Purpose: Fractionated radioimmunotherapy may improve therapeutic outcome by decreasing heterogeneity of the dose delivered to the tumor and by decreasing hematologic toxicity, thereby allowing an increased amount of radionuclide to be administered. Because humanized anti-CD22 epratuzumab can be given repeatedly, a single-center study was conducted to establish the feasibility, safety, optimal dosing, and preliminary efficacy of weekly administrations of 90Y-labeled 1,4,7,10-tetra-azacyclodecane-N,N′,N″,N‴-tetraacetic acid–conjugated epratuzumab.

Experimental Design: Cohorts of three to six patients with B-cell lymphoma received 185 MBq/m2 [90Y]epratuzumab with unconjugated epratuzumab (total protein dose 1.5 mg/kg) once weekly for two to four infusions, with [111In]epratuzumab coadministered at first infusion for scintigraphic imaging and dosimetry.

Results: Sixteen patients received treatment without significant infusional reactions. The overall objective response rate was 62% (95% confidence interval, 39-86%) in both indolent (75%) and aggressive disease (50%). Complete responses (CR/CRu) occurred in 25% of patients and were durable (event-free survival, 14-41 months). Two patients receiving four infusions had hematologic dose-limiting toxicity. Serum epratuzumab levels increased with each weekly dose. Of 13 patients with tumor cell CD22 expression determined by flow cytometry, seven of eight with strongly positive results had objective responses, versus one of five with negative or weakly positive results (P = 0.032).

Conclusions: Radioimmunotherapy with weekly 185 MBq/m2 [90Y]epratuzumab achieved a high objective response rate (62%) across lymphoma subtypes, including durable CRs. The findings that three weekly infusions (555 MBq/m2, total dose) can be administered safely with only minor toxicity, that antibody levels increased during treatment weeks, and that therapeutic response predominantly occurs in patients with unequivocal CD22 tumor expression provide guidance for future studies.

Two radioimmunoconjugates are now available in the United States, and one in Europe, for treatment of advanced non-Hodgkin's lymphoma (NHL). On the basis of efficacy and safety data reported in multiple clinical trials, [90Y]ibritumomab tiuxetan (Zevalin, Biogen IDEC Pharmaceuticals, San Diego, CA) was approved for the treatment of relapsed or refractory follicular/low-grade or transformed NHL, including rituximab-refractory follicular NHL (14), whereas [131I]tositumomab (Bexxar, Glaxo SmithKline, Philadelphia, PA) was subsequently approved for the treatment of patients with CD20-positive, follicular NHL, with and without transformation, whose disease is refractory to rituximab and has relapsed following chemotherapy (59). In both regimens, the therapeutic dose is delivered as a single administration. There are, however, compelling arguments for a fractionated schedule of dose delivery, because fractionation would better deal with the problem of heterogeneity in absorbed dose, as well as decreasing hematologic toxicity, thereby allowing increased amount of radionuclide to be administered (10, 11). There are also experimental data indicating that therapeutic response can be improved by splitting a large single dose of radiolabeled antibody into a number of smaller administrations (12). Further, both approved radioconjugates involve labeled murine antibodies. The [90Y]ibritumomab tiuxetan regimen also requires two additional 250 mg/m2 infusions of chimeric rituximab, whereas the [131I]tositumomab regimen also requires two additional 450 mg infusions of unlabeled murine tositumomab. Although radioimmunotherapy approaches with two or more infusions have been explored clinically using murine antibodies in B-cell malignancies (13, 14), immunogenicity might limit the potential for continued application, and there have been no reports of fractionated studies extending these treatment regimens.

Epratuzumab is a humanized antibody in which the parent antibody has been replaced with ∼90% human IgG1 sequences and the only remaining murine immunoglobulin sequences restricted to the complementarity-determining regions of the binding site (15). As such, epratuzumab is not expected to evoke human anti-human antibodies (HAHA), which makes it suitable for repeated dosing. Epratuzumab binds specifically to the third immunoglobulin domain of CD22, a B-cell–restricted, lineage-dependent antigen that is rapidly internalized upon antibody binding (16, 17). CD22 seems to be involved in the regulation of B-cell activation as well as cell adhesion (18) and in vitro studies of epratuzumab with normal as well as neoplastic B cells support the ability to stimulate internalization as well as promote CD22 signaling (19). Epratuzumab has shown efficacy and safety in clinical studies of B-cell malignancies (20, 21), including retreatment (22) and when administered in combination with rituximab (23).

