab′)2fragment rabbit anti mouse IgG, IgA, IgM (h+l)
(Zymed Laboratories, South San Francisco, CA, USA) diluted
1:200 in PBS containing 0.5 mg ml–1 bovine serum albumin (BSA)
and 0.05% Tween-20. Cells were incubated for 2 h at room
temperature in the dark then washed 3 times with PBS containing
0.05% Tween-20 and added 0.1 ml of 0.4 mg ml–1 of freshly
prepared o-phenylenediamine dissolved in 0.05 Msodium citrate,
0.15 Msodium phosphate, pH 6, containing 32% vol/vol of 30%
hydrogen peroxide and incubated for a further hour at room
temperature in the dark. Absorbance was read at 492 nm with an
enzyme-linked immunosorbent assay (ELISA) reader (500 EIA,
Bio-Rad Laboratories, Hercules, CA, USA).
Figure 1 Structures of the PICs. 17.1A-pl-c
e6
has primary amino groups which give it a polycationic charge, while 17.1A-pl-c
e6
-succ has carboxylic groups
which give it polyanionic charge
58
M Del Governatore et al
British Journal of Cancer (2000) 82(1), 56–64 © 2000 Cancer Research Campaign
Two-colour direct/indirect immunofluorescence
Approximately 3 ×105cells were plated in 35-mm tissue culture
dishes, for 24 h with 2 ml of medium, containing coverslips that
were previously washed with 95% ethanol and flamed. After 24 h
of incubation in 2 ml of medium, cells were washed with PBS
(23) and fixed with 2% formaldehyde at room temperature for
5 min. Cells were washed with PBS/1% BSA (33), and incubated
for 1 h at room temperature with the mAb or conjugate, the
amount of mAb in both cases was 2 µg ml–1. After this time they
were gently washed with PBS/1% BSA (33) and incubated for 1
h with or without fluorescein isothiocyanate-conjugated goat anti-
mouse IgG (FITC-GAM, Sigma) diluted 1:128 with PBS/1%
BSA. Cells were washed with PBS/1% BSA (33) and mounted
on glass microscope slides using GelMount (Biomeda Corp.,
Foster City, CA, USA). An epi-illumination microscope (Model
WL, Zeiss, Oberkochen, Germany) equipped with a CCD camera
(TM 745, Pulnix, Sunnyvale, CA, USA), image intensifier
(M942, Litton Electron Devices, Tempe, AZ, USA), video
monitor and computer was used to capture digital images. Two
different combinations of filters were used. The first set used an
excitation bandpass filter at 450–490 nm and emission 514–530
nm bandpass filter designed for visualizing fluorescein fluores-
cence. The second set used an excitation bandpass filter at
402–447 nm and emission 580 nm longpass filter designed for
visualizing ce6 fluorescence. The images obtained with the ce6
filter combination viewing cells incubated with unconjugated
17.1A and FITC-GAM were totally negative, while those
obtained with cells incubated with a ce6 conjugate without FITC-
GAM and the fluorescein filter set gave a faint image.
Cellular uptake
Twelve-well plates containing 90% confluent cells had 1 ml
medium containing 10% FCS and conjugates added. After the
completion of the incubation time cells were washed with PBS
(33) and incubated with trypsin (0.25%)/EDTA (0.02%) (1 ml) at
37°C for 20 min. The suspension was centrifuged and the resulting
cell pellet dissolved for 48 h in 1.5 ml of 0.1 Msodium hydroxide
(NaOH); 1% sodium dodecyl sulphate (SDS) to give a homo-
geneous solution. Fluorescence was measured with a fluorometer
(Fluorolog 2, Spex Industries, Edison, NJ, USA) (excitation at 400
nm, emission scanned from 580 to 720 nm). The trypsin super-
natant was checked for the presence of fluorescence, which was
always less than 10% of the cell extract. The cell digest was then
assayed for the amount of cell protein by a modified Lowry pro-
cedure (Larson et al, 1986). Quantitation of ce6 concentration in the
cell extracts was obtained by comparing the fluorescence of stan-
dard solutions of the same conjugate of known concentrations in
0.1 MNaOH 1% SDS. The fluorescence of the cell extracts was
always within the linear part of the standard curve. Uptake ex-
periments at 4°C were carried out by adding pre-cooled medium
containing conjugates to the wells in 12-well plates, which were
then wrapped in aluminium foil and incubated in crushed ice for
6h.
Competition
Studies were conducted in which the cells were preincubated with
a saturating concentration of unmodified 17.1A, to see if the
binding of the PICs was blocked. Since 1 ml of 1 µM ce6 equiva-
lent PIC contains approximately 7.5 µg mAb, a fivefold excess of
competing 17.1A or rabbit IgG (37.5 µg) was employed. The
conjugate was added at a concentration of 1 µM ce6 equivalent and
incubated for 2 h. The uncompeted uptake was compared to the
uptake found by the addition of 17.1A or rabbit IgG (37.5 µg ml–1)
for 1 h, followed by washing and addition of PIC (1 µM ce6 equiv-
alent) for 1 h.
