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Clinical Trials |
Departments of Otolaryngology/Head and Neck Surgery [J. W. G. S., J. J. Q., G. B. S., G. A. M. S. v. D.], Nuclear Medicine [J. C. R.], Pharmacy [B. J. W.], and Radiology [J. A. C.], Free University Hospital, 1081 HV Amsterdam, the Netherlands; Boehringer Ingelheim Research and Development, A-1121 Vienna, Austria [M. S., K-H. H., R. M., G. R. A.]; and Boehringer Ingelheim B.V., 1822 BJ, Alkmaar, the Netherlands [M. O. K.]
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ABSTRACT |
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INTRODUCTION |
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5% of all malignant neoplasms in Europe and the United
States. Worldwide, 500,000 new cases are projected annually, and this
incidence is rising. For 1998, it was estimated that 41,400 Americans
would develop HNSCC, and 12,300 would die from it (1)
.
Despite improvements in locoregional treatment modalities,
i.e., surgery and radiotherapy, for stages III/IV (70%)
there is still a high failure rate, either locally or at distant sites.
An effective adjuvant systemic treatment is therefore needed to improve
survival rates in this patient group. In this respect, application of
(neo)adjuvant chemotherapy, with its unfavorable therapeutic index, has
mostly failed to accomplish any improvement in survival
(2, 3, 4)
. Considering the clinical need and the inherent radiosensitivity of HNSCC (5) , our department has put effort on the development of MAbs capable of targeting radionuclides to HNSCC (6) . In particular with regard to the MAb called U36, in vivo imaging/biodistribution trials revealed favorable biodistribution patterns with selective tumor targeting and high tumor uptake of 20.4 ± 12.4% of the ID/kg of tissue (7) .
The antigen recognized by U36 appeared to be identical to the keratinocyte-specific CD44 splice variant epican, which contains the variant exons v3–v10. By screening overlapping synthetic peptides of the epican-specific region encoded by exons 7–11 (v3–v7) the corresponding epitope was mapped, revealing its localization in the v6 domain (8) . Expression of v6-containing CD44 variants has been observed in several types of tumors including SCCs of the head and neck, lung, skin, esophagus and cervix, as well as adenocarcinoma of breast, colon, lung, and stomach (6 , 9) . Among normal tissues, expression was observed only in a subset of epithelial tissues, e.g., skin keratinocytes, breast and prostate myoepithelium, and bronchial epithelium (6 , 9) . Besides that, soluble v6-containing CD44 variants have been detected in the blood of control volunteers as well as of cancer patients (10) . Possible molecular functions of CD44 isoforms are defined currently as adhesion molecules (11) , signal transducers (12) , regulators of cell migration (13) , and as tumor metastasis-promoting proteins (14) . Interestingly, v6-containing CD44 isoforms appeared to be capable of conferring metastatic potential on originally nonmetastatic tumor cells in a rat pancreatic carcinoma model (14) . Moreover, MAbs against CD44v6 were able to prevent outgrowth of metastases in a syngeneic rat model (15) . Overexpression of CD44v6 in tumors was shown to correlate with reduced survival of patients with breast and colon cancer and with non-Hodgkin’s lymphomas (16, 17, 18) .
Alternative MAbs recognizing v6-containing CD44 isoforms have been developed, including MAb 17, MAb Var3.1, and MAb VFF18 (MAb BIWA 1; Refs. 9 , 19 , 20 ). BIWA 1 was selected from a panel of CD44v6-specific MAbs because of its high affinity to human tumor cells (9) . Although BIWA 1 resembles U36, it binds to a different epitope and with a 35-fold higher affinity. According to the numbering of Kugelman et al. (21) , the epitope recognized by U36 consists of amino acids 365–376, and the epitope recognized by BIWA 1 consists of amino acids 360–370. Because high-affinity MAbs may be better suited for tumor targeting (22 , 23) , we decided to evaluate 99mTc-labeled BIWA 1 in a similar clinical RIS setting as performed previously with 99mTc-labeled U36 (7) . As a prelude to RIT, this report describes the results of a first Phase I clinical trial aiming to determine safety, kinetics, tissue distribution, tumor uptake, and diagnostic effectiveness of 99mTc-labeled BIWA 1 in 12 patients with HNSCC. These objectives were evaluated at three different BIWA 1 dose levels.
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MATERIALS AND METHODS |
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. The protocol was approved by the Institutional Review Board. Patients
who participated in this study planned to undergo a resection of the
primary tumor and a unilateral or bilateral neck dissection. Surgery
was performed 40 h after administration of
99mTc-labeled BIWA 1. For one patient, surgery
was canceled because of disclosure of multiple metastases in the lungs
and possibly in the liver. Prior to enrollment, a biopsy of the primary
tumor had to show BIWA 1 binding to >50% of the tumor cells, as
determined by immunohistochemistry, which consistently turned out to be
>95% of cells.
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For the assessment of tumor volumes, MRI data on optical disc were processed by in-house developed software run on a standard Sparc 10 workstation (SUN Microsystems, Palo Alto, CA). Tumor areas were calculated after displaying separate MRI images on a monitor on which visible primary tumor was manually enclosed into a region. Surfaces of each region were multiplied by slice thickness plus interslice gap, thus obtaining total tumor volume.
MAb.
Characterization of the CD44v6-specific murine IgG1 MAb BIWA 1
(Boehringer Ingelheim, Vienna, Austria) and generation of the
BIWA 1 hybridoma cell line (also called VFF-18) have been described
before (9)
. Clinical grade material was produced by
Bio-Intermediair Europe BV, Groningen, the Netherlands. BIWA 1 (2 mg)
was labeled with 99mTc using the chelate
S-benzoylmercaptoglycylglycylglycine
(S-benzoyl-MAG3; Mallinckrodt Medical B.V., Petten, the
Netherlands), and the immunoreactive fraction of each radiolabeled BIWA
1 preparation was tested as described previously (7
,). As
determined by a modified Lineweaver-Burk plot, the immunoreactive
fraction of 99mTc-labeled BIWA 1 was 88.3 ±
5.5% at infinite antigen excess. TLC demonstrated a mean of 97.0 ± 1.6% of 99mTc to be bound to BIWA 1.
Unlabeled BIWA 1 was added to adjust the amount of MAb for the 2-, 12-,
and 52-mg dose groups. BIWA 1 was subsequently injected i.v. over a
5-min period. The low-, middle-, and high-dose patient groups received
2.0 ± 0.2, 10.7 ± 1.0, and 54.5 ± 5.0 mg of BIWA 1,
respectively, labeled with a mean activity of 768.5 ± 45.9,
777.7 ± 15.2, and 728.9 ± 28.1 MBq of
99mTc, respectively.
Imaging.
All patients were preoperatively examined by palpation, CT, and MRI of
the neck, as reported previously (7)
. At 0–1 and 21 h after injection, planar whole body images (anterior and posterior
projection) and 21 h postinjection planar spot images of the head
and neck (anterior, posterior, and lateral projections) were acquired.
An ADAC dual-headed gamma camera equipped with a low-energy collimator
was used. SPECT images of the head and neck were obtained 21 h
after injection. From the one patient whose distant metastases were
disclosed shortly before the actual surgical procedure, additional
SPECT images were obtained of chest and upper abdomen, 21 h after
injection.
CT, MRI, and RIS images were each reviewed by the same experienced examiner, blinded to the results of other examinations and the pathological outcome, although notified of the primary site. Criteria for the optimal assessment of cervical lymph node metastases by CT or MRI were applied (25) . Interpretation of RIS images was based upon asymmetry and retention.
Diagnostic effectiveness of RIS and conventional anatomical imaging modalities were compared. To this end, diagnostic results of palpation, CT, MRI, and RIS were evaluated per neck side and lymph node level, as described previously (7) . Histopathological examination of the neck dissection specimens was used as the gold standard. Hereto, detectable lymph nodes were dissected from each surgical specimen and examined by a pathologist. All diagnostic modalities were correlated with the histopathological examination, and their diagnostic effectiveness was expressed in terms of sensitivity and specificity. Sensitivity is defined as TP/(TP + FN) and specificity as TN/(TN + FP), where TP is a true-positive, FN a false-negative, TN a true-negative, and FP a false-positive observation.
Biodistribution.
Biopsies were taken from tumor tissue (primary site and lymph node
metastases) and from various normal tissues, present in the surgical
specimen. Blood, bone, and bone marrow were obtained immediately before
surgery under general anesthesia. From the patient not proceeding to
surgery, only a sample of tumor and normal mucosa was obtained. Tissues
and blood samples were weighed, the amount of
99mTc was measured in a gamma-well counter (1282
Compugamma, Wallac, Turku, Finland), and data were converted to %ID/kg
tissue, as described previously (7)
. Tumor:nontumor ratios
were calculated using matched uptake values of one patient. If in a
patient several biopsies of one kind of tissue were taken, the mean
uptake in this tissue was calculated and used for further analysis.
After counting, all biopsies were evaluated histopathologically to
determine the presence or absence of HNSCC.
ROIs.
Uptake of MAb in tissues not present in the surgical specimen was
assessed by drawing ROIs on planar anterior and posterior whole-body
images, obtained 0–1 and 21 h after injection. Regions included
whole-body, liver, left kidney, spleen, two lumbar vertebrae, heart
(left ventricle), and right lung. Geometric means were calculated.
Standards were used to correct for decay of activity and camera
efficiency. Geometric mean activity within the whole-body region 0–1 h
after administration was designated the ID. Activity within each ROI
was corrected for background and used to obtain the relative organ
activity, expressed as %ID per 50 pixels. By conversion to the total
pixels for these organs, the activity was also expressed as %ID/whole
organ.
Immunohistochemistry.
Microscopic distribution of BIWA 1 throughout the tumor was assessed by
immunohistochemical analysis of tumor tissue obtained from the surgical
specimen. Frozen, acetone-fixed serial sections were stained applying
the biotin-avidin-peroxidase method after incubation with biotinylated
secondary antibody [F(ab')2 rabbit antimouse
IgG, DAKO, Denmark]. For assessment of maximal binding, sections were
incubated with BIWA 1, followed by the second antibody. The staining
intensity was categorized into: negative, weak, and moderate to strong.
Moreover, the percentage of CD44v6-positive cells having bound
administered BIWA 1 was estimated.
Pharmacokinetics.
Serial blood samples were drawn at various time points after completion
of infusion. Urine was collected in portions of 24 h until the
moment of surgery to determine renal excretion of
99mTc. The amount of radioactivity in blood,
plasma, and urine was assessed in a gamma-well counter and expressed as
%ID/kg as described previously (9)
. Noncompartmental
pharmacokinetic parameters were calculated from concentration
versus time data using the WinNonLin computer program,
version 1.5 (Scientific Consulting, Inc). Size exclusion chromatography
(silica-based gel filtration high-performance liquid
chromatography) was applied to assess the percentages of radiolabeled
free BIWA 1 (Rf 15.9 ± 0.2 min) and complexed BIWA 1 (Rf
11.1 ± 1.1 min), essentially as described formerly
(26)
.
In addition, the concentration of total circulating BIWA 1 was measured by a murine IgG serum ELISA. This assay does not distinguish free from complexed BIWA 1. Furthermore, by means of an ELISA using plates coated with GST-CD44v3-v10 (Boehringer Ingelheim), the concentration of circulating immunoreactive BIWA 1 was assessed. Both ELISAs detect radiolabeled as well as unlabeled BIWA 1.
Levels of sCD44v6 in serum were assessed by use of a commercially available sandwich type ELISA (Bender MedSystems, Vienna, Austria) within 2.5 h before administration, 1 week after injection and 6 weeks after injection. A MAb specific for an epitope of the CD44 standard molecule served as the capturing MAb absorbed to microtiter plates. BIWA 1 coupled to horseradish peroxidase was used for signal generation by the peroxidase/tetramethyl-benzidine system. The ELISA test was performed according to the manufacturer’s instructions as described by Kittl et al. (27) .
HAMA Assay.
A baseline plasma HAMA titer was determined in all patients, with
follow-up at 1 and 6 weeks after injection, using a HAMA titer assay
(28)
. HAMA titers 
500 were arbitrarily considered to be
positive.
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RESULTS |
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Imaging.
At 21 h after injection, planar whole-body images did not
demonstrate unexpected uptake in normal tissues. Besides activity
uptake in tumor tissue, only minimal accumulation of activity was
observed in mouth, lung, spleen, kidney, bone marrow, and scrotal area.
Of 12 primary tumors, 11 were depicted by RIS at 21 h after
injection, 8 on planar images, and another 3 with SPECT. Tumors
measured 2.5–30.2 cm3
(mean, 19.4 ± 9.1
cm3
; Table 1
). The only tumor not visualized had
a size of 26.4 cm3
.
In 12 patients, 19 neck dissected sides contained 89 neck levels.
According to histopathological examination, 10 sides and 18 levels
contained metastases of HNSCC. Diagnostic results of the four
modalities were analyzed per neck side and lymph node level, with
histopathology as the gold standard (Table 2)
. RIS correctly detected lymph node metastases in 6 of 10 sides
(sensitivity, 60%) and 9 of 18 levels (sensitivity 50%). Detected
lymph nodes measured 0.8 x 2.5 up to 4.5 x 5.5 cm. Four
affected sides containing 12 tumor-positive lymph nodes distributed
over seven levels were not disclosed by RIS. Eleven of these lymph
nodes, sizes 0.7 x 1.5 to 2.4 x 3.4 cm, were reexamined
histopathologically and appeared to contain either substantial
necrosis, keratinization, and/or fibrosis (n = 8) or
micrometastasis (n = 3). Besides these localizations,
another two tumor-involved lymph node levels (containing three
metastases with minimal diameters <1 cm) were not detected by RIS.
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In one patient, multiple metastases in both lungs (10; diameters <1
cm) and possibly in the liver were disclosed by CT just prior to the
arranged surgical procedure. Additional SPECT imaging of chest and
upper abdomen were performed. None of the lesions was visualized on
SPECT images; however, these clinical metastases were never
histopathologically confirmed.
Biodistribution.
Uptake of radioactivity in biopsied tumors and normal tissues at each
dose level is shown in Table 3
. From the one inoperable patient, only tumor, mucosa, and blood samples
could be obtained. Corresponding tumor:nontumor ratios are shown in
Table 4
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ROIs.
Upon an increase of the MAb dose, activity within the whole body at
21.5 h after injection (range, 19.5 to 25.2 h) appeared
similar: 91.6 ± 1.9, 93.8 ± 3.6, and 95.8 ± 5.6%ID.
In Table 5
, the relative organ activity per 50 pixels as well as per whole organ
at 21 h after injection is illustrated for liver, lumbar
vertebrae, kidney, spleen, heart, and lung. These appeared to be
comparable for each dose group.
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, A and B). An increase in antibody uptake was
found in the intermediate- and high-dose groups. In these two groups,
more cells were stained (±30–40%, Fig. 1
, C–F)
than in the low-dose group, whereas staining was more intense. Upon
dose increase from 12 to 52 mg, no obvious improvement of staining
patterns was found. Therefore, although >95% of the tumor cells did
express the antigen, maximally 40% of the cells had been targeted
in vivo. In general, the MAb uptake appeared higher at the
tumor-stroma interface and lower in the center of tumor islands.
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On HPLC analyses, each radioimmunoconjugate batch prepared for
injection showed a monomeric IgG peak (Rf 16.0 ± 0.4 min). In
plasma samples obtained immediately after administration, another peak
was consistently detected at Rf 11.1 ± 1.1 min, representing a
high molecular weight substance. Most likely, BIWA 1 had formed a
complex with sCD44v6 present in the circulation. Concentrations of free
and complexed BIWA 1 at different time intervals after MAb
administration are shown for the three MAb dose groups in Fig. 3
.
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A). The
percentage of complexed IgG increased from 63.1 ± 12.2% at
0.5 h after injection to 92.8 ± 2.0% at 43.4 h. After
injection of 12 mg 99mTc-labeled BIWA 1,
relatively more BIWA 1 remained in free form (Fig. 3
B). For
patients receiving 52 mg of 99mTc-labeled BIWA 1,
free IgG was even more prevalent (Fig. 3
C). In the 12-mg
dose group, the mean percentages of complex formed increased from
13.0 ± 4.3% at 0.5 h after injection to 24.2 ± 10.5%
at 43.3 h after injection and in the 52-mg dose group from
4.3 ± 0.6% at 0.5 h to 5.6 ± 1.8% at 43.2 h.
For all MAb doses, a more rapid blood clearance of free compared with
complexed BIWA 1 was observed. HPLC analysis did not reveal the
presence of small radioactive metabolic products in any of the samples.
In addition to HPLC analyses, ELISA analyses were performed to assess
concentrations of total as well as immunoreactive BIWA 1. Assessment of
the total BIWA 1 levels with ELISA revealed, as expected, similar
values as derived from HPLC analyses (Fig. 3)
. Also concentrations of
immunoreactive BIWA 1 appeared to be similar. This latter observation
seems to be remarkable because massive complex formation had been
observed, especially in the 2-mg dose group.
To obtain further insight in the phenomenon of BIWA 1 complexation, the serum levels of soluble CD44v6 were assessed by ELISA. Prior to administration of BIWA 1, concentrations ranged from 111 to 365 ng/ml. In sera taken 1 week after injection, sCD44v6 levels seemed consistently reduced (range, 43–170 ng/ml). This reduction appeared to be related to the BIWA 1 dose administered: 39.0 ± 23.6%, 43.8 ± 27.6%, and 58.4 ± 10.1% in the low-, intermediate-, and high-dose group, respectively. Six weeks after injection, sCD44v6 levels had approximately returned to their preinjection values (range, 120–226 ng/ml).
HAMA Assay.
No HAMAs were detected prior to injection of BIWA 1. Eleven patients
developed elevated HAMA titers (up to 2567), irrespective of the BIWA 1
dose administered. Mean titers 6 weeks after injection measured
1159 ± 943, 1005 ± 398, and 912 ± 834 for the low-,
intermediate-, and high-dose group, respectively. One patient developed
her highest titer 1 week after injection and demonstrated a slight
decrease at 6 weeks after injection, whereas in 10 patients, HAMAs were
detected at 6 weeks but not at 1 week after injection. In two patients,
additional samples were obtained at 12 weeks after injection. In both,
a slightly diminished HAMA level in comparison to 6 weeks after
injection was found.
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DISCUSSION |
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Preferential BIWA 1 uptake was observed in tumor tissue because RIS with radiolabeled BIWA 1 clearly depicted 11 of 12 primary tumors. Biopsies of the only primary lesion not depicted demonstrated an uptake level of 13.3% ID/kg, whereas 30% of the cells had been targeted by the MAb. This MAb uptake was similar to that of visualized tumors, whereas the size of this tumor was relatively large (26.4 cm3 ). An explanation for this diagnostic failure might be that in this particular patient, the MAb uptake in normal mucosa was relatively high, resulting in a low tumor:mucosa ratio of 1.2.
For the detection of lymph node metastases, RIS alone did not demonstrate any diagnostic gain in comparison with the conventional imaging techniques CT and MRI. Sensitivity of RIS, palpation, MRI, and CT per lymph node level appeared to be 50, 44, 61, and 67%, respectively. A similar sensitivity rate was established for RIS with U36 (50%; Ref. 7 ). However, a rather high rate of false-positive observations was found in this study for each diagnostic modality. Moreover, small distant metastases in one patient were not detected by SPECT.
Substantial improvements are needed to make RIS with BIWA 1 of clinical value. 99mTc is not the ideal radionuclide to be used in combination with whole IgG because of the short half-life of 99mTc (t1/2, 6 h), which necessitates imaging to be performed within the first 24 h after administration, before optimal tumor:nontumor ratios are achieved. In a recently initiated RIT study with 186Re-labeled MAb (t1/2 186Re, 91 h), images are acquired up to 14 days after injection. In time, background activity diminishes, resulting in improved tumor detection.3
Measurement of activity in biopsies from the surgical specimen showed higher uptake levels in primary tumor tissue than in any other tissue evaluated. Uptake in primary tumor tissue compared with normal mucosa proved to be significantly higher (P < 0.025). This is thought to be attributable to a better accessibility of antigenic sites in tumor tissue because the literature does not provide any evidence of higher CD44v6 expression in comparison with normal mucosa. Tumor-infiltrated lymph nodes contained only slightly more activity than tumor-negative lymph nodes (5.3 versus 3.5%ID/kg; P > 0.1). Underestimation of uptake in pathological lymph nodes may have occurred because of contamination of samples with lymphoid tissue and necrosis. Immunohistochemical comparison of pathological lymph nodes and primary tumors revealed similar levels of BIWA 1 accumulation.
Radioactivity levels in blood appeared to be similar to the levels in
bone marrow. Activity mainly resided in plasma, as became apparent upon
centrifugation. This is of paramount importance because bone marrow
toxicity is expected to become dose-limiting in RIT with BIWA 1,
especially because of the long half-life time of the MAb in blood. In
other normal tissues, low radioactivity levels were found (Table 3)
.
Measurements of radioactivity in biopsies as well as within ROIs did
not reveal selective MAb uptake in tissues other than normal oral
mucosa. Tumor:normal mucosa ratios at 40 h after injection were
2 for the 2- and 12-mg BIWA 1 dose groups. Moreover,
immunohistochemical analyses revealed that BIWA 1 binding was
restricted to the basal cell layers (Fig. 2)
. Despite this high uptake
in the basal cell layers, it has to be seen whether mucosal toxicity
will occur when using BIWA 1 for clinical RIT. Maraveyas et
al. (29)
reconstructed a theoretical phantom of the
larynx and derived local dosimetric data for the selection of
ß-emitting radionuclides. In their dosimetric calculations, the
authors take into account the fact that some of the disintegration
energy dissipates outside the distribution volume of the tissue. For
186Re, the absorbed fraction in tumors was
calculated to be 1.6 times larger than in the normal mucosa, which
leads to a greater tumor:mucosa dose advantage.
Although the number of patients in this study is small, tumor uptake
levels were not significantly influenced by tumor volume nor by levels
of circulating CD44v6. Data may suggest a tendency toward dose
dependency with lower tumor uptake levels and tumor:normal mucosa
ratios at 52 mg (Tables 3
and 4)
. Most likely, observed differences can
be attributed to statistical variation in view of the small number of
patients/group.
Immunohistochemical analyses showed heterogeneous distribution of the injected MAb throughout the tumor, irrespective of the dose administered. Although >95% of the tumor cells expressed the antigen, maximally 40% of the cells had been targeted by BIWA 1. No obvious improvement was observed upon dose increase from 12 to 52 mg. It has been suggested for high-affinity MAbs that antibody-antigen interaction at vascular entry sites of tumors may impose a binding site barrier that retards MAb percolation, thereby restricting uniform distribution (30, 31, 32, 33) .
It might seem remarkable that the tumor uptake levels in the 2- and 12-mg group were similar, because a much larger proportion of MAb became complexed in the 2-mg group than in the 12-mg group. Apparently, complexed IgG is still capable of binding to antigen present in the tumor. Evidence for this possibility was also found in the ELISA assays performed. With one assay, the total BIWA 1 concentration in the serum was measured, whereas with the other assay, only immunoreactive BIWA 1 was measured. Remarkably, even serum samples containing >50% complexed IgG yielded similar results in both assays, indicating that complexed BIWA 1 in the serum is still able to bind to immobilized antigen. Possibly, one of the antigen binding sites of the bivalent IgG is still available for tumor binding after complexation. Moreover, BIWA 1 may have a higher avidity and affinity for membrane-bound, v6-containing CD44 variants than for soluble CD44v6 variants. The high density of CD44v6 on tumor cell membranes may allow bivalent binding, whereas bivalent binding to soluble CD44v6 variants may be restricted by the low concentration of these molecules in the circulation. Furthermore, v6-containing CD44 variants present on tumor cell membranes may have better interaction with BIWA 1 than the variants present in the circulation because of conformational differences. Reduced affinity for binding to circulating antigen in comparison to immobilized antigen has also been described for other antibodies, including MAbs directed against carcinoembryonic antigen (34 , 35) .
Levels of sCD44v6 apparently fell during the first week after BIWA 1 administration. This does not mean that sCD44v6 is removed from the circulation. It can also be attributable to complex formation between antigen and antibody. BIWA 1 bound to sCD44v6 may compete with the horseradish-peroxidase conjugated BIWA 1 used in the ELISA test. By such competition, sCD44v6 levels will become underestimated.
Because complex formation (a) does not result in an
obviously diminished uptake of BIWA 1 in tumor tissue (Table 3)
and
(b) does not result in an increased uptake in specific
normal tissues, e.g., the liver (Table 5)
and (c)
can be kept relatively low by using high MAb concentrations, the
presence of soluble antigen in the circulation is not a limiting factor
for the therapeutic applicability of BIWA 1.
As outlined in the "Introduction," similar studies have been performed with another CD44v6-specific MAb, called U36. Although U36 resembles BIWA 1, it binds to a different epitope, with a 35-fold lower affinity. The tumor uptake of U36 was not significantly influenced by the MAb dose and certainly not lower than the tumor uptake of BIWA 1. Immune complexes were barely formed, whereas a homogeneous MAb distribution throughout the tumor was achieved when increasing the MAb dose to 52 mg. This indicates that high-affinity BIWA 1 does not result in improved tumor targeting when compared with U36.
In conclusion, CD44v6-specific BIWA 1 is a promising MAb because it can be safely administered to HNSCC patients, and it shows high and selective tumor uptake. RIS with 99mTc-labeled BIWA 1 is as reliable as other imaging techniques, although it was performed under suboptimal conditions (21 h after injection). However, BIWA 1 is immunogenic, forms complexes, and shows a heterogeneous accumulation throughout the tumor, suggesting that BIWA 1 is not able to penetrate deeper cell layers. Because in RIT targeting of the whole tumor cell population is to be preferred and possibly repeated antibody infusions are required, several chimeric and humanized MAb BIWA versions with different affinities have been constructed for further clinical evaluation. Preclinical in vitro and in vivo studies revealed less complex formation and more efficient tumor targeting when the affinity of CD44v6-specific MAbs was diminished.4 Chimeric or humanized BIWA with an optimal affinity will be labeled to an escalating dose of 186Re in a subsequent Phase I RIT trial with HNSCC patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Otolaryngology/Head and Neck Surgery, Free
University Hospital, De Boelelaan 1117, 1081 HV Amsterdam, The
Netherlands. Phone: (0) 20-4440953; Fax.: (0) 20-4443688; E-mail: gams.vandongen{at}azvu.nl 
2 The abbreviations used are: HNSCC, head and neck
squamous cell carcinoma; MAb monoclonal antibody; %ID, percentage of
injected dose; RIS, radioimmunoscintigraphy; RIT, radioimmunotherapy;
CT, computed tomography; MRI, magnetic resonance imaging; SPECT, single
photon emission computerized tomography; ROI, region of interest;
sCD44v6, soluble CD44v6; HAMA, human antimouse antibody.
4 D. R. Colnot, J. J. Quak, J. C. Roos, A. van
Hingen, A. J. Wilhelm, G. van Kamp, P. C. Hüygens, G. B. Snow,
and G. A. M. S. van Dongen. Phase I therapy study of
rhenium-186-labeled chimeric monoclonal antibody U36 in patients with
squamous cell carcinoma of the head and neck. J. Nucl. Med., in press,
2000.
Received 7/28/99; revised 4/20/00; accepted 4/28/00.
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