Treatment of Refractory Hodgkin's Lymphoma Patients With an Iodine-131–Labeled Murine Anti-CD30 Monoclonal Antibody
http://www.100md.com
《临床肿瘤学》
the Department of Internal Medicine I, Department of Nuclear Medicine, University of Cologne, Germany
Hopital Huriez, Lille, France
Department of Radioimmunotherapy, City of Hope National Medical Center, Duarte, CA
ABSTRACT
PURPOSE: Hodgkin's lymphoma (HL) has been demonstrated to be a good target for immunotherapy since lymphocyte activation markers such as CD30 are expressed in high numbers on the malignant cells. Thus, we developed a new radioimmunoconjugate consisting of the murine anti-CD30 monoclonal antibody (MAb) Ki-4 labeled with iodine-131 (131I).
PATIENTS AND METHODS: The biodistribution of 131I–Ki-4 was assessed via dosimetry after preinfusion of 5 mg native Ki-4 followed by 250 to 300 MBq 131I-labeled Ki-4. Whole-body scintigraphy was performed 1 hour, 24 hours, 48 hours, 72 hours, and 6 days after the infusion. Dosimetry was calculated using the programs NucliDose ICON-IDL (version 5.0.2; Siemens, Erlanger, Germany) and MIRDOSE (version 3.1; Oak Ridge National Laboratories; Oak Ridge, TN). The therapeutic dose was given on day 8 after preinfusion of unlabeled Ki-4.
RESULTS: We treated 22 patients with relapsed or refractory CD30-positive HL. Preinfusion of 5 mg native Ki-4 was sufficient to bind the soluble CD30. Imaging demonstrated localization of involved areas measuring 5 cm in diameter or more in four patients and 2.5 cm in one patient. Patients received total body doses of 0.035 Gy to 0.99 Gy. Acute toxicity was mild with grade 1 fatigue in 19 of 22 assessable patients. Seven patients experienced grade 4 degrees hematotoxicity 4 to 8 weeks after treatment. Response included one complete remission, five partial remissions, and three minor responses.
CONCLUSION: Treatment with 131I–Ki-4 is effective but can be associated with severe hematotoxicity.
INTRODUCTION
Hodgkin's lymphoma (HL) has become a curable disease after the introduction of improved radiation techniques and polychemotherapy regimens.1-5 Though most patients even in advanced stages can be cured with standard approaches, only 30% to 50% of those who relapse attain durable disease-free remission after second-line treatment.6-8 The outcome is worse for those with primary refractory disease.9 Data from HL and non-HL (NHL) suggest that small numbers of residual tumor cells remaining after first-line treatment can give rise to late relapses.10,11 Thus, eliminating residual malignant lymphoma cells after first-line treatment might further improve outcome in these diseases. In addition, a combined immuno-/chemotherapy might help to reduce the amount of cytotoxic drugs to be given, resulting in less toxicity of the currently used regimens.
HL seems to be an ideal candidate for monoclonal antibody (MAb) -based immunotherapy since Hodgkin-Reed/Sternberg cells express specific surface antigens such as CD2512 and CD3013 in large amounts. Most MAb-based clinical trials evaluated constructs directed against CD30, which is a 120-kDa transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) –receptor superfamily. Physiologically, expression of CD30 is found on virus-infected lymphocytes and on a small subset of activated T cells. Furthermore, this receptor is involved in the negative selection process of auto-reactive lymphocytes.14 The CD30 ligand (CD30L) is present on activated T cells, resting B cells, and granulocytes. The biologic function of CD30/CD30L interaction is not yet fully understood, but both stimulation of cell growth as well as induction of apoptosis have been described.15 Similar to other members of the TNF family, CD30 seems to be involved in the cell cycle regulation.16,17 CD30 is expressed on a variety of other neoplasms such as mediastinal B-cell lymphoma, anaplastic large-cell lymphoma, peripheral T-cell lymphoma, and embryonal carcinoma.18-21 In respect to a potential clinical use, a vast number of murine anti-CD30 MAbs have been developed. So far, different approaches available to eradicate tumor cells using MAbs were evaluated in HL: immunotoxins,22-26 bispecific constructs,27-29 and more recently, "naked" humanized or fully human MAbs.30-32 So far, radioimmunotherapeutic approaches in HL had been studied using polyclonal antiferritin antibodies labeled with iodine-131 (131I) or Yttrium-90 (90Y).33-37 Since HL is very sensitive to radiotherapy, it is reasonable to investigate anti-CD30 radioimmunoconstructs in HL patients. The most commonly used radionuclide for radioimmunotherapy is 131I. This radionuclide was selected for the presented trial since it is commercially available and allows dosimetry after trace doses of the radioimmunoconjugate. Here we report the first specific treatment of refractory HL patients with an 131I-labeled murine anti-CD30 MAb (Ki-4).
PATIENTS AND METHODS
Patients
Twenty-two patients with histologically-confirmed diagnosis of HL were treated at the University of Cologne. All patients were refractory to conventional therapy or had relapsed. To be eligible, patients older than 15 years had to have measurable disease, a Karnofsky performance status of at least 50%, creatinine less than 2.0 mg/100 mL, ALT less than 3x the upper limit of normal, total bilirubin less than 1.5 mg/mL, if not disease related. In addition, more than 30% of H-RS cells in a prior biopsy had to stain for CD30. Exclusion criteria were uncontrollable infectious disease, collagen vascular disease, vasculitis, active second malignancy, chemotherapy or radiotherapy within 4 weeks, significant impairment of pulmonary function, granulocytes less than 1,500/μL, platelets less than 80,000/μL or any other major organ dysfunction unrelated to lymphoma, bone marrow infiltration greater than 25%, pregnancy, and presence of antibodies against mouse immunoglobulin more than 1 μg/mL. Concomitant administration of steroids was permitted if patients had been treated with steroids for at least 4 weeks before radioimmunotherapy.
Pathology and Eligibility
To analyze the expression of CD30, the last available biopsy derived from lymph nodes or other involved tissue was used. Bone marrow biopsies or aspiration were performed in each patient to examine possible infiltration.
Preparation and Iodination of the Anti-CD30 Antibody Ki-4
Labeling of the anti-CD30 MAb Ki-4. Radio-iodination of the anti-CD30 MAb Ki-4 (IgG1, directed against cluster A)38 was carried out by the Chloramin T method.39 The reaction mixture was then purified on a PD10 desalting column (Pharmacia Biotech, Piscataway, NJ).