Although the mouse parental antibody (mLL2) labeled with 131I has shown efficacy in various subtypes of B-cell lymphoma (14, 24), after internalization the 131I-labeled antibody is dehalogenated and the radionuclide is released from the cell. Subsequent radioimmunotherapy trials with epratuzumab used 90Y, a residualizing radiometal that is retained in the cell upon internalization (25) and distributes β emission over a longer tissue range than 131I, thus potentially extending therapeutic efficacy in regions of poorly vascularized tumor. For radiolabeling, epratuzumab was conjugated with the macrocyclic chelating agent 1,4,7,10-tetra-azacyclodecane-N,N′,N″,N‴-tetraacetic acid (DOTA), and binding studies of DOTA-conjugated epratuzumab labeled with 90Y ([90Y]epratuzumab) showed little loss of isotope over at least 1 week (26). Safety and efficacy data from [90Y]epratuzumab administered as single dose therapy suggested that 740 MBq/m2 (20 mCi/m2) could be safely tolerated by patients with advanced NHL who did not have prior high-dose chemotherapy (27). Here, we present final results from our study evaluating fractionated dose therapy with [90Y]epratuzumab administered according to a once weekly schedule. Interim findings from this study have previously been reported in abstract form (28).

This single-center, open-label, dose-escalation, phase I/II study evaluated the feasibility, safety, optimal dosing, and preliminary efficacy of [90Y]epratuzumab administered once weekly for an increasing number of 2 to 4 consecutive weekly infusions (Fig. 1). This study was approved by the Ethics Committee for Lund University Hospital and was conducted between December 1999 and November 2001. Written informed consent was obtained from all patients.

Fig. 1.

Schematic of treatment schedule per each dose level.

Fig. 1.

Schematic of treatment schedule per each dose level.

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The study population comprised males or females, ≥18 years old, with histologically confirmed B-cell NHL (any histologic grade by Revised European-American Lymphoma classification) who have failed ≥1 regimen of standard chemotherapy. Patients had at least one confirmed site of tumor by computed tomography or other pretherapy imaging showing uptake of [111In]epratuzumab following the first infusion. They had adequate performance status (Karnofsky ≥70%, or Eastern Cooperative Oncology Group 0-2) with a minimal life expectancy of 3 months; were ≥4 weeks beyond any prior chemotherapy, any major surgery, or any radiation therapy to the index lesion; ≥2 weeks beyond change in dosage of corticosteroids; and patients who received an investigational agent must have completed follow-up and be off-study. Required laboratories were as follows: serum creatinine ≤1.5 mg/dL (or creatinine clearance ≥50 mL/min), bilirubin ≤2 mg/dL, WBC ≥3,000/mm3, granulocytes ≥1,500/mm3, and platelets ≥100,000/mm3. Exclusion criteria included central nervous system lymphomatous involvement, ≥25% B cells in unseparated bone marrow by flow cytometry, radiation to specific organs or areas at the maximum tolerated level or to >25% red marrow, splenomegaly, pregnancy, and premenopausal women unwilling to practice adequate birth control.

Both DOTA-conjugated epratuzumab and unconjugated epratuzumab were supplied by Immunomedics, Inc. (Morris Plains, NJ). DOTA-epratuzumab was supplied in 12.0 mg vials (10 mg/mL) for radiolabeling with either 90YCl3 (MAP Medical Technologies OY, Tikkakoski, Finland) or 111InCl3 (Mallinckrodt Medical B.V., Petten, the Netherlands) according to procedures described by Griffiths et al. (26). After a 15-minute incubation at 45°C, diethylenetriaminepentaacetic acid was added to quench the reaction and bind any remaining free isotope. Patient administration required at least 85% of the isotope must be antibody bound, and binding results for the radiolabeling procedure were determined by instant thin-layer chromatography to be 92.7 ± 3.0% for 90Y and 97.0 ± 1.1% for 111In. Patient doses used 185 MBq/m2 (5 mCi/m2) [90Y]epratuzumab measured by a dose calibrator that was calibrated against a 90Y standard with injected activity determined by the difference in syringe measurement before and after injection, whereas 150 MBq (4 mCi) [111In]epratuzumab was used when imaging was done. Radiolabeled epratuzumab was diluted in 10 mL sterile saline containing 1% human serum albumin, and unlabeled epratuzumab added to achieve a total antibody dose of 1.5 mg/kg. After ∼10% of the dose was infused i.v., with no significant reaction and stable vital signs, the infusion was to be completed over ∼30 minutes.

Toxicity was scored according to the National Cancer Institute Common Toxicity Criteria, version 2.0. Dose-limiting toxicity was defined as hematologic toxicity grade 3 lasting >28 days or grade 4 >14 days, whereas any grade 3 nonhematologic toxicity of any duration was regarded as dose-limiting toxicity. For dose escalation, patients were entered in dose cohorts of three to six patients, with the first cohort receiving 2 consecutive weekly infusions, the second cohort 3 weeks, and the third 4 weeks. If three patients in a cohort did not encounter dose-limiting toxicity, dose escalation was to proceed, but if one patient encountered dose-limiting toxicity, the cohort was to be expanded up to six patients, with escalation proceeding only if no additional patient encountered dose-limiting toxicity.

