Safety and Biodistribution of Technetium-labeled Anti-CD44v6 Monoclonal Antibody BIWA 1 in Head and Neck Cancer Patients

The CD44 protein family consists of isoforms, encoded by standard exons and up to nine alternatively spliced variant exons (v2–v10), which are expressed in a tissue-specific way. Expression of v6-containing variants (CD44v6) has been related to aggressive behavior of various tumor types and was shown to be particularly high in squamous cell carcinoma (SCC). Therefore, CD44v6 might be a suitable target for radioimmunoscintigraphy (RIS) and therapy. The present study evaluates the novel high-affinity murine antiCD44v6 monoclonal antibody (MAb) BIWA 1 for its safety and targeting potential in patients with SCC of the head and neck (HNSCC). Twelve HNSCC patients, who had planned to undergo resection of the primary tumor and neck dissection, were included. Preoperatively, 2, 12, or 52 mg of Tclabeled MAb BIWA 1 was administered. RIS results obtained 21 h after injection were compared with palpation, computed tomography, and magnetic resonance imaging, with histopathology as the gold standard. Moreover, biodistribution of BIWA 1 was evaluated by radioactivity measurement in blood and bone marrow and in biopsies from the surgical specimen obtained 40 h after injection. The distribution of BIWA 1 in tumor biopsies was analyzed by immunohistochemistry. BIWA 1 integrity in the blood was assessed by high-performance liquid chromatography and related to soluble CD44v6 levels in serum samples. No drugrelated adverse events were observed. Human antimouse antibody responses were observed in 11 patients. The diagnostic efficacy of RIS appeared to be comparable for the three BIWA 1 dose levels and for the four diagnostic methods. Besides activity uptake in tumor tissue, minimal accumulation of activity was observed in mouth, lungs, spleen, kidney, bone marrow, and scrotal area. Analysis of tissue biopsies revealed high uptake in tumors, with a mean value of 14.2 6 8.4% of the injected dose/kg tumor tissue and a mean tumor:blood ratio of 2.0 6 1.4 at 40 h after injection. Differences among the three dose groups were not statistically significant, although a trend toward lower uptake in the highest dose group was noted. Distribution of BIWA 1 throughout the tumor was heterogeneous for all dose groups, which might be related to the high affinity of the MAb. The mean biological half-life in blood (34.5 6 6.1 h) was not dose dependent. Extensive complex formation of BIWA 1 was observed in the 2-mg group, most probably with soluble CD44v6 present in the blood, and complex formation relatively diminished upon increase of the MAb dose. BIWA 1 is a promising MAb for targeting HNSCC because it can be safely administered to HNSCC patients, while it shows high and selective tumor uptake. However, BIWA 1 is immunogenic, and therefore a chimerized or humanized derivative of BIWA 1 with intermediate affinity will be used in future clinical trials. INTRODUCTION HNSCCs account for ;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 (2–4). Considering the clinical need and the inherent radiosensiReceived 7/28/99; revised 4/20/00; accepted 4/28/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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) 204440953; Fax.: (0) 20-4443688; E-mail: [email protected]. 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. 3046 Vol. 6, 3046–3055, August 2000 Clinical Cancer Research Research. on January 20, 2018. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from tivity 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 6 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 (16–18). 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 Tc-labeled BIWA 1 in a similar clinical RIS setting as performed previously with Tc-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 Tc-labeled BIWA 1 in 12 patients with HNSCC. These objectives were evaluated at three different BIWA 1 dose levels. MATERIALS AND METHODS Patient Population. Twelve patients (5 women and 7 men; age range, 41–76 years) with histologically proven advanced HNSCC (4 patients had stage III disease; 8 had stage IV) were entered after informed consent was obtained (Table 1). 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 Tc-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. A large number of hematological and biochemical parameters were measured from blood and urine samples obtained ,3 weeks before injection, as described previously (7). To assess toxicity, these were compared with values obtained 21 and 40 h and 1 week after injection. Vital signs (i.e., blood pressure, pulse rate, breathing rate, and temperature) were recorded immediately before administration of Tc-labeled BIWA 1 and each 20 min up to 2 h after injection. 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 Table 1 Patient characteristics Dose and patient no. Age (yr) Sex HNSCC site TNM stage Tumor volume (cm) 2 mg BIWA 1 1 53 F Floor of mouth pT3N0Mx 2.8 2 61 F Base of tongue pT4N2bMx 17.3 3 54 M Supraglottic larynx pT4N2aMx 13.2 4 59 M Retromolar area pT4N2bMx 27.4 12 mg BIWA 1 5 71 F Post-cricoid region pT3N0M0 26.4 6 41 M Vallecula pT3N2cM0 30.2 7 48 M Lateral tongue pT3N0Mx 21.7 8 76 F Alveolar process cT2N1M1 2.5 52 mg BIWA 1 9 74 M Supraglottic larynx pT4N2cM0 19.3 10 60 M Base of tongue pT3N0Mx 27.1 11 51 F Supraglottic larynx pT3N2cMx 20.8 12 72 M Tonsil pT3N0Mx 23.6 a According to Union International Contre Cancer classification (24). 3047 Clinical Cancer Research Research. on January 20, 2018. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 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 Tc using the chelate S-benzoylmercaptoglycylglycylglycine (S-benzoylMAG3; 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 Tc-labeled BIWA 1 was 88.3 6 5.5% at infinite antigen excess. TLC demonstrated a mean of 97.0 6 1.6% of Tc 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 6 0.2, 10.7 6 1.0, and 54.5 6 5.0 mg of BIWA 1, respectively, labeled with a mean activity of 768.5 6 45.9, 777.7 6 15.2, and 728.9 6 28.1 MBq of Tc, 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 1 FN) and specificity as TN/(TN 1 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 Tc 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(ab9)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 Tc. 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 highperformance liquid chromatography) was applied to assess the percentages of radiolabeled free BIWA 1 (Rf 15.9 6 0.2 min) and complexed BIWA 1 (Rf 11.1 6 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 3048 Targeting Head and Neck Cancer with MAb BIWA 1 Research. on January 20, 2018. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 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.