Quality control. Size exclusion high-performance liquid chromatography (TosoHaas TSKgel G 2000 SW, 7.5 x 300 mm, 10 μm, eluent: isotonic saline, 1 mL/min) was used for quality control. The labeled MAb was eluted after 6 minutes; free iodine, after 11.5 minutes. The amount of free iodine was usually less than 4% for various labelings. The complex dissociation constant of 131I–Ki-4 with L540 cells as determined by Scatchard plot analysis demonstrated high affinity of the labeled antibody (KD = 7.54 x 10–8 mol/L).40
Protocol Design
After a 30-minute preinfusion of 5 mg native Ki-4, the labeled MAb (250-300 MBq 131I, 1.25 ± 0.78 mg CD30 MAb, average and standard deviation [SD]) was administered as a 30-minute infusion. Patients received iodide tablets (100 mg) starting 24 hours before treatment and continuing for 14 days to minimize the thyroid uptake of free iodine. No premedication was administered. Patients were monitored for alterations in vital signs and adverse reactions during and for up to 2 hours after antibody infusion. Eight days later, a second infusion of 5 mg native Ki-4 and a therapeutic dose of the labeled MAb (2,332 ± 812 MBq 131I, average and SD; range, 743 to 3,823 MBq 131I, 6.49 ± 2.89 mg CD30 MAb) was administered using the same schedule (Fig 1).
The study design was in accordance with the declaration of Helsinki. This trial was approved by the Ethics Committee at the University of Cologne. Before treatment, all patients gave written informed consent. According to the German federal guidelines, the radioimmunoconjugate was registered with the Paul Ehrlich Institute, Berlin, Germany. The protocol was approved by the Bundesamt fuer Strahlenschutz, Salzgitter, Germany. It was intended to conduct a classical dose-escalating phase-I trial. Since patients presented in extremely variable individual conditions we decided to perform dose-escalations on an individual patient data basis. Thus, the maximum-tolerated dose was not the primary end point for this trial but feasibility.
Quantitative Imaging, Biodistribution, and Dosimetry
Biodistribution data and dose factors for the total body were collected by simultaneous anterior and posterior whole-body scans obtained 1 hour, 24 hours, 48 hours, 72 hours, and 144 hours post infusionem, using a dual-head gamma camera (E.CAM; Siemens, Erlangen, Germany) equipped with high-energy collimators. For quantification of each study, a standard capsule of 131I (approx. 2 MBq) was positioned beside the patients' ankles.
Using NucliDose version 5.0.2 on a Siemens ICON computer, regions of interest for the whole-body, background and standard capsule defined whole-body time-activity curves from which the residence times were derived. The whole-body residence times were entered into MIRDOSE 3.1, yielding the total absorbed radiation dose to the whole-body per injected activity (dose factor).
To estimate the radiation dose delivered to the red marrow blood samples were taken in five dosimetric and three therapeutic studies at the time of the scans. Dose coefficients for red marrow were calculated from the blood time-activity curves using the formula given in Shen et al.41
Assessment of Toxicity
Adverse events were graded according to the National Cancer Institute toxicity criteria (NCI Cancer Therapy Evaluation Program Common Toxicity Criteria, version 2.0).
Evaluation of Response
Clinical staging was performed according to the Ann Arbor system. Restaging computed tomography CT scans of involved areas were performed 6 weeks after the start of treatment. A complete response (CR) was defined as the absence of any clinical or radiological evidence of active disease over a period of 4 weeks. A partial remission (PR) was defined as a 50% or greater reduction in the sum of the products of the maximal and perpendicular diameters of all measurable lesions for at least 4 weeks; a minor response (MR) as a decrease of 25% to 50% of the measurable tumor mass for at least 4 weeks. Stable disease was defined when the criteria of CR, PR, MR or progressive disease (PD) were not met. PD was defined as enlargement of measurable tumor by more than 25% or appearance of any new lesion.
Detection of Human Antimouse Antibodies
Human antimouse antibodies (HAMA) against Ki-4 were measured as previously described23 before dosimetry, before treatment and on day 8 of treatment.
Flow Cytometry Analyses
Blood counts, with differentiation and analysis of circulating CD30-positive peripheral mononuclear blood cells (PMNBC; FACScan; Becton Dickinson, Mountain View, CA) were performed before dosimetry and before treatment.
Soluble CD30
Soluble CD30 (sCD30) was analyzed by standard ELISA methods: sCD30 (Dako, Hamburg, Germany). Analyses were performed before initiating treatment, and on study day 1 and 8 before and after administration of the unlabeled Ki-4.
Statistical Methods
A descriptive analysis was used to evaluate demographics, disease characteristics, adverse events and toxicity and degree of remission. As a measure of goodness of fit, R2 was calculated by using Pearson's correlation coefficient.
RESULTS
Patients Demographs
Twenty-two patients were treated in this trial (Table 1) . All biopsies were strongly positive for CD30. The median age was 31 years (range, 15 to 43 years). Most patients had advanced disease (19 of 22 patients) and were heavily pretreated with a median of four different prior chemotherapies (range, two to six) including high-dose chemotherapy (HDCT) and autologous peripheral stem-cell transplantation (APSCT) in 16 of 22 patients. Seventeen patients had received prior radiotherapy (Table 2). Eight patients had primary progressive disease (ie, relapse within 3 months after first-line treatment), and eight patients presented with early relapse within 12 months after initial treatment. At the start of dosimetry, the median performance status as measured by the Karnofsky index was 70 (range, 60 to 90). Platelet count before treatment was 85,000/μL in one patient, between 100,000/μL and 150,000/μL in two patients, and greater than 150,000 in 19 patients.
Imaging and Dosimetry
Dosimetry and treatment were performed in the time-frame as planned in the protocol in 20 of 22 patients. Due to disease-related infectious complications, therapeutic doses were postponed until recovery in patient 17 on day 24 and in patient 20 on day 32.
Early gamma camera images showed mainly blood pool activity, which was present up to 144 hours after administration. Specific uptake of the radioimmunoconjugate in lymphoma sites or in pulmonary infiltrates was clearly delineated on the scans 24 hours until 144 hours post infusionem during dosimetry and therapy in four patients with tumor masses measuring more than 5 cm and in one patient with a thoracic mass of 2.5 cm in diameter (Table 3). Figure 2 shows representative sets of whole-body scans. In general, tumor contrast was low, likely due to very low accumulation of 131I–Ki-4 in the tumor lesions. Tumor visualization was particulary difficult for lesions 3 cm in diameter due to limited resolution of the gamma camera for small lesions with modest uptake and often adjacent blood pool activity.