Phototoxicity
Phototoxicity was measured by a colony forming assay. In P35
dishes cells were grown to 70% confluence in 2 ml medium
containing 10% FBS. Cells were washed with PBS and 1 µM ce6
equivalent of PICs and PS in serum containing medium was
added. After 6 h incubation at 37°C, the medium was removed,
cells were washed twice with PBS, and 2 ml of fresh serum
containing medium was added. The dishes were illuminated with
666 nm light for ce6 conjugates or 654 nm light for ce6 at a power
density of 48 mW cm–2, measured with a power meter (Model 210,
Coherent Inc., Palo Alto, CA, USA). An argon-pumped dye laser
(Innova 100 and 599 Dye, Coherent) was focused through a 310
microscope lens onto the end of a 1 mm diameter optical fibre
which delivered light through an inverted 34 microscope lens (No
774317, Olympus, Tokyo, Japan) to give a 35 mm diameter spot
for irradiation. Controls were as follows: no conjugate and no light
remaining in incubator throughout, no conjugate and no light but
plates wrapped in foil for duration of the irradiation time out of the
incubator, conjugate given and no light wrapped in foil, no conju-
gate and irradiated. At the completion of irradiation the cells were
given fresh medium containing FCS and returned to the incubator
for 24 h. The cells were then washed with medium and any
detached cells aspirated off. The remaining cells were detached
with trypsin/EDTA (0.5 ml) and an aliquot counted for viable cells
using the trypan blue exclusion assay and a haemocytometer. The
cell suspension was diluted with medium and plated in P60 dishes
containing 4 ml of medium at densities of 50, 100, 150 and 200
cells per plate. When the colonies had formed (9 days later) the
cells were fixed with 0.2% formalin (vol/vol) in MeOH and
stained with crystal violet. The number of colonies, which
contained 50 or more cells, was then counted. The survival frac-
tion was calculated by multiplying the fraction of viable cells at
the counting stage compared to controls (sensitizer, dark, out of
incubator), together with the fraction of colonies formed by treated
cells compared to controls.
RESULTS
Absorbance spectroscopy
The absorption spectra of the cationic and anionic PICs together
with unconjugated ce6 are shown in Figure 2. The conjugates have
a distinct absorbance at 280 nm due to the protein in the IgG, the
intensity of the Soret band is somewhat reduced, and the long
wavelength Q band is red shifted to 666 nm as opposed to 654 nm.
It can be calculated assuming extinction coefficients of 1.5 ×105
lmol–1 cm–1 for ce6 at the Soret band and 2.4 ×105lmol–1 cm–1 for
IgG at 280 nm, that the anionic 17.1A-pl-ce6-succ had 8–9 ce6
molecules attached to each IgG (two polylysine chains per IgG),
while the cationic 17.1A-pl-ce6 had a lower loading of 4–5 ce6
molecules per IgG (one polylysine chain per IgG).
17.1A photoimmunoconjugates
59
© 2000 Cancer Research Campaign
ELISA
Results of ELISA binding assays using fixed cells and assaying
binding of 17.1A mAb, 17.1A-pl-ce6, 17.1A-pl-ce6-succ and non-
specific mouse IgG are shown in Figure 3. The unmodified mAb
exhibited a typical binding curve with reduction in binding over the
concentration range 10–0.1 µg ml–1 protein. The anionic 17.1A-pl-
ce6-succ showed a somewhat reduced affinity, while the cationic
17.1A-pl-ce6 showed a slightly higher affinity compared to native
17.1A. As expected, non-specific mouse IgG showed no binding.
Two-colour direct/indirect immunofluorescence
microscopy
Two-colour direct/indirect immunofluorescence microscopy was
carried out by treating fixed cells with a PIC, which binds to
membrane antigens, and then adding a FITC-conjugated goat anti-
mouse second antibody that recognizes the murine IgG of the first
mAb. Fluorescent images were obtained with a dual filter system
capable of isolating green fluorescence (510 nm) emitted by FITC,
from red fluorescence (670 nm) emitted by ce6. Non-matching
filter/fluorophore combinations, i.e. green emission filter and ce6
alone, and red emission filter and FITC alone gave negative images
(data not shown). Images with similar appearance were obtained
with both direct ce6 and indirect FITC fluorescent emissions from
17.1A-pl-ce6 bound to fixed HT29 cells (Figure 4 A, B). In a similar
fashion images obtained from 17.1A-pl-ce6-succ with both ce6 and
FITC emissions were strikingly similar (Figure 4 C, D).
Cellular binding and uptake
The uptake of ce6 per mg cell protein from the 17.1A-pl-ce6 and
17.1A-pl-ce6-succ in a range of concentrations of PICs (measured
as ce6 equivalent in the medium) is shown in Figure 5. The uptake
of ce6 equivalent from the cationic PIC is up to 4 times higher than
that obtained from the anionic PIC after 6 h incubation, and both
show linear relationships with increasing concentration. This
linear increase is consistent with internalization of the conjugates.
A concentration of 3 µMce6 equivalent is equivalent to approxi-
mately 300–500 nM17.1A, which is much higher than the binding
constant of the typical mAb. Thus if there was no internalization
some saturation of uptake should have been observed. The cellular
uptake of ce6 obtained with the 17.1A PICs were compared to that
obtained with non-specific rabbit IgG PICs, and free ce6 (Table 1)
under the same incubation conditions (1 µMce6 equivalent, 6 h
incubation at 37°C in serum containing medium). Free ce6 gave the
lowest uptake followed by rabbit IgG-pl-ce6-succ and 17.1A-pl-ce6-
succ. The cationic rabbit IgG-pl-ce6 gave roughly twice the uptake
of its anionic counterpart. The 17.1A conjugates gave the highest
uptakes among conjugates bearing the same charge, and the
cationic 17.1A-pl-ce6 gave 3 times the uptake of the anionic 17.1A-
pl-ce6-succ.