Baseline investigations were done within 4 weeks of initiating treatment and included physical examination; computed tomography scans of the thorax, abdomen, and pelvis; bone marrow biopsy; routine laboratories; and a blood sample for determination of HAHAs. During the 12 weeks posttreatment evaluation period, blood samples for hematology were obtained at least weekly and increased to twice to thrice per week in the event of grade 3 toxicity; serum chemistries were obtained at 1 and 4 weeks; blood samples for HAHA at 2, 4, 8, and 12 weeks; and physical examinations at 4 and 12 weeks. Evaluation of treatment response after the last weekly infusion included clinical examination and computed tomography scans at 4 to 6 weeks and 12 weeks and thereafter at least every 3 months until recurrence. Bone marrow biopsy was repeated if bone marrow had been involved previously. The treatment could be repeated once after 3 months provided there was neither severe toxicity nor progression of the disease.

The first infusion also included [111In]epratuzumab. Blood samples for pharmacokinetics and calculation of bone marrow absorbed dose (29) were to be obtained at 5 minutes after the first weekly infusion, then at 1 hour; 2 to 4 hours; 1, 2, and 3 to 5 days; and at 7 days. In addition to counting 111In and 90Y serum radioactivity, serum samples were shipped to Immunomedics for determination of antibody levels using an ELISA assay. Scintigraphic imaging for tumor targeting and dosimetry was done following the first infusion, then at 1, 2, 3 to 5, and 6 to 7 days. Imaging sessions included anterior and posterior whole-body images as well as planar views of the head/neck, chest, abdomen, and pelvis, with at least one single-photon emission computed tomography session including regions of suspected or confirmed tumor masses. The conjugate view method was used for activity quantification (30). Anterior and posterior regions of interest were drawn around organs and tumors, and activity calculated from the geometric mean of region of interest counts. After correcting for background, attenuation, and camera sensitivity, 111In activity was converted to 90Y activity by correcting for differences in physical decay. Using a monoexponential or biexponential curve fit to organ time-activity, the total cumulated activity was calculated as the area under the curve extrapolated to infinite time. Organ and tumor absorbed doses (the cumulated activity times the S value) were calculated using medical internal radiation dose formulas (31).

Tumor responses were assessed by International Workshop criteria (32) as CR (complete disappearance of all disease-related radiologic abnormalities and other assessable disease), CRu (one or more residual tumors >1.5 cm in diameter that regressed by >75% in the sum of the products of the two longest perpendicular diameters, SPD), partial response (50% or greater reduction in SPD of the six largest measurable sites, and no new lesions), progressive disease (>25% increase from the nadir value in the SPD of measurable lesions or the appearance of a new lesion), or stable disease (<50% reduction or <25% increase from the nadir in the SPD of measurable lesions, with no new lesions). An objective response is either a complete response (CR/Cru) or partial response with duration measured from onset of objective response to progressive disease. Intervals measured in all patients include time to progression (start of treatment to progression or death), event-free survival (start of treatment to death from any cause or failure in patients with an objective response), or time-to-treatment failure (start of treatment to death, progression, or change in therapy from any cause).

CD22 expression levels on lymphoma cells isolated from selected biopsy, bone marrow, or blood specimens were assessed by flow cytometry using FITC-labeled anti-CD22 monoclonal antibody M0738 (clone 4KB128; DAKO, Copenhagen, Denmark), which binds domain 2 of CD22 without interference from epratuzumab. CD22 expression was analyzed by quadrant analysis and described in a semiquantitative manner (Fig. 2). Negative expression was defined using the non–B cells among the analyzed cells. CD22 expression was described as “definitely positive” when virtually all tumor cells were positive, as “weakly positive” when more than a few percentage of the cells were not clearly positive, and as “negative” when less than a few percentage or no cells were positive.

Fig. 2.

CD22 expression levels for three different patients obtained from fine-needle aspirate or peripheral blood samples. Top left, definitely positive tumor cells from fine-needle aspirate. Top right, weakly positive tumor cells from fine-needle aspirate. Bottom, peripheral blood sample showing negative tumor cells in the top-left quadrant with positive polyclonal B cells in the top-right quadrant.

Fig. 2.

CD22 expression levels for three different patients obtained from fine-needle aspirate or peripheral blood samples. Top left, definitely positive tumor cells from fine-needle aspirate. Top right, weakly positive tumor cells from fine-needle aspirate. Bottom, peripheral blood sample showing negative tumor cells in the top-left quadrant with positive polyclonal B cells in the top-right quadrant.

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Patient characteristics and responses. Patient characteristics for the 16 patients treated in this study are summarized in Table 1. Initially, three patients were treated at dose level 1 (2consecutive weekly doses) and three patients at dose level 2 (3 consecutive weekly doses) without more than grade 2 toxicity. After treatment was advanced to dose level 3 (4 consecutively weekly doses), two patients experienced dose-limiting toxicity and the remaining five patients were then studied at dose level 2.

Table 1.

Patient characteristics (N = 16)

Age (y)  
    Median (range) 60 (44-79) 
Sex  
    Male/female 11/5 
Diagnosis to radioimmunotherapy (mo)  
    Median (range) 42 (13-196) 
Prior treatment regimens  
    Median (range) 2 (1-7) 
Prior rituximab  
    Yes/no 8/8 
Clinical characteristics  
    Aggressive/indolent 8/8 
Histology (REAL)  
    Follicular, grade 2 
    Follicular, grade 3 
    Follicular, transformed 
    DLBCL 
    Mantle cell 
    MALT 
    CLL 
Disease extent at study entry  
    Local/disseminated 8/8 
Serum lactate dehydrogenase  
    Normal/elevated 2/14 
Maximum tumor size  
    <5 cm/>5 cm 9/7 
Tumor burden  
    <500 g/>500 g 14/2 
Bone marrow involvement  
    Yes/no 7/9 
Age (y)  
    Median (range) 60 (44-79) 
Sex  
    Male/female 11/5 
Diagnosis to radioimmunotherapy (mo)  
    Median (range) 42 (13-196) 
Prior treatment regimens  
    Median (range) 2 (1-7) 
Prior rituximab  
    Yes/no 8/8 
Clinical characteristics  
    Aggressive/indolent 8/8 
Histology (REAL)  
    Follicular, grade 2 
    Follicular, grade 3 
    Follicular, transformed 
    DLBCL 
    Mantle cell 
    MALT 
    CLL 
Disease extent at study entry  
    Local/disseminated 8/8 
Serum lactate dehydrogenase  
    Normal/elevated 2/14 
Maximum tumor size  
    <5 cm/>5 cm 9/7 
Tumor burden  
    <500 g/>500 g 14/2 
Bone marrow involvement  
    Yes/no 7/9 

Abbreviations: REAL, Revised European-American Lymphoma classification; DLBCL, diffuse large B-cell lymphoma; MALT, mucosa-associated lymphoid tissue; CLL, chronic lymphocytic leukemia.

Treatment response data are given for each of the 16 patients in Table 2. Ten patients (62%; 95% confidence interval, 39-86%) had an objective response, including four (25%) with CR/CRu. For these 10 patients, the time to best treatment response was 6 to 22 weeks (median 8 weeks) and the median event-free survival was 7 months (26 months for the four patients with CR/CRu). Objective responses occurred in five of the six patients with indolent follicular lymphoma, with the other patient found to have a transformed lymphoma at a posttreatment biopsy, and each of the five other objective responses involved other lymphoma histologies: aggressive (i.e., grade 3) follicular, transformed follicular, mantle cell, mucosa-associated lymphoid tissue, and diffuse large B-cell lymphoma. Objective responses (all partial responses) occurred for four of seven patients with at least one tumor >5 cm in one dimension, both patients with a tumor burden >500 g, and two patients with pleural effusion (with resolution of the effusion in one patient).

Table 2.

Patient treatment response data

Patient no.Histology, REAL classificationPrior treatments
Radioimmunotherapy
Overall survival (mo)
RegimensLast regimenResponseTTF (mo)No. 90Y injectionsResponseTime to best response (wk)EFS (mo)TTF (mo)
Follicular transformed Fludarabine SD PR 3.5 3.5 11 
DLBCL CHOP + XRT CR 40 CR 32 32 42+ 
Follicular grade 2 Rituximab 8 injection PR 11 CR 18 19 19 39+ 
MALT Idarubicine fludarabine CRu 16 CRu 22 41 41 42+ 
Follicular grade 2 Chlorambucil NA 22 PD — — 1.5 24 
Follicular grade 2 Mitoxantrone fludarabine PR PR 38+ 
Mantle Cytosar PR 3* SD — — 36+ 
Follicular transformed CHOP SD 10 PD — — 10 
Atypical B-CLL MIME SD 16 SD — — 22 35+ 
10 Mantle Cytosar PD CR 13 14 14 34+ 
11 DLBCL DHAP PD 1.5 PD — — 1.5 
12 Follicular grade 2 CHOP PR PR 31+ 
13 Follicular transformed DHAP PD PD — — 31 
14 Follicular grade 2 Chlorambucil OR* PR 23+ 
15 Follicular grade 2 Cyclophosphamide fludarabine PR 12 PR 25 
16 Follicular grade 3 ICE PD PR 13 6.5 6.5 20+ 
Patient no.Histology, REAL classificationPrior treatments
Radioimmunotherapy
Overall survival (mo)
RegimensLast regimenResponseTTF (mo)No. 90Y injectionsResponseTime to best response (wk)EFS (mo)TTF (mo)
Follicular transformed Fludarabine SD PR 3.5 3.5 11 
DLBCL CHOP + XRT CR 40 CR 32 32 42+ 
Follicular grade 2 Rituximab 8 injection PR 11 CR 18 19 19 39+ 
MALT Idarubicine fludarabine CRu 16 CRu 22 41 41 42+ 
Follicular grade 2 Chlorambucil NA 22 PD — — 1.5 24 
Follicular grade 2 Mitoxantrone fludarabine PR PR 38+ 
Mantle Cytosar PR 3* SD — — 36+ 
Follicular transformed CHOP SD 10 PD — — 10 
Atypical B-CLL MIME SD 16 SD — — 22 35+ 
10 Mantle Cytosar PD CR 13 14 14 34+ 
11 DLBCL DHAP PD 1.5 PD — — 1.5 
12 Follicular grade 2 CHOP PR PR 31+ 
13 Follicular transformed DHAP PD PD — — 31 
14 Follicular grade 2 Chlorambucil OR* PR 23+ 
15 Follicular grade 2 Cyclophosphamide fludarabine PR 12 PR 25 
16 Follicular grade 3 ICE PD PR 13 6.5 6.5 20+ 