Excluding the two patients whose therapy was postponed, the average diagnostic residence time was 72.6 ± 19.1 hours (median, 70.3 hours), respectively. The average dose factor for the calculated total body dose was 0.20 ± 0.05 mGy/MBq (median, 0.21 mGy/MBq). Imaging during treatment showed an average residence time of 78.0 ± 24.1 hours (median, 78.4 hours). The average dose factor for the calculated total body dose was 0.22 ± 0.08 mGy/MBq (median 0.22 mGy/MBq). The correlation between the residence time during dosimetry and during therapy was 0.58 (Fig 3) and the correlation of the dose factor for the total body dose during dosimetry and during therapy was 0.81. The two patients who had a longer interval from dosimetry to therapy showed intensive accumulation in liver and spleen. The therapeutic dose factor for the calculated total body dose dropped to 0.037 and 0.047 mGy/MBq, respectively.
The dose factor for the calculated red marrow dose was 0.50 ± 0.24 mGy/MBq (median, 0.48 mGy/MBq) during dosimetry and 0.29 ± 0.10 mGy/MBq (median, 0.35 mGy/MBq) during treatment. The intraindividual ratio between the red marrow dose factor calculated from the blood samples to the total body dose factor calculated from the images was 2.60 ± 0.64 (median, 2.65; dosimetry) and 2.31 ± 0.68 (median, 2.64; treatment), respectively.
Toxicity
Infusion of 131I–Ki-4 was well-tolerated with no adverse reactions. The most common nonhematologic toxicity was grade 1 fatigue in 20 of 22 patients. Fatigue started a few hours after infusion of 131I–Ki-4 and lasted for a period of 24 to 72 hours. Five patients experienced mild nausea (grade 1); one patient, pain (grade 1) in involved lymph node areas; and one patient, mild gastritis (grade 1). In one patient, an allergic reaction with urticaria occurred 1 week after treatment. One patient developed a probably treatment-related skin ulcer in the heavily preirradiated right neck area 4 weeks after treatment. A similar ulcer had been present in the left neck area before treatment with 131I–Ki-4. In another patient a hemorrhagic colitis occurred 4 weeks after 131I–Ki-4 administration that was not clearly related to treatment.
The main toxicity was hematologic (Table 4). One patient was not assessable due to rapid disease progression of pulmonary HL and death within 3 weeks. Of 21 assessable patients, seven (33%) experienced a grade 4 hematotoxicity. Grade 4 thrombocytopenia was seen in seven patients and grade 3 in two patients. Grade 4 neutropenia occurred in three patients and grade 3 in one patient. Anemia grade 4 was observed in one patient and grade 3 in three patients. The median time to nadir for thrombocyte and absolute neutrophil count was 5 weeks (range, 4 to 8 weeks). Three patients remained thrombocytopenic grade 4 until death 8 to 15 weeks after start of treatment. Deaths were not related to bleeding complications but to disease progression. The median time to thrombocyte recovery to grade 1 thrombocytopenia in these patients was 8 weeks. There was neither a correlation between toxicity and number of pretreatments (R2, 0.13), number of treatments per year (R2, 0.00), administered whole-body dose (R2, 0.19), platelet count before treatment (R2, –0.30), hemoglobin (R2, –0.26), LDH (R2, 0.29), C-reactive protein (R2, 0.29), or sCD30 (R2, 0.00). The strongest impact on toxicity was observed for Karnofsky index (R2, –0.41) and the blood sedimentation rate (R2, 0.61). Importantly, three of four patients with lesions in the pelvis and vertebrae, consistent with HL involvement experienced a grade 4 hematotoxicity. None of six patients who were not treated with HDCT and PSCT had a grade 4 hematotoxicity.
HAMA
Antibody response against Ki-4 (>1 μg/mL) developed in four of 22 patients on day 7 after treatment. In two of these patients treatment was postponed and HAMA (1.4 μg/mL and 1.7 μg/mL) were already detectable but not analyzed before treatment. Treatment delay and HAMA had a significant impact on the residence time during the treatment period by reducing the residence time from 68.2 hours and 71.7 hours (dosimetry) to 11.6 hours and 18.6 hours (treatment), respectively.
sCD30 and Peripheral CD30-Positive Blood Cells
Eleven patients were analyzed for CD30-positive PMNBC. Before dosimetry, two patients showed less than 1% CD30-positive PMNBC; and one patient, 2% CD30-positive PMNBC, whereas eight patients were negative. In none of 11 patients were CD30-positive PMNBCs detectable before treatment.
sCD30 (median, 65 U/mL; range, 1 to 531 U/mL; normal, 0 to 20 U/mL42) was detectable in all 22 patients. Seven patients had serum levels above 100 U/mL. Immediately after preinfusion of native Ki-4 Mab, sCD30 was no longer detectable in 14 patients, and six patients had residual sCD30 (1 to 9 U/mL). In two patients, the preinfusion of 5 mg Ki-4 led to a reduction of sCD30 (28% and 88%) with persistence of higher levels (526 and 31 U/mL). Thus, preinfusion of 5 mg Ki-4 is sufficient to block the complete or nearly complete sCD30 in 91% of patients.
Response
One patient with small cervical, axillary, and retroperitoneal lymph nodes (maximum diameter of 2 cm) experienced a CR lasting 5 months. A PR was diagnosed in five patients lasting up to 6 months (median, 4 months). Figure 4 illustrates the marked reduction of a pulmonary Hodgkin's tumor after administration of 131I–Ki-4 resulting in a good PR for a period of 5 months. Overall, a total of 27% of patients responded to treatment. Responding patients had relapses in nonirradiated as well as in both, irradiated and nonirradiated areas. The whole body dose in responding patients varied from 0.169 to 0.989 Gy (median, 0.621 Gy). Only one responding patient showed a positive imaging of tumor localization. Three patients had MR, and one patient had stable disease with significant improvement of symptoms (paravertebral pain). Twelve patients (57%) experienced progressive disease.
DISCUSSION
In this trial, we evaluated the first anti-CD30 radioimmunoconjugate (131I–Ki-4) in relapsed and refractory HL patients. The following major findings emerge from this trial: (1) Only a minority of patients (23%) showed visualization of tumor masses. (2) Acute toxicity was mild with transient fatigue in 86% and nausea in 23% of patients. (3) The most relevant toxicity in this heavily pretreated patient population was severe myelosuppression in 33%. (4) The best predictors of hematotoxicity were blood sedimentation rate (R2, 0.61) and Karnofsky index (R2, –0.41). (5) Response included one CR, five PRs, and three MRs, which lasted for a median period of 4 months.
Although HL is sensitive for radiotherapy, there are only a limited number of approaches using targeted radiation clinically: One trial analyzed low-dose radioimmunotherapy in HL patients using 131I-labeled polyclonal ferritin-directed antibodies.35 Ferritin is a tumor-associated protein that was described in HL and other tumors.43,44 Antiferritin Abs did not bind to the tumor cells but to histiocytes instead.45 Patients received a total of 50 mCi, resulting in 40% tumor regression. Side effects were related to bone marrow toxicity with grade 4 thrombocytopenia in 10% and grade 4 neutropenia in 5%. Compared to our trial, these patients were less heavily pretreated with no HDCT and APSCT, which might explain the relatively low toxicity reported.
A phase I/II study with 90Y-labeled polyclonal antiferritin immunoglobulins and ABMT for refractory HL was performed by Vriesendorp et al.36 Nineteen patients received doses ranging from 20 to 50 mCi followed by ABMT with an overall response rate (ORR) of 65%. Sixteen patients with bone marrow involvement or unsuccessful marrow harvest were treated with a reduced activity (20 mCi), achieving an ORR of 58%. In general, responses were better in patients with smaller tumor burden (< 30 cm3) as compared with large tumor masses (> 500 cm3). When the ORRs were compared, 20 mCi 90Y-labeled antiferritin was as effective as 40 mCi, while myelosuppression was reduced with the lower activity. An update of this trial including 39 patients evaluable for response reported 10 CRs and 10 PRs, with nine CRs occurring in patients receiving more than one cycle of treatment.34
Positive tumor imaging was not predictive of good response in our trial. Only one patient with a positive scintigraphy (lesion of 2.5 cm in diameter) responded to treatment, whereas the remaining 4 patients had PD. In contrast, 80% of responding patients had a negative imaging. To our knowledge, only one other group investigated in vivo targeting of H-RS cells in patients using the 131I-labeled anti-CD30 MAb Ber-H2.46 Of six evaluated patients, a positive imaging was seen in 50%, with optimal images at 48 to 72 hours. In contrast, immunohistological studies of tissue biopsies, following the MAb administration, showed staining of H-RS cells at all tumor sites, including the lesions not imaged by immunoscintigraphy. The proportion of 90% to 100% tumor targeting with 111In- or 131I-labeled polyclonal antiferritin Abs35,36,47 might be due to variable antigen distribution of CD30 and ferritin in HL.45 Interestingly, some patients had no tumor imaging using the 111In monoclonal antiferritin Abs but became positive when receiving the polyclonal counterpart.36 This suggests different targets for polyclonal and monoclonal antiferritin Abs. Nevertheless, lesions smaller than 2 cm were not visible by scintigraphy.
Another clinical trial evaluated the administration of polyclonal 90Y-labeled antiferritin Abs in 90 HL patients in fractionated (2 x 0.25 mCi/kg) or unfractioned (0.3-0.5 mCi/kg) design.37 The most significant toxicity was thrombocytopenia, with a median grade 3 in the 0.3 and 0.4 mCi/kg group, and grade 4 in the patients receiving 0.5 or 2 x 0.25 mCi/kg. Thus, fractionation did not result in reduced hematologic toxicity. Tumor response was activity-related, with response rates of 22% in the 0.3 mCi/kg dose group, 60% in patients treated with 0.4 mCi/kg, 86% in the 0.5 mCi/kg group, and 45% using the fractionated schedule. A total of 15 of 90 CRs and 29 of 90 PRs, with a median duration of 6 months, were observed.
The population treated in the latter study is comparable to our patients since 50% of the patients had advanced disease with a median of four prior chemotherapies. Seventy-five percent of patients had failed ABMT or APSCT. Their treatment regimen was more myelosuppressive compared with our trial. Very similar, platelet values at study entry were not predictive for the severity of toxicity experienced after 90Y-labeled antiferritin Abs. Two of 50 re-treated patients developed human anti-Ab antibodies resulting in a completely different biodistribution of the Abs with increased liver-uptake and rapid blood clearance. Thus, the development of HAMA or other antibodies is not of clinical importance except for those patients with postponed treatment or re-treatment. Two of our patients also developed HAMA within 8 days after treatment. However, the percentage of patients with relevant HAMA titers is similar to those observed in other trials.47-49
The response rate of 27% with a median duration of 4 months compares to what was reported in the literature. However, 90Y-based constructs might be more promising since 90Y transfers a higher beta-energy with a longer pathway compared with 131I. This could be an advantage, especially in patients with larger tumor masses. Nevertheless, there is no significant difference in NHL with 90Y ibritumomab tiuxetan or 131I tositumomab.50 This might be due to the fact that in contrast to CD30, CD20 is not internalized and iodinated MAbs will not become dehalogenated.
Our results suggest that treatment with 131I-Ki-4 radioimmunotherapy of multiple relapsed patients refractory to APSCT is effective but associated with unpredictable myelosuppression. It might thus be more reasonable to combine radioimmunoconstructs in HL with HDCT followed by APSCT to treat selected patients (ie, primary refractory patients). This approach gave promising results in NHL51 but was disappointing in HL using 20 to 30 mCi polyclonal 90Y-labeled antiferritin in combination with HDCT.33 As well, it might be reasonable to focus on target antigens distinct from CD30 that are not restricted to H-RS cells since the number of H-RS cells is small, thus delivering only limited radioactivity to tumor sites. An alternative antigen could be CD25, which is expressed on H-RS cells and bystander T cells.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported in part by Deutsche Krebshilfe, grant 10-1899-En3, and Koeln Fortune program, Faculty of Medicine, University of Cologne, grant 149/2001.
Presented, in part, as oral presentation at the 42nd Annual Meeting of the American Society of Hematology, Philadelphia, PA, December 6-10, 2002, and the 16th Annual Congress of the European Association of Nuclear Medicine, Amsterdam, the Netherlands, August 23-27, 2003.
R.S. and M.D. contributed equally to this work.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Horn-Lohrens O, Tiemann M, Lange H, et al: Shedding of the soluble form of CD30 from the Hodgkin-analogous cell line L540 is strongly inhibited by a new CD30-specific antibody (Ki-4). Int J Cancer 60:539-544, 1995
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Hopital Huriez, Lille, France
Department of Radioimmunotherapy, City of Hope National Medical Center, Duarte, CA
ABSTRACT
PURPOSE: Hodgkin's lymphoma (HL) has been demonstrated to be a good target for immunotherapy since lymphocyte activation markers such as CD30 are expressed in high numbers on the malignant cells. Thus, we developed a new radioimmunoconjugate consisting of the murine anti-CD30 monoclonal antibody (MAb) Ki-4 labeled with iodine-131 (131I).
PATIENTS AND METHODS: The biodistribution of 131I–Ki-4 was assessed via dosimetry after preinfusion of 5 mg native Ki-4 followed by 250 to 300 MBq 131I-labeled Ki-4. Whole-body scintigraphy was performed 1 hour, 24 hours, 48 hours, 72 hours, and 6 days after the infusion. Dosimetry was calculated using the programs NucliDose ICON-IDL (version 5.0.2; Siemens, Erlanger, Germany) and MIRDOSE (version 3.1; Oak Ridge National Laboratories; Oak Ridge, TN). The therapeutic dose was given on day 8 after preinfusion of unlabeled Ki-4.
RESULTS: We treated 22 patients with relapsed or refractory CD30-positive HL. Preinfusion of 5 mg native Ki-4 was sufficient to bind the soluble CD30. Imaging demonstrated localization of involved areas measuring 5 cm in diameter or more in four patients and 2.5 cm in one patient. Patients received total body doses of 0.035 Gy to 0.99 Gy. Acute toxicity was mild with grade 1 fatigue in 19 of 22 assessable patients. Seven patients experienced grade 4 degrees hematotoxicity 4 to 8 weeks after treatment. Response included one complete remission, five partial remissions, and three minor responses.
CONCLUSION: Treatment with 131I–Ki-4 is effective but can be associated with severe hematotoxicity.
INTRODUCTION
Hodgkin's lymphoma (HL) has become a curable disease after the introduction of improved radiation techniques and polychemotherapy regimens.1-5 Though most patients even in advanced stages can be cured with standard approaches, only 30% to 50% of those who relapse attain durable disease-free remission after second-line treatment.6-8 The outcome is worse for those with primary refractory disease.9 Data from HL and non-HL (NHL) suggest that small numbers of residual tumor cells remaining after first-line treatment can give rise to late relapses.10,11 Thus, eliminating residual malignant lymphoma cells after first-line treatment might further improve outcome in these diseases. In addition, a combined immuno-/chemotherapy might help to reduce the amount of cytotoxic drugs to be given, resulting in less toxicity of the currently used regimens.
HL seems to be an ideal candidate for monoclonal antibody (MAb) -based immunotherapy since Hodgkin-Reed/Sternberg cells express specific surface antigens such as CD2512 and CD3013 in large amounts. Most MAb-based clinical trials evaluated constructs directed against CD30, which is a 120-kDa transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) –receptor superfamily. Physiologically, expression of CD30 is found on virus-infected lymphocytes and on a small subset of activated T cells. Furthermore, this receptor is involved in the negative selection process of auto-reactive lymphocytes.14 The CD30 ligand (CD30L) is present on activated T cells, resting B cells, and granulocytes. The biologic function of CD30/CD30L interaction is not yet fully understood, but both stimulation of cell growth as well as induction of apoptosis have been described.15 Similar to other members of the TNF family, CD30 seems to be involved in the cell cycle regulation.16,17 CD30 is expressed on a variety of other neoplasms such as mediastinal B-cell lymphoma, anaplastic large-cell lymphoma, peripheral T-cell lymphoma, and embryonal carcinoma.18-21 In respect to a potential clinical use, a vast number of murine anti-CD30 MAbs have been developed. So far, different approaches available to eradicate tumor cells using MAbs were evaluated in HL: immunotoxins,22-26 bispecific constructs,27-29 and more recently, "naked" humanized or fully human MAbs.30-32 So far, radioimmunotherapeutic approaches in HL had been studied using polyclonal antiferritin antibodies labeled with iodine-131 (131I) or Yttrium-90 (90Y).33-37 Since HL is very sensitive to radiotherapy, it is reasonable to investigate anti-CD30 radioimmunoconstructs in HL patients. The most commonly used radionuclide for radioimmunotherapy is 131I. This radionuclide was selected for the presented trial since it is commercially available and allows dosimetry after trace doses of the radioimmunoconjugate. Here we report the first specific treatment of refractory HL patients with an 131I-labeled murine anti-CD30 MAb (Ki-4).
PATIENTS AND METHODS
Patients
Twenty-two patients with histologically-confirmed diagnosis of HL were treated at the University of Cologne. All patients were refractory to conventional therapy or had relapsed. To be eligible, patients older than 15 years had to have measurable disease, a Karnofsky performance status of at least 50%, creatinine less than 2.0 mg/100 mL, ALT less than 3x the upper limit of normal, total bilirubin less than 1.5 mg/mL, if not disease related. In addition, more than 30% of H-RS cells in a prior biopsy had to stain for CD30. Exclusion criteria were uncontrollable infectious disease, collagen vascular disease, vasculitis, active second malignancy, chemotherapy or radiotherapy within 4 weeks, significant impairment of pulmonary function, granulocytes less than 1,500/μL, platelets less than 80,000/μL or any other major organ dysfunction unrelated to lymphoma, bone marrow infiltration greater than 25%, pregnancy, and presence of antibodies against mouse immunoglobulin more than 1 μg/mL. Concomitant administration of steroids was permitted if patients had been treated with steroids for at least 4 weeks before radioimmunotherapy.
Pathology and Eligibility
To analyze the expression of CD30, the last available biopsy derived from lymph nodes or other involved tissue was used. Bone marrow biopsies or aspiration were performed in each patient to examine possible infiltration.
Preparation and Iodination of the Anti-CD30 Antibody Ki-4
Labeling of the anti-CD30 MAb Ki-4. Radio-iodination of the anti-CD30 MAb Ki-4 (IgG1, directed against cluster A)38 was carried out by the Chloramin T method.39 The reaction mixture was then purified on a PD10 desalting column (Pharmacia Biotech, Piscataway, NJ).
Quality control. Size exclusion high-performance liquid chromatography (TosoHaas TSKgel G 2000 SW, 7.5 x 300 mm, 10 μm, eluent: isotonic saline, 1 mL/min) was used for quality control. The labeled MAb was eluted after 6 minutes; free iodine, after 11.5 minutes. The amount of free iodine was usually less than 4% for various labelings. The complex dissociation constant of 131I–Ki-4 with L540 cells as determined by Scatchard plot analysis demonstrated high affinity of the labeled antibody (KD = 7.54 x 10–8 mol/L).40
Protocol Design
After a 30-minute preinfusion of 5 mg native Ki-4, the labeled MAb (250-300 MBq 131I, 1.25 ± 0.78 mg CD30 MAb, average and standard deviation [SD]) was administered as a 30-minute infusion. Patients received iodide tablets (100 mg) starting 24 hours before treatment and continuing for 14 days to minimize the thyroid uptake of free iodine. No premedication was administered. Patients were monitored for alterations in vital signs and adverse reactions during and for up to 2 hours after antibody infusion. Eight days later, a second infusion of 5 mg native Ki-4 and a therapeutic dose of the labeled MAb (2,332 ± 812 MBq 131I, average and SD; range, 743 to 3,823 MBq 131I, 6.49 ± 2.89 mg CD30 MAb) was administered using the same schedule (Fig 1).
The study design was in accordance with the declaration of Helsinki. This trial was approved by the Ethics Committee at the University of Cologne. Before treatment, all patients gave written informed consent. According to the German federal guidelines, the radioimmunoconjugate was registered with the Paul Ehrlich Institute, Berlin, Germany. The protocol was approved by the Bundesamt fuer Strahlenschutz, Salzgitter, Germany. It was intended to conduct a classical dose-escalating phase-I trial. Since patients presented in extremely variable individual conditions we decided to perform dose-escalations on an individual patient data basis. Thus, the maximum-tolerated dose was not the primary end point for this trial but feasibility.
Quantitative Imaging, Biodistribution, and Dosimetry
Biodistribution data and dose factors for the total body were collected by simultaneous anterior and posterior whole-body scans obtained 1 hour, 24 hours, 48 hours, 72 hours, and 144 hours post infusionem, using a dual-head gamma camera (E.CAM; Siemens, Erlangen, Germany) equipped with high-energy collimators. For quantification of each study, a standard capsule of 131I (approx. 2 MBq) was positioned beside the patients' ankles.
Using NucliDose version 5.0.2 on a Siemens ICON computer, regions of interest for the whole-body, background and standard capsule defined whole-body time-activity curves from which the residence times were derived. The whole-body residence times were entered into MIRDOSE 3.1, yielding the total absorbed radiation dose to the whole-body per injected activity (dose factor).
To estimate the radiation dose delivered to the red marrow blood samples were taken in five dosimetric and three therapeutic studies at the time of the scans. Dose coefficients for red marrow were calculated from the blood time-activity curves using the formula given in Shen et al.41
Assessment of Toxicity
Adverse events were graded according to the National Cancer Institute toxicity criteria (NCI Cancer Therapy Evaluation Program Common Toxicity Criteria, version 2.0).
Evaluation of Response
Clinical staging was performed according to the Ann Arbor system. Restaging computed tomography CT scans of involved areas were performed 6 weeks after the start of treatment. A complete response (CR) was defined as the absence of any clinical or radiological evidence of active disease over a period of 4 weeks. A partial remission (PR) was defined as a 50% or greater reduction in the sum of the products of the maximal and perpendicular diameters of all measurable lesions for at least 4 weeks; a minor response (MR) as a decrease of 25% to 50% of the measurable tumor mass for at least 4 weeks. Stable disease was defined when the criteria of CR, PR, MR or progressive disease (PD) were not met. PD was defined as enlargement of measurable tumor by more than 25% or appearance of any new lesion.
Detection of Human Antimouse Antibodies
Human antimouse antibodies (HAMA) against Ki-4 were measured as previously described23 before dosimetry, before treatment and on day 8 of treatment.
Flow Cytometry Analyses
Blood counts, with differentiation and analysis of circulating CD30-positive peripheral mononuclear blood cells (PMNBC; FACScan; Becton Dickinson, Mountain View, CA) were performed before dosimetry and before treatment.
Soluble CD30
Soluble CD30 (sCD30) was analyzed by standard ELISA methods: sCD30 (Dako, Hamburg, Germany). Analyses were performed before initiating treatment, and on study day 1 and 8 before and after administration of the unlabeled Ki-4.
Statistical Methods
A descriptive analysis was used to evaluate demographics, disease characteristics, adverse events and toxicity and degree of remission. As a measure of goodness of fit, R2 was calculated by using Pearson's correlation coefficient.
RESULTS
Patients Demographs
Twenty-two patients were treated in this trial (Table 1) . All biopsies were strongly positive for CD30. The median age was 31 years (range, 15 to 43 years). Most patients had advanced disease (19 of 22 patients) and were heavily pretreated with a median of four different prior chemotherapies (range, two to six) including high-dose chemotherapy (HDCT) and autologous peripheral stem-cell transplantation (APSCT) in 16 of 22 patients. Seventeen patients had received prior radiotherapy (Table 2). Eight patients had primary progressive disease (ie, relapse within 3 months after first-line treatment), and eight patients presented with early relapse within 12 months after initial treatment. At the start of dosimetry, the median performance status as measured by the Karnofsky index was 70 (range, 60 to 90). Platelet count before treatment was 85,000/μL in one patient, between 100,000/μL and 150,000/μL in two patients, and greater than 150,000 in 19 patients.
Imaging and Dosimetry
Dosimetry and treatment were performed in the time-frame as planned in the protocol in 20 of 22 patients. Due to disease-related infectious complications, therapeutic doses were postponed until recovery in patient 17 on day 24 and in patient 20 on day 32.
Early gamma camera images showed mainly blood pool activity, which was present up to 144 hours after administration. Specific uptake of the radioimmunoconjugate in lymphoma sites or in pulmonary infiltrates was clearly delineated on the scans 24 hours until 144 hours post infusionem during dosimetry and therapy in four patients with tumor masses measuring more than 5 cm and in one patient with a thoracic mass of 2.5 cm in diameter (Table 3). Figure 2 shows representative sets of whole-body scans. In general, tumor contrast was low, likely due to very low accumulation of 131I–Ki-4 in the tumor lesions. Tumor visualization was particulary difficult for lesions 3 cm in diameter due to limited resolution of the gamma camera for small lesions with modest uptake and often adjacent blood pool activity.
Excluding the two patients whose therapy was postponed, the average diagnostic residence time was 72.6 ± 19.1 hours (median, 70.3 hours), respectively. The average dose factor for the calculated total body dose was 0.20 ± 0.05 mGy/MBq (median, 0.21 mGy/MBq). Imaging during treatment showed an average residence time of 78.0 ± 24.1 hours (median, 78.4 hours). The average dose factor for the calculated total body dose was 0.22 ± 0.08 mGy/MBq (median 0.22 mGy/MBq). The correlation between the residence time during dosimetry and during therapy was 0.58 (Fig 3) and the correlation of the dose factor for the total body dose during dosimetry and during therapy was 0.81. The two patients who had a longer interval from dosimetry to therapy showed intensive accumulation in liver and spleen. The therapeutic dose factor for the calculated total body dose dropped to 0.037 and 0.047 mGy/MBq, respectively.
The dose factor for the calculated red marrow dose was 0.50 ± 0.24 mGy/MBq (median, 0.48 mGy/MBq) during dosimetry and 0.29 ± 0.10 mGy/MBq (median, 0.35 mGy/MBq) during treatment. The intraindividual ratio between the red marrow dose factor calculated from the blood samples to the total body dose factor calculated from the images was 2.60 ± 0.64 (median, 2.65; dosimetry) and 2.31 ± 0.68 (median, 2.64; treatment), respectively.
Toxicity
Infusion of 131I–Ki-4 was well-tolerated with no adverse reactions. The most common nonhematologic toxicity was grade 1 fatigue in 20 of 22 patients. Fatigue started a few hours after infusion of 131I–Ki-4 and lasted for a period of 24 to 72 hours. Five patients experienced mild nausea (grade 1); one patient, pain (grade 1) in involved lymph node areas; and one patient, mild gastritis (grade 1). In one patient, an allergic reaction with urticaria occurred 1 week after treatment. One patient developed a probably treatment-related skin ulcer in the heavily preirradiated right neck area 4 weeks after treatment. A similar ulcer had been present in the left neck area before treatment with 131I–Ki-4. In another patient a hemorrhagic colitis occurred 4 weeks after 131I–Ki-4 administration that was not clearly related to treatment.
The main toxicity was hematologic (Table 4). One patient was not assessable due to rapid disease progression of pulmonary HL and death within 3 weeks. Of 21 assessable patients, seven (33%) experienced a grade 4 hematotoxicity. Grade 4 thrombocytopenia was seen in seven patients and grade 3 in two patients. Grade 4 neutropenia occurred in three patients and grade 3 in one patient. Anemia grade 4 was observed in one patient and grade 3 in three patients. The median time to nadir for thrombocyte and absolute neutrophil count was 5 weeks (range, 4 to 8 weeks). Three patients remained thrombocytopenic grade 4 until death 8 to 15 weeks after start of treatment. Deaths were not related to bleeding complications but to disease progression. The median time to thrombocyte recovery to grade 1 thrombocytopenia in these patients was 8 weeks. There was neither a correlation between toxicity and number of pretreatments (R2, 0.13), number of treatments per year (R2, 0.00), administered whole-body dose (R2, 0.19), platelet count before treatment (R2, –0.30), hemoglobin (R2, –0.26), LDH (R2, 0.29), C-reactive protein (R2, 0.29), or sCD30 (R2, 0.00). The strongest impact on toxicity was observed for Karnofsky index (R2, –0.41) and the blood sedimentation rate (R2, 0.61). Importantly, three of four patients with lesions in the pelvis and vertebrae, consistent with HL involvement experienced a grade 4 hematotoxicity. None of six patients who were not treated with HDCT and PSCT had a grade 4 hematotoxicity.
HAMA
Antibody response against Ki-4 (>1 μg/mL) developed in four of 22 patients on day 7 after treatment. In two of these patients treatment was postponed and HAMA (1.4 μg/mL and 1.7 μg/mL) were already detectable but not analyzed before treatment. Treatment delay and HAMA had a significant impact on the residence time during the treatment period by reducing the residence time from 68.2 hours and 71.7 hours (dosimetry) to 11.6 hours and 18.6 hours (treatment), respectively.
sCD30 and Peripheral CD30-Positive Blood Cells
Eleven patients were analyzed for CD30-positive PMNBC. Before dosimetry, two patients showed less than 1% CD30-positive PMNBC; and one patient, 2% CD30-positive PMNBC, whereas eight patients were negative. In none of 11 patients were CD30-positive PMNBCs detectable before treatment.
sCD30 (median, 65 U/mL; range, 1 to 531 U/mL; normal, 0 to 20 U/mL42) was detectable in all 22 patients. Seven patients had serum levels above 100 U/mL. Immediately after preinfusion of native Ki-4 Mab, sCD30 was no longer detectable in 14 patients, and six patients had residual sCD30 (1 to 9 U/mL). In two patients, the preinfusion of 5 mg Ki-4 led to a reduction of sCD30 (28% and 88%) with persistence of higher levels (526 and 31 U/mL). Thus, preinfusion of 5 mg Ki-4 is sufficient to block the complete or nearly complete sCD30 in 91% of patients.
Response
One patient with small cervical, axillary, and retroperitoneal lymph nodes (maximum diameter of 2 cm) experienced a CR lasting 5 months. A PR was diagnosed in five patients lasting up to 6 months (median, 4 months). Figure 4 illustrates the marked reduction of a pulmonary Hodgkin's tumor after administration of 131I–Ki-4 resulting in a good PR for a period of 5 months. Overall, a total of 27% of patients responded to treatment. Responding patients had relapses in nonirradiated as well as in both, irradiated and nonirradiated areas. The whole body dose in responding patients varied from 0.169 to 0.989 Gy (median, 0.621 Gy). Only one responding patient showed a positive imaging of tumor localization. Three patients had MR, and one patient had stable disease with significant improvement of symptoms (paravertebral pain). Twelve patients (57%) experienced progressive disease.
DISCUSSION
In this trial, we evaluated the first anti-CD30 radioimmunoconjugate (131I–Ki-4) in relapsed and refractory HL patients. The following major findings emerge from this trial: (1) Only a minority of patients (23%) showed visualization of tumor masses. (2) Acute toxicity was mild with transient fatigue in 86% and nausea in 23% of patients. (3) The most relevant toxicity in this heavily pretreated patient population was severe myelosuppression in 33%. (4) The best predictors of hematotoxicity were blood sedimentation rate (R2, 0.61) and Karnofsky index (R2, –0.41). (5) Response included one CR, five PRs, and three MRs, which lasted for a median period of 4 months.
Although HL is sensitive for radiotherapy, there are only a limited number of approaches using targeted radiation clinically: One trial analyzed low-dose radioimmunotherapy in HL patients using 131I-labeled polyclonal ferritin-directed antibodies.35 Ferritin is a tumor-associated protein that was described in HL and other tumors.43,44 Antiferritin Abs did not bind to the tumor cells but to histiocytes instead.45 Patients received a total of 50 mCi, resulting in 40% tumor regression. Side effects were related to bone marrow toxicity with grade 4 thrombocytopenia in 10% and grade 4 neutropenia in 5%. Compared to our trial, these patients were less heavily pretreated with no HDCT and APSCT, which might explain the relatively low toxicity reported.
A phase I/II study with 90Y-labeled polyclonal antiferritin immunoglobulins and ABMT for refractory HL was performed by Vriesendorp et al.36 Nineteen patients received doses ranging from 20 to 50 mCi followed by ABMT with an overall response rate (ORR) of 65%. Sixteen patients with bone marrow involvement or unsuccessful marrow harvest were treated with a reduced activity (20 mCi), achieving an ORR of 58%. In general, responses were better in patients with smaller tumor burden (< 30 cm3) as compared with large tumor masses (> 500 cm3). When the ORRs were compared, 20 mCi 90Y-labeled antiferritin was as effective as 40 mCi, while myelosuppression was reduced with the lower activity. An update of this trial including 39 patients evaluable for response reported 10 CRs and 10 PRs, with nine CRs occurring in patients receiving more than one cycle of treatment.34
Positive tumor imaging was not predictive of good response in our trial. Only one patient with a positive scintigraphy (lesion of 2.5 cm in diameter) responded to treatment, whereas the remaining 4 patients had PD. In contrast, 80% of responding patients had a negative imaging. To our knowledge, only one other group investigated in vivo targeting of H-RS cells in patients using the 131I-labeled anti-CD30 MAb Ber-H2.46 Of six evaluated patients, a positive imaging was seen in 50%, with optimal images at 48 to 72 hours. In contrast, immunohistological studies of tissue biopsies, following the MAb administration, showed staining of H-RS cells at all tumor sites, including the lesions not imaged by immunoscintigraphy. The proportion of 90% to 100% tumor targeting with 111In- or 131I-labeled polyclonal antiferritin Abs35,36,47 might be due to variable antigen distribution of CD30 and ferritin in HL.45 Interestingly, some patients had no tumor imaging using the 111In monoclonal antiferritin Abs but became positive when receiving the polyclonal counterpart.36 This suggests different targets for polyclonal and monoclonal antiferritin Abs. Nevertheless, lesions smaller than 2 cm were not visible by scintigraphy.
Another clinical trial evaluated the administration of polyclonal 90Y-labeled antiferritin Abs in 90 HL patients in fractionated (2 x 0.25 mCi/kg) or unfractioned (0.3-0.5 mCi/kg) design.37 The most significant toxicity was thrombocytopenia, with a median grade 3 in the 0.3 and 0.4 mCi/kg group, and grade 4 in the patients receiving 0.5 or 2 x 0.25 mCi/kg. Thus, fractionation did not result in reduced hematologic toxicity. Tumor response was activity-related, with response rates of 22% in the 0.3 mCi/kg dose group, 60% in patients treated with 0.4 mCi/kg, 86% in the 0.5 mCi/kg group, and 45% using the fractionated schedule. A total of 15 of 90 CRs and 29 of 90 PRs, with a median duration of 6 months, were observed.
The population treated in the latter study is comparable to our patients since 50% of the patients had advanced disease with a median of four prior chemotherapies. Seventy-five percent of patients had failed ABMT or APSCT. Their treatment regimen was more myelosuppressive compared with our trial. Very similar, platelet values at study entry were not predictive for the severity of toxicity experienced after 90Y-labeled antiferritin Abs. Two of 50 re-treated patients developed human anti-Ab antibodies resulting in a completely different biodistribution of the Abs with increased liver-uptake and rapid blood clearance. Thus, the development of HAMA or other antibodies is not of clinical importance except for those patients with postponed treatment or re-treatment. Two of our patients also developed HAMA within 8 days after treatment. However, the percentage of patients with relevant HAMA titers is similar to those observed in other trials.47-49
The response rate of 27% with a median duration of 4 months compares to what was reported in the literature. However, 90Y-based constructs might be more promising since 90Y transfers a higher beta-energy with a longer pathway compared with 131I. This could be an advantage, especially in patients with larger tumor masses. Nevertheless, there is no significant difference in NHL with 90Y ibritumomab tiuxetan or 131I tositumomab.50 This might be due to the fact that in contrast to CD30, CD20 is not internalized and iodinated MAbs will not become dehalogenated.
Our results suggest that treatment with 131I-Ki-4 radioimmunotherapy of multiple relapsed patients refractory to APSCT is effective but associated with unpredictable myelosuppression. It might thus be more reasonable to combine radioimmunoconstructs in HL with HDCT followed by APSCT to treat selected patients (ie, primary refractory patients). This approach gave promising results in NHL51 but was disappointing in HL using 20 to 30 mCi polyclonal 90Y-labeled antiferritin in combination with HDCT.33 As well, it might be reasonable to focus on target antigens distinct from CD30 that are not restricted to H-RS cells since the number of H-RS cells is small, thus delivering only limited radioactivity to tumor sites. An alternative antigen could be CD25, which is expressed on H-RS cells and bystander T cells.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported in part by Deutsche Krebshilfe, grant 10-1899-En3, and Koeln Fortune program, Faculty of Medicine, University of Cologne, grant 149/2001.
Presented, in part, as oral presentation at the 42nd Annual Meeting of the American Society of Hematology, Philadelphia, PA, December 6-10, 2002, and the 16th Annual Congress of the European Association of Nuclear Medicine, Amsterdam, the Netherlands, August 23-27, 2003.
R.S. and M.D. contributed equally to this work.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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