1.2
1
0.8
06
0.4
0.2
0
200 300 400 500 600 700
Wavelength (nm)
Absorbance
17.1A-pl- c
e6
17.1A-pl- c
e6
-succ
c
e6
17.1A mAb
17.1A Cationic PIC
17.1A Anionic PIC
mouse IgG
0.64
0.56
0.48
0.4
0.32
0.24
0.16
0.08
00.01 0.1 1 10
mAb (µg ml–1)
Absorbance at 492 nm
Figure 2 Absorption spectra. Conjugates and free c
e6
were dissolved in
0.1 MNaOH/1% SDS at a concentration of 7 µM c
e6
equivalent Figure 3 ELISA assays on fixed cells. Fixed cells were incubated with a
dilution of mAb or PIC, then treated with HRP-conjugated rabbit anti-mouse
F(ab′)2 fragment and colour developed with o-phenylenediamine and H2O2.
Each point is the mean of six wells and bars are the s.e.m.
Table 1 Comparison of uptake after incubation at 37°C and 4°C
Uptake at 37°C Uptake at 4°C Ratio
17.1A-pl-ce6 2.4 ±0.17 1.4 ±0.053 1.71
17.1A-pl-ce6-succ 0.73 ±0.037 0.23 ±0.006 3.17
Rabbit IgG-pl-ce6 1.1 ±0.064 0.65 ±0.035 1.53
Rabbit IgG-pl-ce6-succ 0.32 ±0.022 0.071 ±0.003 4.5
ce6 0.063 ±0.02 0.025 ±0.002 2.47
Uptake was determined after incubation with 1 µMc
e6
equivalent
concentration for 6 h. Concentrations are expressed as nmol c
e6
equivalent/mg cell protein. Each value is the mean of values from two
separate experiments each containing three wells ±s.e.m.
British Journal of Cancer (2000) 82(1), 56–64
Effect of incubation temperature on uptake
In order to gain additional information on the degree to which the
PIC which is bound to the cells is internalized, the uptake after
incubation at 37°C was compared to that found after incubation at
4°C. The concentration was 1 µMce6 equivalent and the incubation
time was 6 h. The results are given in Table 1. It can be seen that
the cationic species have about 65% of the uptake remaining at
4°C, while the anionic conjugates have only 30% remaining.
However, the absolute amount of the conjugate internalized
(difference between uptakes at 37°C and 4°C is greater for the
cationic species than the anionic species. Note that the uptake of
free ce6 is also significantly greater at 37°C than 4°C.
Comparison of uptake with non-target cell line
OVCAR-5 human ovarian carcinoma cell line was used as a non-
target control compared to HT29 cells. Indirect immunofluores-
cence using 17.1A mAb and FITC-goat anti-mouse IgG on fixed
OVCAR-5 cells gave a negative fluorescence image (data not
shown) indicating that OVCAR-5 cells expressed very low levels
of EpCAM, but the two-colour direct/indirect immunofluores-
cence procedure with both the cationic and anionic 17.1A PICs
gave weak images for both ce6 and FITC fluorescence (data not
shown) indicating a small amount of non-specific binding of the
PICs to OVCAR-5 cells.
The uptake of ce6 by live OVCAR-5 and HT29 cells from the
cationic and anionic 17.1A PICs and from cationic and anionic
non-specific rabbit IgG PICs was compared and the results are
shown in Table 2. Almost no difference was found between target
and non-target cell line using either the cationic and anionic non-
specific rabbit IgG PICs as expected, whereas the uptake from
both the cationic and anionic 17.1A PIC showed twofold selec-
tivity for the target HT29 cells compared to the non-target
OVCAR-5 cells.
Competition of uptake by unmodified proteins
In order to further understand to what extent the observed uptake
was due to antigen binding and what extent to non-specific charge
interaction, uptake of ce6 from cationic and anionic 17.1A PICs by
HT29 cells was compared with and without saturation of the
antigens by preincubation with unmodified 17.1A (Figure 6). The
uptake of ce6 from the cationic 17.1A-pl-ce6 was reduced to 30% of
the control level by preincubation with 17.1A, while that of the
anionic 17.1A-pl-ce6-succ was reduced to 10% of the control level.
That this reduction was due to blocking of the antigenic binding
sites by 17.1A is confirmed by the observation that preincubation
with rabbit IgG had no effect on subsequent binding of 17.1A PICs.
As expected the uptake of the cationic PIC had a greater contribu-
tion from non-antigen mediated charge interactions than the anionic
PIC.
60
M Del Governatore et al
British Journal of Cancer (2000) 82(1), 56–64 © 2000 Cancer Research Campaign
ABCD
Figure 4 Two-colour direct/indirect immunofluorescence microscopy with fixed HT29 cells incubated with PIC at 2 µg ml-1 protein concentration,
followed by FITC-conjugated goat anti-mouse second antibody and imaged with a filter set which isolated green from red fluorescence. Bar
represents 20 µm. (A, B) 17.1A-pl-c
e6
,
(A) c
e6
fluorescence, (B) FITC fluorescence. (C, D) 17.1A-pl-c
e6
-succ, (C) c
e6
fluorescence, (D) FITC
fluorescence
0 0.5 1 1.5 2 2.5 3 3.5
7
6
5
4
3
2
1
0
Nmol c
e 6
/mg cell protein
Concentration c
e 6
equivalent (µm)
Figure 5 Concentration dependence of cellular uptake of c
e6
from cationic
17.1A-pl-c
e6
and anionic 17.1A-pl-c
e6
-succ. Cells were incubated for 6 h at
37°C for each PIC and cellular fluorescence was measured after extraction
into 0.1 MNaOH/1% SDS and expressed in nmol c
e6
per mg cell protein.
Each point is the mean of values from two separate experiments each
containing three wells and bars are s.e.m.
Table 2 Selectivity of conjugates for HT29 cells compared to OVCAR-5 cells
Conjugate HT29 cells OVCAR-5 cells Selectivity for HT29
17.1A-pl-c
e6
2.45 ±0.17 1.3 ±0.06 1.9 : 1 (
P
< 0.01)
17.1A-pl-c
e6
-succ 0.74 ±0.04 0.34 ±0.02 2.2 : 1 (
P
< 0.01)
Rabbit IgG-pl-c
e6
- 1.0 ±0.06 0.9 ±0.01 1.1 : 1 (n.s.)
Rabbit IgG-pl-c
e6
-succ 0.29 ±0.01 0.34 ±0.02 0.9 : 1 (n.s.)
Uptake was determined after incubation at 1 µMc
e6
equivalent concentration
for 6 h at 37°C using non-specific rabbit IgG anionic and cationic PICs and
17.1A cationic and anionic PICs. Cellular fluorescence was measured after
extraction into 0.1 MNaOH/1% SDS and expressed in nmol c
e6
equivalent/mg cell protein. Each value is the mean of values from two
separate experiments each containing three wells ±s.e.m. Significance was
assessed by two-tailed unpaired Student’s
t
-test.
Phototoxicity
Cells were illuminated after 6 h incubation with 1 µMce6 equiva-
lent of all the compounds. The phototoxicity was measured by
using a colony forming assay that combines short-term and long-
term damage to the cells. The 17.1A cationic and anionic PICs
(with HT29 target cells) showed fluence-dependent phototoxicity
with greater than 99.9% killing after 10 J cm–2 666 nm red light
(Figure 7A). To compare the phototoxicities of different pairings
of cells and PICs we carried out experiments at a constant fluence
of 3 J cm–2 and the results are shown in Figure 7B. Under these
conditions 10% of HT29 cells survived after treatment with
17.1A-pl-ce6 and 27% survived after PIT with 17.1A-pl-ce6-succ.
Comparing these results with the killing produced by the non-
specific rabbit IgG PICs of the same overall charges the difference
is seen to be significant; four times the number of surviving of
cells with the rabbit IgG cationic PIC compared to the 17.1A
cationic PIC, and three times more cells surviving after PIT with
the anionic rabbit IgG PIC compared to the 17.1A anionic PIC.
The free ce6 had a very low phototoxicity compared to both the
17.1A PICs. Cell type selectivity of the 17.1A PICs was shown by
using OVCAR-5 cells as a non target cell line; the survival was
3–4 times higher than the target HT29 cell line gave. Interestingly
OVCAR-5 cells were killed by PIT with the 17.1A cationic and
anionic conjugates to almost exactly the same extent as the non-
specific rabbit IgG PICs killed HT29 cells as shown in Figure 7B.
DISCUSSION
Additional therapies to target colorectal cancer metastases in the
liver are urgently needed (Van Cutsem, 1996). Existing treatments
include surgery (Steele and Ravikumar, 1989), chemoemboliza-
tion (Sanz-Altamira et al, 1997), regional chemotherapy
(McMurrick and Nelson, 1997) and cryotherapy (Yeh et al, 1997).
Among investigative treatments some workers are exploring the
role of PDT (Van Hillegersberg et al, 1992a). A drawback of PDT
in the liver is the high accumulation of most free PS in normal liver
(Van Hillegersberg et al, 1992b). The preparation of PICs that
could target tumour-associated antigens on the colorectal cancer
metastasis as opposed to normal liver tissue may increase the
specificity of PDT for liver metastases. In addition the dissemi-
nated intraperitoneal spread of colorectal cancer may also be
treated by PDT (Veenhuizen et al, 1996), but again additional
selectivity for the tumour is necessary to prevent unwanted
17.1A photoimmunoconjugates
61
British Journal of Cancer (2000) 82(1), 56–64
© 2000 Cancer Research Campaign
1.5
1
0.5
017.1A-pl- c
e6
17.1A- pl- c
e6
-succ
nmol c
e 6
/mg cell protein
PIC only
MAb+ PIC
Rabbit IgG + PIC
Figure 6 Inhibition of uptake by preincubation of cells with unmodified Mab.
Cells were incubated for 2 h at 37°C with the 17.1A cationic or anionic PICs
at the concentration of 1 µMc
e6
equivalent that contains 7.5 µg 17.1A mAb
(PIC only). Other dishes of cells were incubated for the first hour with the
unmodified 17.1A mAb or unmodified rabbit IgG at the amount of 37.5 µg
(fivefold excess over the protein contained in the PICs) and for a further hour
with 17.1A cationic or anionic PICs (mAb+PIC) or (rabbit IgG + PIC). Cellular
fluorescence was measured after extraction into 0.1 MNaOH/1% SDS and
expressed in nmol c
e6
/mg cell protein. Each point is the mean of values from
two separate experiments each containing three wells and bars are s.e.m.
100
10
1
0.1
0.01 024681012
J cm-2 of 666 nm light
17.1A-pl-c
e 6
17.1A-pl-c
e 6
-succ
Surviving cells (% of control)
A
10
c
e 6
Cells surviving (% of controls)
BHT 29
OVCAR- 5
100
RIgG-pl-c
e 6
RIgG-pl-c
e 6
-succ
17.1A-pl- c
e 6
-succ
17.1A-pl- c
e 6
Figure 7 Phototoxicity of conjugates. (A) Fluence response survival
curve for HT29 cells treated with 17.1A-pl-c
e6
and 17.1A-pl-c
e6
-succ.
(B) Comparison of survival of HT29 cells and OVCAR-5 cells treated with
specific and non-specific PICs at a fluence of 3 J cm–2. Cells were
incubated for 6 h at 37°C with 1 µMc
e6
equivalent of: free c
e6
, non-specific
cationic rabbit IgG-pl-c
e6
and anionic rabbit IgG-pl-c
e6
-succ, cationic 17.1A-
pl-c
e6
and anionic 17.1A-pl-c
e6
-succ. Cells were illuminated in a P35 dishes
using 666 nm light (conjugates) and 654 nm (c
e6
). Results were measured
by a colony forming ability after 9 days and are expressed in % cells
surviving compared to dark controls treated with PS or conjugate. For each
point three separate plates of cells were given PIT and each plate further
plated for colonies in four dilutions. Values are the means of survival
fractions obtained and bars are s.e.m.
damage to intestines and other intraperitoneal organs (Veenhuizen
et al, 1997). There are many reports in the literature which confirm
the selectivity of the 17.1A murine mAb toward gastrointestinal
tumours and in particular to colorectal cancer (Martin et al, 1986;
Pierce et al, 1990; Buchsbaum et al, 1993b; Meredith et al, 1995).
In order for PIT to be effective each PIC molecule must deliver as
much PS as possible to the tumour, without sacrificing unduly the
specificity and affinity of the PIC for its antigen. One way of
accomplishing this is to use a polymeric linker to attach several PS
molecules in a site-specific manner to the mAb, and this linker
may bear positive, negative or neutral charge (Hamblin et al,
1996). Variation in the overall charge may affect the binding of the
PIC to its target antigen, its intracellular location and its photo-
toxicity. A previous publication reported the effect of charge on
the selectivity, uptake and phototoxicity of OC125 F(ab′)2PICs
constructed in a similar fashion to the present 17.1A PICs
(Hamblin et al, 1996). Alterations in the charge borne by mAb
conjugates can also lead to wide variations in the biodistribution
and pharmacokinetics (Slinkin et al, 1993; Duska et al, 1997). The
object of this study was to investigate the binding (specificity and
affinity) of cationic and anionic 17.1A PICs to HT29 target human
colorectal cancer cells, and their consequent phototoxicity.
The results from the ELISA and two-colour direct/indirect
immunofluorescence studies on fixed cell, showed that the
capacity of the two differently charged PICs to recognize the
membrane antigen expressed on HT29 cells, compared reasonably
with that of unmodified 17.1A. Since these cells were fixed, the
subsequent endocytosis of bound PICs was not an issue. Although
the affinity of the anionic PIC was somewhat reduced as measured
by the ELISA it was still relatively high. The fluorescence
microscopy showed that with fixed cells the 17.1A and the ce6
delivered by the PIC had very similar localizations making it
likely that the great majority of ce6 delivered to the cells was cova-
lently linked to the mAb.
When the PICs were administered to living cells considerable
differences in the uptake of ce6 between the opposite charged PICs
were observed. The cationic PIC delivered an average of 3.5 times
more ce6 than the anionic PIC. This is a similar but smaller
multiple to that found with cationic and anionic OC125 F(ab′)2
PICs and their target OVCAR-5 cells in a previous study (Hamblin
et al, 1996), where the enhanced ce6 uptake from the cationic PIC
was attributed to increased rates of internalization of polycationic
mAbs compared to uncharged or polyanionic species. The relative
ce6 uptake of the cationic species relative to free ce6 was 38:16:1 for
17.1A PIC, rabbit IgG PIC and ce6 respectively, and for the anionic
species was 11.5:5.5:1 respectively. It seems therefore that the
antigen mAb recognition at least doubles the uptake compared to
other conjugates of the same charge. The data comparing uptake at
37°C and 4°C show that the cationic conjugates bind much better
than the anionic ones to the membrane at 4°C, and the additional
internalized uptake at 37°C is only another 50%; while for the
anionic conjugates the additional uptake at 37°C was 200–300%
of that at 4°C. This suggests that a higher proportion of the anionic
17.1A PIC was internalized than for the cationic 17.1A PIC,
although the overall uptake was less. Uptake of ce6 from both the
cationic and anionic 17.1A PICs was also approximately twofold
higher by target HT29 colorectal cancer cells than by non-target
OVCAR-5 ovarian cancer cells, while the non-specific rabbit IgG
PICs showed no difference in ce6 uptake between cell lines.
However, since the EpCAM antigen is a common antigen over-
expressed on cancer cells it is possible that OVCAR-5 cells also
expressed this antigen, although the indirect immunofluorescence
was negative. Additional evidence of the retention of antigen
recognition in the PICs was provided by the experiments in which
the antigen was saturated by preincubation with unmodified mAb.
It should be noted that it is difficult to get any selectivity between
two epithelial human cancer cell lines using unconjugated PS in
vitro.
One of the critical issues to be considered in immunoconjugate
therapy is the penetration of the immunoconjugate into the tumour
tissue (Jain, 1990). It has been shown (Saga et al, 1995) that high
affinity mAbs penetrate less well than those of lower affinity, and
therefore a conjugation process which reduces the affinity of the
mAb for target antigen expressing cells, may be quite acceptable.
The question then arises, to what extent do the values for ce6
uptake delivered by the various conjugates correlate with the
phototoxicity? Under the conditions where the uptake of ce6 from
the cationic conjugates relative to unconjugated ce6 was 38:16:1
for 17.1A and rabbit IgG, the relative phototoxicity (1/survival
fraction) compared to unconjugated ce6 with 3 J cm–2 666 nm light
was 6.5:1.4:1. Similarly for the anionic series where the relative
uptakes were 11.5:5.5:1, the relative phototoxicities were
2.4:0.8:1. To determine which of these conjugates is inherently the
most effective photosensitizer, we can divide the relative photo-
toxicity by the uptake in nmol ce6 equivalent per mg cell protein
and the resulting numbers are shown in Table 2. Free ce6 would
appear to have more phototoxic potential than any of the conju-
gates per unit ce6 uptake, but since the uptake is exceptionally low
this is not of much relevance. This is in agreement with other
reports (Bachor et al, 1991) that free ce6 has such low cellular
uptake that it is difficult to get any phototoxicity in vitro. The
remaining conjugates have roughly similar values but the values
obtained when mAb conjugates interact with target cells are
consistently higher than non-matching pairs. These results can be
compared with those obtained (Hamblin et al, 1996) with poly-
cationic and polyanionic OC125F(ab′)2conjugates prepared in a
similar manner and directed towards target OVCAR-5 cells. In this
previous study the charge effect was found to be of larger magni-
tude but in the same direction as the present study, with the poly-
cationic PIC having 6 times the uptake and 10 times the
phototoxicity of the polyanionic PIC. This difference in magnitude
between charge effects may be attributed to differences in the
magnitude of the negative charge expressed on the outside of the
cells (Bischoff et al, 1981), or to differences in the extent to which
polycations stimulate endocytosis between cell lines (Duncan et
al, 1979). It is accepted that imparting cationic charge to a protein
62
M Del Governatore et al
British Journal of Cancer (2000) 82(1), 56–64 © 2000 Cancer Research Campaign
Table 3 Relative phototoxicity per unit cellular uptake of c
e6
delivered by
conjugates and free c
e6
Conjugate HT29 cells OVCAR-5 cells
17.1A-pl-c
e6
4.17 ±0.56 2.17 ±0.06
17.1A-pl-c
e6
-succ 5.07 ±0.11 4.79 ±0.07
Rabbit IgG-pl-c
e6
2.17 ±0.07
Rabbit IgG-pl-c
e6
-succ 3.59 ±0.07
c
e6
24.5 ±1.31
The phototoxicity (1/survival fraction) after incubation at 37°C for 6 h at 1 µM
c
e6
equivalent concentration and 3 J cm–2 666 or 654 nm light, was divided by
the cellular uptake in nmol c
e6
equivalent per mg cell protein. Errors are the
s.e.m. of the ratio of the means calculated in quadrature.
17.1A photoimmunoconjugates
63
British Journal of Cancer (2000) 82(1), 56–64
© 2000 Cancer Research Campaign
(Shen and Ryser, 1978), receptor ligand (Cotten et al, 1990), mAb
(Pardridge et al, 1994) or immunoconjugate (Hamblin et al, 1996)
increases the absolute uptake and the degree to which it is internal-
ized in vitro. It is thought that the mechanism of increased uptake
is due to non-clathrin-coated pit-mediated endocytosis leading to
endosomal processing (Hansen et al, 1993), and to accumulation
in lysosomes where proteolysis may take place. In vivo, however,
polycationic moieties have high and fast uptake in the liver and
kidney when administered i.v. (Clegg et al, 1990), which suggest
that polycationic PICs would be better suited to intracavitary
administration (Hamblin et al, 1996). This hypothesis was
confirmed with a series of biodistribution experiments conducted
with OC125F(ab′)2PICs prepared in a similar manner to the
present PICs and injected i.p. in nude mice bearing i.p. OVCAR-5
tumours (Duska et al, 1997). However, in order to effectively
target colorectal tumour cells growing in the liver where the PIC
must be administered i.v., it is likely that a polyanionic PIC will
outperform a polycationic species. This hypothesis will be tested
in a forthcoming report.
In conclusion we have demonstrated advantages of conjugating
ce6 to mAb 17.1A by a site-specific synthetic route. The
immunoreactivity is preserved, the PICs show selectivity to target
colorectal cancer cells over non-target ovarian cancer cells, and
the absolute uptake by tumour cells is very much higher for both
charges than that given by the free ce6. In vitro there is little differ-
ence in the amount of killing per molecule of ce6 delivered by poly-
cationic and polyanionic PICs, thus leaving the choice of charge
borne by the PIC for in vivo PIT to be made on the basis of biodis-
tribution and pharmacokinetic data. While these initial data on the
preferential photodestruction of target cells are encouraging, there
remain many questions which will only be answered by in vivo
experiments. Will the PICs be able to penetrate solid tumours after
i.v. administration? In addition to binding to tumour cells, will the
PICs be taken up by cells of the monocyte/macrophage lineage
which are especially prevalent in liver? Will the pharmacokinetics
and biodistribution of the PICs be suitable for effective photo-
destruction of tumours in vivo? These questions will be addressed
in forthcoming reports.
ACKNOWLEDGEMENTS
This work was supported by NIH Grant number R01 AR 40352
and facilities by the ONR-MFEL program contract number N 00
014-94-1-0927.
We thank Dr K Tanabe for his generous gift of HT-29 cells,
Centacor Inc. for the gift of 17.1A mAb, and MP Bamberg for
technical assistance.
REFERENCES
Bachor R, Shea CR, Gillies R and Hasan T (1991) Photosensitized destruction of
human bladder carcinoma cells treated with chlorin e6-conjugated
microspheres. Proc Natl Acad Sci USA 88: 1580–1584
Barr H, Krasner N, Boulos PB, Chatlani P and Bown SG (1990) Photodynamic
therapy for colorectal cancer: a quantitative pilot study. Br J Surg 77: 93–96
Benotti P and Steele G (1992) Patterns of recurrent colorectal cancer and recovery
surgery. Cancer 70: 1409–1413
Bischoff P, Robert F and Donner M (1981) A comparative study of some growth
characteristics and cell-surface properties of neoplastic cells. Br J Cancer 44:
545–552
Buchsbaum DJ, Langmuir VK and Wessels BW (1993a) Experimental
radioimmunotherapy. Med Phys 20: 551–567
Buchsbaum DJ, Lawrence TS, Roberson PL, Heidorn DB, Ten Haken RK and
Steplewski Z (1993b). Comparison of 131I- and 90Y-labeled monoclonal
antibody 17-1A for treatment of human colon cancer xenografts. Int J Radiat
Oncol Biol Phys 25:629–638
Clegg JA, Hudecz F, Mezo G, Pimm MV, Szekerke M and Baldwin RW (1990)
Carrier design: biodistribution of branched polypeptides with a poly(L-lysine)
backbone. Bioconjug Chem 1: 425–430
Cotten M, Langle-Rouault F, Kirlappos H, Wagner E, Mechtler K, Zenke M, Beug H
and Birnstiel ML (1990) Transferrin-polycation-mediated introduction of DNA
into human leukemic cells: stimulation by agents that affect the survival of
transfected DNA or modulate transferrin receptor levels. Proc Natl Acad Sci
USA 87: 4033–4037
Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J
and Peng Q (1998) Photodynamic therapy. J Natl Cancer Inst 90: 889–905
Duncan R, Pratten MK and Lloyd JB (1979) Mechanism of polycation stimulation
of pinocytosis. Biochim Biophys Acta 587: 463–475
Duska LR, Hamblin MR, Bamberg MP and Hasan T (1997) Biodistribution of
charged F(ab′)2photoimmunoconjugates in a xenograft model of ovarian
cancer. Br J Cancer 75: 837–844
Goff BA, Bamberg M and Hasan T (1991) Photoimmunotherapy of human ovarian
carcinoma cells ex vivo. Cancer Res 51: 4762–4767
Goff BA, Hermanto U, Rumbaugh J, Blake J, Bamberg M and Hasan T (1994)
Photoimmunotherapy and biodistribution with an OC125-chlorin
immunoconjugate in an in vivo murine ovarian cancer model. Br J Cancer 70:
474–480
Goff BA, Blake J, Bamberg MP and Hasan T (1996) Treatment of ovarian cancer
with photodynamic therapy and immunoconjugates in a murine ovarian cancer
model. Br J Cancer 74: 1194–1198
Hamblin MR and Newman EL (1994) On the mechanism of the tumour-localising
effect in photodynamic therapy. J Photochem Photobiol B 23: 3–8
Hamblin MR, Miller JL and Hasan T (1996) The effect of charge on the interaction
of site-specific photoimmunoconjugates with human ovarian cancer cells.
Cancer Res 56: 5205–5210
Hansen SH, Sandvig K and van Deurs B (1993) Molecules internalized by clathrin-
independent endocytosis are delivered to endosomes containing transferrin
receptors. J Cell Biol 123: 89–97
Hasan T (1992) Photosensitizer delivery mediated by macromolecular carrier
systems. In Photodynamic Therapy: Basic Principles and Clinical Applications,
Henderson B and Dougherty T (eds), pp. 187–200. Marcel Dekker: London
Hasan T and Parrish JA (1996) Photodynamic therapy of cancer. In: Cancer
Medicine, Holland JF, Frei EI, Bast RCJ, Kufe DW, Morton DL and
Weichselbaum RR (eds), pp. 739–751. Williams & Wilkins: Baltimore
Henderson BW and Dougherty TJ (1992) How does photodynamic therapy work?
Photochem Photobiol 55: 145–157
Houston LL (1993) Targeted delivery of toxins and enzymes by antibodies and
growth factors. Curr Opin Biotechnol 4: 739–744
Jain RK (1990) Physiological barriers to delivery of monoclonal antibodies and
other macromolecules in tumours. Cancer Res 50: 814s–819s
Johnson DA, Briggs SL, Gutowski MC and Barton R (1995) Anti-tumor activity of
CC49-doxorubicin immunoconjugates. Anticancer Res 15: 1387–1393
Kornguth SE, Kalinke T, Robins HI, Cohen JD and Turski P (1989) Preferential
binding of radiolabeled poly-L-lysines to C6 and U87 MG glioblastomas
compared with endothelial cells in vitro. Cancer Res 49: 6390–6395
Larson E, Howlett B and Jagendorf A (1986) Artificial reductant enhancement of the
Lowry method for protein determination. Anal Biochem 155: 243–248
Litvinov SV, Velders MP, Bakker HA, Fleuren GJ and Warnaar SO (1994) Ep-CAM:
a human epithelial antigen is a homophilic cell–cell adhesion molecule. J Cell
Biol 125: 437–446
McMurrick PJ and Nelson H (1997) Liver-directed therapies for gastrointestinal
malignancies. Curr Opin Oncol 9: 367–372
Martin EW, Jr, Tuttle SE, Rousseau M, Mojzisik CM, O’Dwyer PJ, Hinkle GH,
Miller EA, Goodwin RA, Oredipe OA, Barth RF & et al. (1986)
Radioimmunoguided surgery: intraoperative use of monoclonal antibody 17-
1A in colorectal cancer. Hybridoma 5: S97–S108
Meredith RF, Khazaeli MB, Plott WE, Spencer SA, Wheeler RH, Brady LW, Woo
DV and LoBuglio AF (1995) Initial clinical evaluation of iodine-125-labeled
chimeric 17-1A for metastatic colon cancer. J Nucl Med 36: 2229–2233
Mew D, Wat CK, Towers GH and Levy JG (1983) Photoimmunotherapy: treatment
of animal tumors with tumor-specific monoclonal antibody-hematoporphyrin
conjugates. J Immunol 130: 1473–1477
Pardridge WM, Bickel U, Buciak J, Yang J and Diagne A (1994) Enhanced
endocytosis and anti-human immunodeficiency virus type 1 activity of anti-rev
antibodies after cationization. J Infect Dis 169: 55–61
64
M Del Governatore et al
British Journal of Cancer (2000) 82(1), 56–64 © 2000 Cancer Research Campaign
Pierce DL, Heindel ND, Schray KJ, Jetter MM, Emrich JG and Woo DV (1990)
Misonidazole conjugates of the colorectal tumor associated monoclonal
antibody 17-1A. Bioconjug Chem 1: 314–318
Pogrebniak HW, Matthews W, Black C, Russo A, Mitchell JB, Smith P, Roth JA and
Pass HI (1993) Targetted phototherapy with sensitizer-monoclonal antibody
conjugate and light. Surg Oncol 2: 31–42
Riethmuller G, Schneider-Gadicke E, Schlimok G, Schmiegel W, Raab R, Hoffken K,
Gruber R, Pichlmaier H, Hirche H, Pichlmayr R & et al. (1994) Randomised trial
of monoclonal antibody for adjuvant therapy of resected Dukes’ C colorectal
carcinoma. German Cancer Aid 17-1A Study Group. Lancet 343: 1177–1183.
Saga T, Neumann RD, Heya T, Sato J, Kinuya S, Le N, Paik CH and Weinstein JN
(1995) Targeting cancer micrometastases with monoclonal antibodies: a
binding-site barrier. Proc Natl Acad Sci USA 92: 8999–9003
Sanz-Altamira PM, Spence LD, Huberman MS, Posner MR, Steele G, Jr, Perry LJ
and Stuart KE (1997) Selective chemoembolization in the management of
hepatic metastases in refractory colorectal carcinoma: a phase II trial. Dis
Colon Rectum 40: 770–775
Schmidt S, Wagner U, Oehr P and Krebs D (1992) Clinical use of photodynamic
therapy in gynecologic tumor patients–antibody-targeted photodynamic laser
therapy as a new oncologic treatment procedure. Zentralbl Gynakol 114: 307–311
Shen WC and Ryser HJ (1978) Conjugation of poly-L-lysine to albumin and
horseradish peroxidase: a novel method of enhancing the cellular uptake of
proteins. Proc Natl Acad Sci USA 75: 1872–1876
Slinkin MA, Curtet C, Faivre-Chauvet A, Sai-Maurel C, Gestin JF, Torchilin VP and
Chatal JF (1993) Biodistribution of anti-CEA F(ab′)2 fragments conjugated
with chelating polymers: influence of conjugate electron charge on tumor
uptake and blood clearance. Nucl Med Biol 20: 443–452
Speiser PP (1991) Nanoparticles and liposomes: a state of the art. Methods Find Exp
Clin Pharmacol 13: 337–342
Steele G, Jr and Ravikumar TS (1989) Resection of hepatic metastases from
colorectal cancer. Biologic perspective. Ann Surg 210: 127–138
Van Cutsem E (1996) A glimpse of the future: new directions in the treatment of
colorectal cancer. Eur J Cancer 32A:S23
Van Hillegersberg R, Marijnissen JP, Kort WJ, Zondervan PE, Terpstra OT and Star
WM (1992a) Interstitial photodynamic therapy in a rat liver metastasis model.
Br J Cancer 66: 1005–1014
Van Hillegersberg R, Van den Berg JW, Kort WJ, Terpstra OT and Wilson JH
(1992b) Selective accumulation of endogenously produced porphyrins in a liver
metastasis model in rats. Gastroenterology 103: 647–651
Veenhuizen RB, Marijnissen JP, Kenemans P, Ruevekamp-Helmers MC, Mannetje
LW, Helmerhorst TJ and Stewart FA (1996) Intraperitoneal photodynamic
therapy of the rat CC531 adenocarcinoma. Br J Cancer 73: 1387–1392
Veenhuizen RB, Ruevekamp MC, Oppelaar H, Ransdorp B, van de Vijver M,
Helmerhorst TJ, Kenemans P and Stewart FA (1997) Intraperitoneal
photodynamic therapy: comparison of red and green light distribution and
toxicity. Photochem Photobiol 66: 389–395
Vrouenraets MB, Visser GW, Stewart FA, Stigter M, Oppelaar H, Postmus PE, Snow
GB and van Dongen GA (1999) Development of meta-
tetrahydroxyphenylchlorin–monoclonal antibody conjugates for
photoimmunotherapy. Cancer Res 59: 1505–1513
Yeh KA, Fortunato L, Hoffman JP and Eisenberg BL (1997) Cryosurgical ablation
of hepatic metastases from colorectal carcinomas. Am Surg 63: 63–68
17.1A photoimmunoconjugates
65
British Journal of Cancer (2000) 82(1), 56–64
© 2000 Cancer Research Campaign