Abbreviations: TTF, time-to-treatment failure; EFS, event-free survival; OR, objective response; PD, progressive disease; SD, stable disease; PR, partial response; XRT, X-ray therapy; NA, not available; CHOP, cyclophosphamide, doxurubicin, vincristine, and prednisone; ICE, ifosfamide, carboplatin, and etoposide; DHAP, dexamethasone, cisplatin, and cytarabine; MIME, methylGAG, ifosfamide, methotrexate, and etoposide.

*

Objective response in patient with only palpable lesion that regressed by palpation.

Table 2 compares best response to last treatment with response data after radioimmunotherapy: Two of four patients with progressive disease achieved objective responses with radioimmunotherapy (including one CR), one of three patients with stable disease achieved a partial response with radioimmunotherapy, four of five patients with partial responses achieved objective responses with radioimmunotherapy (including one CR), and both patients with complete responses (CR or CRu) again had complete responses after radioimmunotherapy. For eight patients, the time-to-treatment failure after radioimmunotherapy was longer than it was following prior treatment. Among the seven patients with response data also available after previous rituximab treatment, all seven patients showed comparable or better response with radioimmunotherapy, including partial responses in two of four patients without any prior objective response to rituximab, and CRs in two of three patients with partial responses after rituximab.

CD22 expression. Lymphoma cells were evaluated by flow cytometry in 13 patients (10 from tumor masses, 2 from involved bone marrow, and 1 from blood). Objective responses occurred in seven of eight patients with definitely positive CD22 expression (including three CRs) and one of four patients with weakly CD22-positive tumor cells who had partial response, whereas the only patient with CD22-negative tumor cells exhibited progressive disease. Thus, response rates seem to be strongly correlated with unequivocal CD22 tumor expression (seven of eight versus one of five, P = 0.032 by Fischer's exact test).

Tumor targeting and dosimetry. Based on [111In]epratuzumab imaging with the first infusion, absorbed radiation doses to normal organs were calculated for all 16 patients for the first 185 MBq/m2 [90Y]epratuzumab infusion (Table 3). Estimates of median cumulative radiation doses in patients treated at each dose level can then be obtained by multiplying the tabulated values by the number of infusions administered. Although such an approach necessarily ignores possible pharmacokinetic and biodistribution changes that may occur with subsequent infusions, the resulting cumulative radiation dose estimates in this study are seen to remain below generally accepted limits of 3 Gy to the red marrow for nonmyeloablative therapy and 20 Gy to the liver, lungs, and kidneys. Fifteen patients showed unequivocal tumor uptake, whereas one patient had equivocal uptake in spite of a CD22-positive tumor. Twelve patients with targeting and adequately determined tumor volumes also had tumor dosimetry estimates calculated from a single 185 MBq/m2 [90Y]epratuzumab administration (Table 3). Median tumor doses for the first fractionated injections were 5.8 Gy (5.7-9.6) for three patients with CRs, compared with 2.2 Gy (1.2-4) for five patients with partial responses and 4.2 Gy (0.9-6.8) for four nonresponders, and 4.6 Gy (2.1-9.6) for seven patients with definitely positive CD22 expression, compared with 3.9 Gy (0.9-6.8) for three patients with weakly positive tumors and 3.6 Gy for the single patient with negative expression.

Table 3.

Absorbed radiation dose estimates for an infusionof 185 MBq/m2 [90Y]epratuzumab

OrgannMedian (Gy)Range (Gy)
Kidney* 16 1.6 1.0-8.9 
Liver 16 1.3 0.8-1.6 
Lungs 16 2.1 1.4-3.3 
Spleen 15 2.4 1.0-4.0 
Red marrow 16 0.44 0.33-1.3 
Total body 16 0.23 0.19-0.26 
Tumor§ 12 3.8 0.9-9.6 
OrgannMedian (Gy)Range (Gy)
Kidney* 16 1.6 1.0-8.9 
Liver 16 1.3 0.8-1.6 
Lungs 16 2.1 1.4-3.3 
Spleen 15 2.4 1.0-4.0 
Red marrow 16 0.44 0.33-1.3 
Total body 16 0.23 0.19-0.26 
Tumor§ 12 3.8 0.9-9.6 
*

Maximum value attributed to patient with overlapping tumor in kidney regions of interest.

One patient had splenectomy.

Determined by the blood method (29).

§

Twelve patients with tumor volume adequately determined for dosimetry. Themedian tumor/bone marrow absorbed dose ratio is 8 (range 2-21).

Toxicity. The only significant toxicity was hematologic, but no patient received either hematopoietic growth factors or platelet transfusions. No patient had any bleeding episode and no patient had a significant infection, except one who developed a Pneumocystis carinii infection, which responded to treatment with trimethoprim-sulfamethoxazole. As summarized in Table 4, for the 10 patients treated at dose levels 1 and 2 (i.e., with 2-3 consecutive weekly doses), eight patients had grade 0 to 2 hematologic toxicity and two patients had grade 3 neutropenia. Median time for recovery was week 9 after treatment. Of five patients at dose level 3, two had dose-limiting hematologic toxicity (grade 4 toxicity for 5 weeks, grade 3 for >7 weeks); both patients had received cytosar (3 g/m2, twice daily for 2 days) as their last chemotherapy infusion within 2 months of the start of radioimmunotherapy.

Table 4.

Patient hematologic toxicity following radioimmunotherapy

Dose levelnPlatelets (Nadir NCI grade)
ANC (Nadir NCI grade)
<1234<1234
1 (2 wk)     
2 (3 wk) 1*   
3 (4 wk)  4   3 
Dose levelnPlatelets (Nadir NCI grade)
ANC (Nadir NCI grade)
<1234<1234
1 (2 wk)     
2 (3 wk) 1*   
3 (4 wk)  4   3 

Abbreviations: ANC, absolute neutrophil count; NCI, National Cancer Institute.

*

One patient with platelet count already rapidly declining at study entry.

Includes one patient who withdrew 2 weeks after last infusion with disease progression with toxicity developing after receiving MIME.

Excluding one patient who received concomitant chemotherapy (MIME) and another with rapidly falling platelets probably due to progressive disease, linear regression of a patient's maximum hematologic toxicity grade with the number of 185 MBq/m2 infusions received (i.e., dose level) resulted in a correlation of R2 = 0.43 (P = 0.01 by Jonckheere-Terpstra test).

Fifteen patients had least one posttreatment serum sample evaluated for HAHA, including 13 patients with samples within 4 weeks, nine patients with samples at 8-week evaluations, and 12 patients with 12-week samples. No patients developed HAHA.

Pharmacokinetics. Epratuzumab peak and trough serum levels were measured immediately preceding and 2 hours following every infusion. As summarized in Table 5, levels increased with each 1.5 mg/kg infusion, with a mean Cmax of 65.4 μg/mL after fourth infusion. In the initial 12 patients, serum samples collected over the week following the first and last infusions were fit with a monoexponential function to determine circulation half-lives. As seen, mean half-lives increased from ∼4.5 days after the first infusion to ∼14 days after the fourth infusion.

Table 5.

Epratuzumab serum pharmacokinetics

InfusionPeak (μg/mL)* n (mean ± SD)Trough (μg/mL)n (mean ± SD)Half-life (h)n (mean ± SD)
16 (40.8 ± 16.6) 12 (107 ± 23) 
16 (52.3 ± 16.6) 16 (12.3 ± 6.0) 12 (211 ± 152) 
12 (64.5 ± 19.2) 12 (26.3 ± 11.2) 8 (211 ± 121) 
5 (65.4 ± 14.7) 5 (28.2 ± 8.0) 4 (334 ± 94) 
InfusionPeak (μg/mL)* n (mean ± SD)Trough (μg/mL)n (mean ± SD)Half-life (h)n (mean ± SD)
16 (40.8 ± 16.6) 12 (107 ± 23) 
16 (52.3 ± 16.6) 16 (12.3 ± 6.0) 12 (211 ± 152) 
12 (64.5 ± 19.2) 12 (26.3 ± 11.2) 8 (211 ± 121) 
5 (65.4 ± 14.7) 5 (28.2 ± 8.0) 4 (334 ± 94) 

NOTE: n specifies number of patients evaluated.

*

Serum sample obtained 2 hours after infusion.

Serum sample obtained immediately before infusion.

Half-lives determined by monoexponential fit to postinfusion serum samples.

Retreatment. Four patients with partial responses at 3 months received 185 MBq/m2 [90Y]epratuzumab coinfused with [111In]epratuzumab. One patient at the first dose level did not show any uptake and received no further infusions. However, all three patients at the second dose level showed tumor uptake and completed two additional weekly infusions, with two patients subsequently improving their response category to CR and CRu. Two patients had tumor dosimetry estimates calculated for the first 185 MBq/m2 [90Y]epratuzumab infusion; both were higher with retreatment than initial treatment (4.5 versus 2.2 Gy; 9.7 versus 4.0 Gy), and one patient also had [111In]epratuzumab administered at the third infusion, which showed a lower tumor uptake (2.0 Gy) compared with the first infusion (4.5 Gy). None of these four patients had more than grade 2 hematologic toxicity or evidence of cumulative toxicity. Three patients had samples evaluated for HAHA following retreatment and none was positive.

Patients in this study received 2 to 4 weekly administrations of 185 MBq/m2 [90Y]epratuzumab coinfused with unconjugated epratuzumab at a 1.5 mg/kg/wk total protein dose. This fractionated schedule of radioimmunotherapy was well tolerated with only minor infusional reactions. Hematologic toxicity was moderate and dose-limiting hematologic toxicity was not encountered until patients received 4 weekly doses. Dosimetry estimates from the first infusions showed that radiation doses to the red marrow and other organs remained within generally accepted limits for up to four administrations of 185 MBq/m2 [90Y]epratuzumab, and none of the patients had any evidence of a HAHA response.

Of the 16 patients, 62% achieved an objective response, including patients with aggressive as well as indolent disease, and with large (>500 mL) tumor burdens. In addition, 25% of patients achieved complete responses (CR/CRu) and these patients had particularly durable responses (event-free survival, 14-41 months). The 62% response rate compares favorably with the 50% response rate to the preceding therapy, and 50% of all patients had longer time-to-treatment failure following radioimmunotherapy than after their previous treatment. Although this was a dose-escalation study enrolling small numbers of patients across various indolent and aggressive histologies, these response rates seem comparable with those obtained in the pivotal trials of [90Y]ibritumomab tiuxetan (80% objective response, 34% CR/CRu) or [131I]tositumomab (65% objective response, 20% CR), both of which were restricted to patients with more favorable histologies (i.e., predominantly indolent follicular lymphoma; refs. 3, 7). In addition to lymphoma subtype, prior therapies may also influence radioimmunotherapy responsiveness, as supported by a recently reported trial demonstrating the ability of radioimmunotherapy administered as initial therapy to induce a superior response rate and prolonged clinical and molecular remissions in patients with advanced follicular lymphoma (33).

A major finding was the correlation between tumor cell CD22 expression and treatment response, because seven of eight patients with unequivocal CD22 expression had objective responses, compared with only one of four patients with weak CD22 expression, whereas the single patient with an apparent CD22-negative tumor exhibited progressive disease. In contrast, the relationship between tumor radiation dose and response remains less clear. This may, in part, reflect the coadministration of unconjugated epratuzumab, which itself has biological activity; however, efficacy is usually obtained in clinical trials repeatedly administering epratuzumab at higher doses (360 mg/m2) compared with the 1.5 mg/kg dosing used here. The 3.8 Gy median tumor dose with first infusion may be sufficient to induce complete responses if delivered by external beam radiotherapy (34). However, although the estimated tumor radiation doses varied almost 10-fold, the difference between median tumor radiation doses for patients achieving complete responses, partial responses, or those without objective responses did not show an entirely consistent pattern.

The relevance of targeting/dosimetry in evaluating tumor response to radioimmunotherapy continues to be a matter of debate (35, 36), reflecting not only limitations in the current methodology used for accurately determining tumor doses from planar images as well as the multifactorial nature of tumors, but also effects from the coadministration of cold antibody as has been directly investigated in a randomized trial, comparing response rates of cold and radiolabeled tositumomab with cold tositumomab alone (37). The lack of clear relationship between dosimetry and response has further been raised in other radioimmunotherapy NHL studies using single dose administration of [90Y]epratuzumab (27). In this study of fractionated radioimmunotherapy, dosimetry estimates are further based only on 111In imaging with the first infusion and a baseline computed tomography scan. For example, two patients had visible and palpable tumors that shrunk during the treatment period, and because absorbed tumor dose is inversely proportional to the tumor mass, the actual radiation dose delivered to their tumors might have been even higher (38, 39). The observation that both patients with repeated tumor dosimetry each had retreatment values twice that of initial treatment indicates the potential usefulness of fractionation.

Whereas the addition of unconjugated antibody (cold monoclonal antibody) to radioimmunotherapy protocols presumably improves dose distribution by blocking unwanted sequestration of the radiolabeled antibody, the amount of cold monoclonal antibody is likely to have an optimum (40) above which the dose distribution is worsened. Cold epratuzumab blood levels increased on the day of second, third, and fourth infusions. This progressive rise in the circulating unconjugated antibodies might interfere with the binding of the radiolabeled antibody at the tumor site, raising the question of the suitability of using 1.5 mg/kg per infusion in this fractionated schedule. A lower amount of cold antibody has been advocated and used in single therapeutic infusion study using epratuzumab (27). The finding that the absorbed tumor dose was lower following the third compared with the first infusion, in the only patient who received [111In]epratumab with these two infusions, is compatible with such an interpretation.

In this study, 3 weekly 185 MBq/m2 [90Y]epratuzumab infusions (cumulative dose, 555 MBq/m2) were determined to be safe, with two patients developing transient grade 3 neutropenia, only one patient with platelets already <100,000/mm3 by first infusion developing grade 3 thrombocytopenia, and no occurrence of grade 4 hematologic toxicity. Although [90Y]epratuzumab has been administered as a single dose at higher levels (740 MBq/m2) without encountering dose-limiting toxicity (27), this likely reflects the poorer status of patients entered in the present study. In fact, a separate ongoing, multicenter [90Y]epratuzumab study in NHL is continuing escalation after recently achieving an even higher cumulative dose (1,110 MBq/m2) with 3 weekly 370 MBq/m2 infusions (37).

In conclusion, this study shows the feasibility of a dose fractionation approach for radioimmunotherapy in NHL. The fractionated schedule used here was well tolerated, and up to 3 consecutively weekly administrations of 185 MBq/m2 [90Y]epratuzumab seems to be a safe and effective dose. In addition, the degree of tumor cell CD22 expression was strongly related to outcome response, an important finding that warrants confirmation in future radioimmunotherapy trials using [90Y]epratuzumab.

Grant support: Swedish Cancer Society, Mrs. Berta Kamprad Foundation, Gunnar, Arvid and Elisabeth Nilsson Foundation, Gustaf V Jubileum Fund, MedicalFaculty at University of Lund, Foundations of Lund's Health District Organization, and Siv-Inger and Per-Erik Andersson's Foundation.

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.

We thank Professors Tor Olofsson and Per-Ola Bendahl for their valuable comments on flow cytometry and statistics, respectively, and Associate Professor Lennart Darte, Karin Wingårdh, Gun. Holmström, and Margareta Persson for excellent technical assistance.

1
Witzig TE, White CA, Wiseman GA, et al. Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20(+) B-cell non-Hodgkin's lymphoma.
J Clin Oncol
1999
;
17
:
3793
–803.
2
Witzig TE, Flinn IW, Gordon LI, et al. Treatment with ibritumomab tiuxetan radioimmunotherapy in patients with rituximab-refractory follicular non-Hodgkin's lymphoma.
J Clin Oncol
2002
;
20
:
3262
–9.
3
Witzig TE, Gordon LI, Cabanillas F, et al. Randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin's lymphoma.
J Clin Oncol
2002
;
20
:
2453
–63.
4
Wiseman GA, Gordon LI, Multani PS, et al. Ibritumomab tiuxetan radioimmunotherapy for patients with relapsed or refractory non-Hodgkin lymphoma and mild thrombocytopenia: a phase II multicenter trial.
Blood
2002
;
99
:
4336
–42.
5
Kaminski MS, Estes J, Zasadny KR, et al. Radioimmunotherapy with iodine (131)I tositumomab for relapsed or refractory B-cell non-Hodgkin lymphoma: updated results and long-term follow-up of the University of Michigan experience.
Blood
2000
;
96
:
1259
–66.
6
Vose JM, Wahl RL, Saleh M, et al. Multicenter phase II study of iodine-131 tositumomab for chemotherapy-relapsed/refractory low-grade and transformed low-grade B-cell non-Hodgkin's lymphomas.
J Clin Oncol
2000
;
18
:
1316
–23.
7
Kaminski MS, Zelenetz AD, Press OW, et al. Pivotal study of iodine I 131 tositumomab for chemotherapy-refractory low-grade or transformed low-grade B-cell non-Hodgkin's lymphomas.
J Clin Oncol
2001
;
19
:
3918
–28.
8
Wahl RL, Zasadny KR, Estes J, et al. Single center experience with iodine-131 tositumomab radioimmunotherapy for previously untreated follicular lymphoma.In: Society of Nuclear Medicine 47th annual meeting, 2000. J Nucl Med 2000;41:78P.
9
Horning SJ, Lucas A, Younes D, et al. Iodine-131 tositumomab for non-Hodgkin's lymphoma (NHL) patients who progressed after treatment with rituximab: results of a multi-center Phase II study. In: The American Society of Hematology 43rd annual meeting and exposition 2000; 2000. p. 508a.
10
O'Donoghue JA. Dosimetric principles of targeted radiotherapy. In: Abrams PG, Fritzberg AR, editors. Radioimmunotherpy of cancer. New York: Marcel Dekker,Inc.; 2000. p. 1–20.
11
DeNardo GL, Schlom J, Buchsbaum DJ, et al. Rationales, evidence, and design considerations for fractionated radioimmunotherapy.
Cancer
2002
;
94
:
1332
–48.
12
Schlom J, