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Experimental Therapeutics |
Tumour Targeting Program, Ludwig Institute for Cancer Research, Melbourne Branch, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia [K. C., F-T. L., F. E. S., A. M. S.]; Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892-100 [M. W. B.]; Ludwig Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, New York, New York 10021 [L. J. O.].
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A number of tumor antigens have been shown to have high
expression on malignant breast cancer cells, including HER2/neu, MUC,
carcinoembryonic antigen, and Ley
(3)
. The Ley antigen is a family
member of the blood group-related difucosylated oligosaccharides with
chemical structure Fuc
1
2Galß14[Fuc13]
GlcNAcß1R(4)
. They are associated with 60–90% of
human carcinomas of epithelial cell origin including breast, colon,
gastric, and lung cancer (5, 6, 7)
. The high frequency of
Ley-expressing tumors, high density of
Ley on tumor cell surface, and relatively
homogeneous expression in primary and metastatic lesions have led to
its selection as an antigenic target.
3S193 is a mAb produced using standard hybridoma techniques following immunization of BALB/c nude mice with Ley-expressing MCF-7 breast cancer cells. The murine antibody has been shown to have high specificity for Ley and reacted strongly in rosetting assays and cytotoxic tests with Ley-expressing cells (8) . The use of murine mAbs, however, is limited by their immunogenicity. Murine antibodies are recognized as foreign, and the immune system mounts a classical humoral immune response producing HAMAs. Although clinical HAMA requires rechallenge to the same antibody, serum sickness reactions can occur after the first or repeated infusions and are usually unrelated to dose or rate of administration and are manifested principally by myalgias and arthralgias (9) . A further limitation of many murine antibodies is their inability to harness the effector and complement response of the human immune system as effectively as humanized antibodies, depending upon the isotype of the antibody (10) .
CDR grafting is one method of humanizing antibodies, combining murine antigen binding regions (CDR) with human V-region framework determinants. CDR grafting aims to make the surfaces of the antibody appear as fully human as possible, but retain the murine antigen-binding packaging and interface reactions determining high-affinity binding (11) . 3S193 has been humanized by CDR grafting, and the reactivity for Ley has been confirmed to be similar to the murine version, and with retained potent effector function of complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (8 , 12) . hu3S193 has only 3–5% murine residues in the antibody variable domain, markedly reducing the potential for an immune response in planned human trials.
The role of conventional radiotherapy in metastatic breast cancer has historically been limited to local palliation. Given that breast cancer is a radiation-sensitive disease (2) , targeted delivery of radiation of effective dose to all metastatic sites using mAbs would be a novel and potentially useful therapeutic modality. Radionuclides are attractive agents for selective targeting to tumors by chemical conjugation to antitumor antibodies. The emitted radiation targeted to tumors can be used to kill tumor cells (therapy) or to detect primary or metastatic tumor (imaging), depending on the isotope selected. Considerations in selecting a radioisotope include type of emission, its range, tumor dose rate and total tumor dose, biological half-life, labeling efficiency, and stability. To be effective, the link between the antibody and the radionuclide must be stable in vivo, and the labeling procedure must not alter the biodistribution or binding characteristics of the antibody. This study explores such properties of hu3S193 bound to 125I, 111In, and 90Y.
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MATERIALS AND METHODS |
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Cell Lines.
MCF-7, a Ley-expressing human breast
adenocarcinoma cell line originally derived from the pleural effusion
of a 69-year-old woman with estrogen receptor-positive metastatic
breast cancer (14)
, was obtained from the American Type
Culture Collection (Rockville, MD). SW1222, a
Ley-negative human colonic cancer cell line, was
a gift from the tumor cell bank of the New York Branch of the Ludwig
Institute and was used as a control cell line.
Cells were grown in 175-cm2 plastic flasks (Nunclon; Nunc, Roskilde, Denmark) and maintained in log-phase growth in RPMI 1640 (Trace Biosciences, Sydney, Australia) supplemented with 10% (MCF-7) or 5% (SW1222) FCS (MultiSer; Trace Biosciences, Sydney, Australia), 100 units/ml penicillin, 100µg/ml streptomycin, 0.25 ml/l insulin, 2 mM glutamine, and essential amino acids. Cells were cultured at 37°C in a 5% CO2 incubator (Forma Scientific, Inc., Marietta, Ohio) and passaged with 0.05% EDTA/PBS (BDH Chemicals, Merck, Melbourne, Australia). Cell viability in all experiments, as determined by trypan blue exclusion, exceeded 90%.
Radiolabeling and Quality Assurance.
hu3S193 and control antibody (huA33) were labeled with three isotopes,
125I, 111In, and
90Y. Isotopes were obtained from DuPont (Life
Science Products, Boston, MA). Radioiodination was performed using a
modification of a previously published chloramine-T reaction
(15)
using a 2-fold molar excess of chloramine-T (Merck,
Darmstadt, Germany) over antibody, dissolved in 0.5 M
potassium phosphate buffer (pH 7). After a brief 2-min incubation
period, the reaction was stopped by adding a 5-fold excess of sodium
metabisulfite, again dissolved in a 0.5 M phosphate buffer,
and then purified through a desalting column (P6DG; Bio-Rad, Sydney,
Australia) equilibrated with PBS.
Antibody labeling with 90Y and 111In was achieved via a bifunctional metal ion chelating agent, CHX-A''-DTPA (16) using a modification of a method published previously (17) . In brief, a dialysis bag with a Mr cutoff of 10,000 (Novex, Sydney, Australia) was treated with 10 mM sodium bicarbonate and soaked in deionized water and 1 mM EDTA (pH 7.0) to remove heavy metals. hu3S193 was dialyzed against a 50 mM sodium bicarbonate buffer (pH 8.6) containing 0.15 M NaCl for 6 h. CHX-A''-DTPA was added in molar excess of 5:1 and incubated at room temperature overnight in the dark. Excess unbound antibody was removed by 8 h dialysis with 20 mM sodium acetate buffer containing 0.15 M NaCl (pH 6.3). 111In or 90Y were bound to CHX-A''-DTPA antibody conjugate under mildly acidic conditions (pH 5.5) for 20 min, and then the pH was raised to 7 by the addition of small aliquots of 2.0 M sodium acetate, followed by 10 mM EDTA. The radiolabeled mixture was purified by centrifugal desalting on a Sephadex G50 column (Pharmacia, Uppsala, Sweden) equilibrated with PBS.
Radiolabeling was performed on the day of injection into mice. Prior to injection, the percentage of unbound radionuclide content was determined by ITLC, and binding ability of the final radiolabeled product was tested by a Ley-positive cell binding (Lindmo) assay as detailed below. Scatchard analysis was used to determine the binding constant (Ka) and number of antibody molecules bound per cell for 125I- and 111In-labeled antibody.
ITLC.
The amount of free versus bound antibody following
radiolabeling was determined by ITLC as described previously (17
, 18)
. Aliquots of radiolabeled hu3S193 were placed on the origin
of standard silica gel impregnated glass fiber ITLC strips (Gelman
Scientific, Ann Arbor, MI). To determine the percentage of bound
111In, ITLC strips were developed using 10
mM EDTA (pH 4.5) and 0.9% saline/10
mM NaOH solution as solvents. In EDTA, hu3S193
remains at the origin, and the CHX-A''-DTPA-111In
or 90Y, and free 111In or
90Y, respectively, migrate with the solvent
front. In 10 mM NaOH solution, hu3S193 and free
radionuclide remain at the origin, and CHX-A''-DTPA-radionuclide
migrates with the solvent front. Strips were developed until the
solvent migrated to the end, and then they were cut in half and counted
on a gamma camera. Radioactivity in the bottom half of the strip was
calculated as a percentage of the entire strip (top plus bottom). The
percentage of free radionuclide was determined by subtraction
(NaOH - EDTA). Results were performed in duplicate. Ten
% trichloroacetic acid was used as the solvent to determine the
percentage of unbound 125I, with free iodine
migrating with the solvent front and bound remaining at the origin. The
percentage of isotope bound to hu3S193 was >90% in all experiments
detailed.
IR.
IR of radiolabeled hu3S193 to MCF-7 target cells was determined by
linear extrapolation to binding at infinite antigen excess using a
"Lindmo" assay (19)
. Fifteen to 20 ng of radiolabeled
hu3S193 were added to a range of MCF-7 cell concentrations and
incubated for 45 min at room temperature with continuous mixing
throughout to keep the cells in suspension. Cells were washed three
times to remove unbound antibody, and pellets were measured in a gamma
counter (Cobra II, Model 5002, Auto-gamma; Packard Instruments,
Canberra, Australia). Three samples of radiolabeled hu3S193, at
the same concentration as that initially added to the cells, were
measured at the same time as cell pellets and the percentage of binding
of hu3S193 to MCF-7 cells was calculated by the formula: (cpm cell
pellet/mean cpm radioactive antibody standards) x 100.
The percentage of binding was graphed against MCF-7 cell concentration
using Sigmaplot for Windows (Jandel Scientific, San Rafael, CA), and IR
was calculated as the Y-intercept of the inverse plot of both values.
Cell pellets to which >100-fold unlabeled hu3S193 was added, prior to
radiolabeled hu3S193, were used as a control to determine background
cpm.
Scatchard analysis was used to calculate the apparent association constant (Ka) and number of antibody molecules bound per cell (19) . Unlabeled hu3S193 at concentrations ranging from 5 to 200 µg/ml were added to 12 x 106/ml MCF-7 cells and mixed, and subsequently 30 ng of labeled hu3S193 were added. After 45 min incubation, the cells were washed and counted as described above for Lindmo assay. The IR fraction was taken into account in calculating the amount of free, reactive antibody [(100 - % bound)/100 x total antibody x IR fraction], and specific binding (nM; total antibody x % bound) was graphed against specific binding/reactive free. The association constant was determined from the negative slope of the line. The number of hu3S193 molecules bound per cell was derived by: [(X-intercept of Scatchard plot (nM)/1000) x (6.023 x 1023)]/number of cells used in the assay (12 x 106).
Animal Model.
In vivo tissue biodistribution studies were performed in
female athymic BALB/c nude mice, 5–6 weeks of age, homozygous for the
nu/nu allele, bred by the SPF Facility, University of South
Australia (Adelaide, Australia). Mice were maintained in
autoclaved microisolator cages housed in a positive pressure
containment rack (Thoren Caging Systems, Inc., Hazelton, PA).
To establish MCF-7 human breast xenografts, mice were supplemented with exogenous estrogen (20 , 21) . After light Ethrane anesthesia, a 60-day slow release estrogen pellet (0.72 mg estradiol/pellet; Innovative Research of America, Sarasota, FL) was inserted using aseptic techniques into a small s.c. pocket fashioned between the shoulder blades. The surgical incision was closed with a single stitch (6-0 absorbable Dacron or silk). MCF-7 cells (25 x 106) in 100–150 µl of medium were subsequently injected s.c. into the left inguinal mammary line. SW1222, non-Ley-expressing, colon cancer xenografts were established by injection of 10 x 106 cells in the right inguinal area of each mouse as a control tumor. The growth rates of both tumors differed. To obtain comparable tumor sizes, the more rapidly growing SW1222 colon cancer cells were injected 4 weeks after inoculation of MCF-7 tumors. Both tumors were of a size appropriate for use 14 days after this time.
Tumor volume was calculated by the formula [(length x width2 /2] (21 , 22) , where length was the longest axis and width the measurement at right angles to the length.
Biodistribution Studies.
Two biodistribution studies were performed. In the first study, 41 mice
were injected i.v. via the retro-orbital plexus with hu3S193 labeled
with 5 µCi of both 125I and
111In CHX-A''-DTPA-hu3S193 (total, 10 µg of
antibody and 10 µCi of radioactivity) suspended in 150 µl of
sterile media. Groups of four to five mice, with a mean (± SD) tumor
volume of 647 mg ± 252, were sacrificed by cervical
dislocation after light ethrane anesthetic at 4, 24, 48, 72, 96, 120,
168, 240, and 360 h after injection of radiolabeled antibody. Mice
were bled via cardiac puncture, and blood was collected into
heparinized tubes. Tumors and organs [skin, liver, spleen, intestine,
stomach, kidneys, brain, bone (femur), lungs, and heart] were
immediately removed, blotted dry, and weighed (Sartorius Basic Balance,
Ratingen Germany). All samples were counted in a dual chamber
gamma scintillation counter (Cobra II,
Auto-gamma; Packard Instruments) using a dual tracer program with
standard windows set for each isotope, 15–75 Kev for
125I and 140–430 Kev for
111In. Standards prepared from the injected
material were counted each time, with tissues and tumors enabling
calculations to be corrected for the physical decay of the isotopes.
Results of labeled antibody distribution over time were expressed as
the % ID/g [(cpm tissue sample/cpm cpm standard) x 100/weight in grams] and as tumor:blood ratios.
A second biodistribution study was also performed to assess the distribution and localization of 90Y-labeled antibody. Twenty µCi of 90Y-CHX-A''-DTPA-hu3S193 were injected into the retro-orbital plexus of 25 mice bearing MCF-7 xenografts. One to four mice were sacrificed at time points 4, 24, 48, 72, 120, 168, 240, 336, 408, and 504 h after injection and blood and tumors removed. At the 72-h time point, spleen, bone, liver, and kidney were also excised. % ID/g was calculated as described above.
Statistical Analysis.
Paired Student t tests were performed on the different
isotope biodistribution time points in each study and individual organ
uptakes to assess any differences observed for statistical
significance.
Pharmacokinetics.
Pharmacokinetics were determined for 125I and
111In radioconjugates using a curve-fitting
program (SAAM II; University of Washington, Seattle, WA) and assuming a
two-compartmental model.
In Vivo Antibody Stability.
The stability of the iodine and indium label in vivo was
assessed by the amount of free radionuclide present over time, as
determined by ITLC. Blood collected from mice at each time point into
heparinized tubes was pooled and spun at 1000 x g
for 15 min to obtain plasma. Two µl of each sample were spotted onto
the origin of two standard ITLC strips. Strips were developed, and the
percentage of free indium and iodine was calculated as described
previously.
A single point immunoreactivity assay was also performed on each pooled plasma sample to determine the in vivo stability of the antibody in terms of binding ability. An aliquot of plasma, volume adjusted to standardize for % ID/g, was added to 12 x 106 MCF-7 cells and incubated for 45 min at room temperature. Excess unbound antibody was removed at the end of the incubation period by washing twice with medium, and radioactivity in the washed pellet was measured with a gamma counter. An equal volume of blood as that added to the assay was kept as a standard, and binding of blood to cells was calculated as a percentage of antibody present in the standard.
Imaging.
To enable qualitative comparison of radiolabeled-antibody localization
over time, a single mouse from the 125I/111In
co-labeled antibody study was anesthetized on four separate occasions.
At time points 4, 24, 72, and 120 h, the animal received i.p.
injections of 0.2 ml of solution containing 18.7 mg/ml tribromoethanol
dissolved in 12.5 µl of isopentyl alcohol and 1 ml of sterile water
for injection. Images were obtained using a dual head gamma camera
("Biad"; Trionix Research Laboratories, Twinsbury, OH)
linked to a Unix computer system with photopeaks set at 173 KeV and 247
KeV with 20% windows. A medium energy collimater was used to acquire
images attributable predominantly to 111In, with
negligible contribution from 125I. Ten-min images
were obtained at each time point. Images were acquired in a
256 x 256-bit matrix, and a standard of known activity
was included in the field of view.
Autoradiography.
Immediately after sacrifice, an MCF-7 tumor from one mouse at time
points 24, 48, 72, and 120 h in the
125I/111In biodistribution
study was excised, frozen, and sectioned on a cryomicrotome (Zeis
Microm HM 5000, Melbourne, Australia). Sequential 5-µm
sections were used for direct autoradiography and for H&E staining.
Tissue sections were placed on silane-coated glass slides and allowed
to dry. Glass slides were placed face down in contact with emulsion of
Hyperfilm-MP film (Amersham Life Science, Little Chalfont,
United Kingdom) or standard radiography film. The glass slides and film
were enclosed in a cassette and stored at -70°C. An image
intensifier (Hyperscreen; Amersham) was used for Hyperfilm samples and
was located directly behind and in contact with the film
(23)
. Slides and film were taken up at varying time
intervals, and film was developed in a standard automatic film
processor. It was not possible to distinguish between
125I and 111In using the
method described.
Immunohistochemistry.
Immunohistochemistry was performed to confirm the presence and
distribution of Ley antigen in tumor xenografts
and to examine normal murine tissues for Ley
antigen expression. Xenografts and normal kidney, liver, lung, spleen,
stomach, duodenum, jejunum, ileum, and large bowel were surgically
removed, embedded in Tissue-Tek OCT Compound (Diagnostic Division,
Elkhart, IN), and cut at 5-µm thickness with a cryomicrotome.
Sections were fixed with acetone at 4°C for 10 min, and nonspecific
binding was blocked using a commercial protein blocking agent (Lipshaw
Immunon, Pittsburgh, PA). Five µg of hu3S193 diluted in 1% BSA-PBS
were applied to sections and incubated at room temperature for 1 h
(primary antibody), rinsed with PBS, and then the secondary antibody
(biotinylated IgG1 goat antihuman 1:300; Sigma Immunochemicals)
was applied for 30 min. After 10 min immersion in 0.3% peroxidase,
streptavidin peroxidase (1:1000) was applied for 30 min, and finally
the chromagen 3-amino-9 ethyl carbazole (Sigma Chemical Co., St. Louis,
MO) for 20 min was used to develop pink coloration to antigen-positive
cells. An isotype-matched huIgG1 control antibody (huA33) and no
primary antibody controls were used for each tissue or time point
analyzed.
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RESULTS |
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Biodistribution.
125I/111In-labeled
biodistribution study results are presented in Table 1
. The comparison of %ID/gram of 125I- and
111In-labeled hu3S193 in MCF-7 and control
tumors, blood, and group size at each time point are detailed. The
%ID/g of both isotopes in MCF-7 xenografts peaked at 48 h after
antibody injection, with the uptake of
111In-labeled hu3S193 being 3-fold greater than
125I (30.1% versus 10%,
respectively) and reaching statistical significance
(P = < 0.001). Uptake of hu3S193 within
breast tumors was superior for the 111In label at
all time points studied, and of note 111In uptake
was >3-fold that of 125I at
t = 48 h to 360 h inclusive (Fig. 1)
. The blood levels of both isotopes closely approximated each other
throughout the study, declining progressively over time. The
tumor:blood ratio for 111In-labeled hu3S193
peaked at 5.15 ± 1.65 at 168 h, compared with
2.83 ± 1.2 at 120 h for
125I.
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. The % ID/g in all normal tissues was <10%. Immunohistochemical
analysis demonstrated Ley expression only in the
gastric glands of the stomach and intestinal glands of the jejunum and
ileum; this result was consistent with the low uptake of hu3S193 in
normal tissues. The uptake of 111In was only
significantly higher than 125I in the kidney
(Ps between < 0.001 and 0.006, depending on
the time point studied).
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3
4)
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, and a comparison to the 111In-hu3S193
biodistribution study is shown in Fig. 3
. 90Y-labeled antibody uptake in MCF-7 tumors
peaked at 22% ID/g (t = 48–72 h), which was
slightly lower but not statistically significantly different
(P = 0.087) from peak
111In tumor uptake. At the last time point at
which a comparison was made between the two isotopes
(t = 360 h), the %ID/g within MCF-7
tumors was 4-fold higher for mice that received the
90Y label, suggesting prolonged retention of
90Y within the tumor. Blood clearance for both
isotopes approximated each other. At 72 h, hematopoietic organs
and kidneys were removed from four mice. Comparison with the results
obtained from the
125I/111In biodistribution
study at the same time points after antibody injection showed higher
uptake of 90Y in kidney, spleen, and bone marrow
(%ID/g 9.6, 6.02, and 1.99, respectively). Differences were
statistically significant in the kidney compared with
125I and 111In
(P = 0.001 and 0.038, respectively) and in
the spleen compared with 111In
(P = 0.009). Differences in bone marrow
uptake between the three isotopes were not statistically significant.
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for
125I-, 111In- and
90Y-labeled 3S193 was 9.3, 7.4, and 7.5 h
and mean T1/2 ß 69.3, 69.3, and
79 h, respectively.
In Vivo Stability.
In vivo stability was analyzed in the
125I-hu3S193 and
111In-hu3S193 study. The percentage of bound
111In, as determined by ITLC, was >99.5% for
all time points up to 7 days at which time 98% bound
111In was present. The percentage of bound
125I was lower at baseline (93.5%) and decreased
to 78% at 7 days. Immunoreactivity for
111In-hu3S193 and
125I-hu3S913 at t = 0 h, as determined by single-point binding assay, was 65 and
51.5%, respectively, and slowly decreased over time, not dropping
markedly until 7 days after injection, at which time the %IR for
111In-hu3S193 was 27.5% and
125I-hu3S913 was 27%.
Imaging.
Localization of 111In-hu3S193 in a single BALB/c
mouse bearing an MCF-7 and control SW-1222 tumor over time, as
determined by gamma camera imaging, is presented in Fig. 4
. At 4 h, no tumor localization was evident, with the image showing
generalized blood pool activity and slightly increased activity in the
central portion of the scan corresponding to the cardiac area. At
24 h, minimal uptake was seen in the control tumor (left side of
image), but there was definite localization of antibody to the MCF-7
Ley-expressing xenograft (right side of image).
Over time with decreasing blood pool activity, the uptake in the MCF-7
tumor was more clearly defined. The mouse was sacrificed after the last
image was obtained at 120 h. Radioactivity was clearly localized
to the MCF-7 tumor, and the tumor:blood ratio was determined to be
6.07:1.
Autoradiography and Immunohistochemistry.
The distribution of radioactivity via autoradiography, the location of
viable tumor cells using hematoxylin staining, and antigen distribution
as determined by immunoperoxidase techniques were compared in an MCF-7
xenograft removed at day 5 in the
125I/111In-hu3S193
biodistribution study. Representative results are shown in Fig. 5
. Hematoxylin staining indicated that the tumor contained viable tissue
peripherally and a small amount in the center of the section, with a
large area of central necrosis and connective tissue (Fig. 5B)
. Antigen (Ley) was distributed
evenly throughout the section in areas corresponding to viable tumor
cells (Fig. 5C)
. Control slides for antigen staining showed
some nonspecific staining in the connective tissue stroma but were
negative in the tumor itself (Fig. 5D)
. Radioactivity,
assessed by autoradiography, was distributed throughout regions of
viable tumor including the central area of viable tumor cells, with
little activity in the central necrotic area (Fig. 5A)
,
indicating specific localization of radiolabeled hu3S193 to tumor
cells. A similar even distribution of radioactivity throughout viable
tumor cells was seen in all other time points studied (results not
shown), and given differences in tumor sections, in particular the
quantity of necrotic or connective tissue, there was no qualitative
difference in distribution of radioisotope over time.
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DISCUSSION |
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In addition, a number of other Ley antibodies have been developed and have reached different stages in preclinical evaluation and clinical testing. These include ABL 364 (BR55-2), an IgG3 antibody derived from the BR55-2 hybridoma (7) . Murine ABL 364 administered to patients with cytokeratin-positive tumor cells in their marrow was shown to reduce or eliminate Ley antigen-cytokeratin (+) cells but not cytokeratin (+) cells in Ley-negative patients (27) . However, a dose escalation study of murine BR55-2 in patients with metastatic breast cancer reported only minor clinical responses in 28.6% of patients, with HAMA limiting repeated cycles of treatment (28) . A Phase I study with the LMB-1 immunotoxin, incorporating the murine anti-Ley monoclonal antibody B3 and recombinant Pseudomonas exotoxin, has also been performed in patients bearing solid epithelial tumors. Responses were observed in 5 of 38 patients studied; however, 90% of patients developed neutralizing antibodies against LMB-1 after one cycle of treatment (29) .
Studies in nude mice bearing human lung carcinoma xenografts of another anti-Ley antibody, BR96, demonstrated that murine and chimeric BR96 were able to prevent or slow growth when administered soon after cell implantation but had only modest effects with staged tumors (22) . In contrast, when chimeric BR96 was conjugated to doxorubicin, complete regressions of xenografted human lung, breast, and colon cancers grown s.c. in nude mice were seen (30) . In Phase I and II clinical trials of chimeric BR96 (IgG1) conjugated to doxorubicin in patients with Ley-positive advanced stage carcinomas, limited response rates were observed, whereas gastrointestinal toxicity, which improved with implementation of steroid premedication, was demonstrated (31 , 32) .
We have examined the in vivo biodistribution and labeling stability of the recently humanized anti-Ley antibody 3S193, comparing 125I-, 111In-, and 90Y-labeled hu3S193 in an MCF-7 xenografted BALB/c nude mouse model. Excellent and specific tumor uptake was observed compared with a Ley-negative control tumor, with the peak tumor (MCF-7) uptake for all three isotopes occurring at 48 h. The %ID/g for both 111In and 90Y were significantly higher than 125I. In addition, both 111In and 90Y were retained within xenografts for a longer period of time than 125I. Lower absolute levels of iodine uptake compared with 111In in xenografts have been demonstrated in other studies (13) , and this together with differences in tumor retention of radiolabel most likely represents differences in cellular processing of radiolabeled 125I as opposed to both 111In and 90Y. Catabolism of 125I linked to tyrosine results in the generation of [125I]mono-iodotyrosine within lysosomes, which rapidly leaves the cell via cell-mediated transport systems (18 , 33 , 34) . In contrast, endocytosed 111In-DTPA-antibodies are delivered to lysosomes and hydrolyzed by lysosomal enzymes into small molecular weight metabolites that are retained within tumor lysosomes (13 , 33 , 34) .
Cellular processing of 90Y by tumor cells in vitro has been shown in other antibody-antigen systems to be similar to 111In (35) and is also reflected in similarities in distribution in vivo (36) . It has been reported that although the pattern of uptake is comparable, superior tumor retention is obtained with the use of 90Y (36) , a conclusion that this current study supports.
Within normal tissues, the uptake of 111I and 90Y in liver, spleen, and kidneys was higher than 125I. The higher uptake values in spleen and liver is consistent with known sequestration of radiometals in reticuloendothelial organs (36) . This uptake in normal organs is not a consequence of specific targeting attributable to an absence of Ley antigen expression in these normal tissues of the mouse but is a reflection of blood flow and catabolism of the radiolabeled conjugates. The increased uptake of 111In- and 90Y-labeled antibody compared with 125I in the kidneys reflects differences in metabolism and excretion of radiometals (37) . However, the increased renal uptake of 90Y compared with 111In may also reflect the conjugation of 90Y-CHX-A''-DTPA-hu3S193, given the observed low immunoreactivity of the labeling experiment presented, and warrants further investigation using chelating agents of enhanced stability for 90Y immunoconjugates (37) . The %ID/g of 90Y in the bone, assumed to represent bone marrow, was approximately double that of 125I and 111In; however, the total amount present was negligible (2% ID/g), and differences were not statistically significant. Other studies have shown that accumulation of 90Y within bone is a concern with evidence that 90Y accumulates within the bone marrow (36) and indicates that 111In may underestimate 90Y bone marrow uptake.
The differences observed between isotopes in radiolabeling techniques and cellular processing were also reflected in the in vivo stability results. The percentage of 125I-bound radioactivity over time, as determined by ITLC, decreased rapidly, consistent with rapid release of free 125I from lysosomes. In contrast, only 2% of free 111In was detected over the same time period, again consistent with current literature suggesting retention of 111In-DTPA-lysine catabolites by tumor lysosomes (13 , 33 , 34) . Retention of antigen binding for up to 5 days in vivo was also observed for both 125I- and 111In-labeled hu3S193.
Although promising, clinical responses with unconjugated mAbs in the therapy of solid tumors have been poorer and less consistent than with hematopoietic neoplasms (38, 39, 40) , and there remains room for improvement. Given the high frequency of epithelial tumors expressing the Ley antigen and the high density of Ley on the surface of tumor cells, hu3S193 has potential therapeutic applications. The in vivo studies presented demonstrate that 111In- and 90Y-radiolabeled hu3S193 have favorable biodistribution characteristics, with high %ID/g uptake of both antibody bound isotopes within Ley-expressing MCF-7 breast xenografts. In contrast, the biological behavior of 125I-hu3S193, as demonstrated in this report, would indicate that 131I would not be the preferred isotope for this antibody-antigen system. Tumor localization of 111In-CHX-A''-DTPA hu3S193 was confirmed visually in imaging studies and by autoradiography, demonstrating good penetration of radionuclide to all areas of viable tumor cells. In addition to the direct targeting of the tumor by hu3S193, our previous studies have described the in vitro immune effector functions mediated by the IgG1 Fc portion of hu3S193 (8 , 12) . The antitumor effect of hu3S193 may be limited in the murine model used in the current study because of restricted murine serum complement activity. The potent in vitro antibody-dependent cellular cytotoxicity of hu3S193 would suggest that, in conjunction with potent complement-dependent cytotoxicity activity in the human, these humoral effects in a clinical setting would augment the therapeutic effects of targeted radiolabeled hu3S193.
hu3S193 has the theoretical advantage of reduced immunogenicity provided by humanization of an antibody, an assumption that has been supported by preliminary data with earlier, fully humanized antibodies (24 , 41 , 42) , allowing multiple courses of treatment that have not been possible with murine or chimeric antibodies. The humanization of 3S913 has yielded a molecule with only 3–5% sequence homology to the parent murine antibody in the immunogenic variable domains. A similar humanization, through CDR grafting, to that of 3S193 has been described for an antihuman CD4 mAb using the same human framework (REI for variable light chain and KOL for variable heavy chain; Ref. 42 ). Preliminary clinical results in patients have been reported and show a lack of immunogenicity (42 , 43) .
The humanization strategy we adopted for hu3S193 and the results of clinical studies using mAbs with similar human frameworks provide evidence of the reduced potential for an immune response to hu3S193 in planned clinical trials.
Some Ley antigen expression was observed in the
gastric glands of the stomach and basal intestinal glands of the small
bowel of the BALB/c mice used in the current study, a similar
distribution pattern to that reported previously in immunohistochemical
studies of normal human tissues with other
anti-Ley mAbs (6
, 44)
. However, the
current biodistribution study did not demonstrate increased specific
targeting to the small bowel of the BALB/c mice (Fig. 2B)
.
The expression of Ley antigen in normal human
tissues (including the GI tract) is of concern in targeting strategies
against this antigen; however, the accessibility of normal tissues to
anti-Ley antibodies remains unclear. Results of
studies with murine and chimeric antibodies against
Ley suggest that gastric toxicity may be relevant
in proposed clinical trials; however, significant toxicity has only
been observed at protein doses >200 mg (31
, 32)
. The
optimal protein dose for effective targeting of
Ley tumors (tumor:blood ratios, normal tissue
uptake) is yet to be defined with carefully designed biodistribution
and biopsy-based trials. In this context, high protein doses of
anti-Ley antibodies reported to cause toxicities
may not be required for effective tumor targeting.
The current model has demonstrated the stability of hu3S193, and its characteristics and properties are well suited to beginning trials in the clinic.
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FOOTNOTES |
|---|
1 To whom requests for reprints should be
addressed, at Ludwig Institute for Cancer Research, Austin and
Repatriation Medical Centre, Heidelberg, Victoria, 3084, Australia.
Phone: (613) 9496-5876; Fax: (613) 9496-5892; E-mail: ams{at}austin.unimelb.edu.au 
2 The abbreviations used are: mAb, monoclonal
antibody; Ley, Lewisy antigen; HAMA, human
anti-mouse antibodies; CDR, complementarity determining region; hu,
humanized; ITLC, instant thin layer chromatography; CHX-A''-DTPA,
C-functionalized
trans-cyclohexyldiethylenetriaminepentaacetic acid;
%ID/g, percent injected dose/gram; IR, immunoreactivity.
Received 12/ 9/99. Accepted 7/ 6/00.
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);
111In-MCF-7 tumor (
); 111In-control tumor
(
); 125I-blood (
); 125I-MCF-7 tumor
(
); and 125I-control tumor (
).
)-labeled hu3S193 in normal murine
tissues, MCF-7 tumors, and blood at 48 h (A) and
72 h (B) after injection of BALB/c nude mice. Data
from groups of five mice are expressed as mean %ID/g; bars, SD.
) and MCF-7 xenografts (
) and MCF-7 xenografts (•)
from BALB/c nude mice. Results represent a comparison of two separate
biodistribution studies with the individual radiolabels. Groups of one
to four mice were sacrificed at time points 4, 24, 48, 72, 120, 168,
240, 336, 408, and 504 h after injection, and blood and tumors
were removed. %ID/g was calculated. Data are expressed as means;
bars, SD.
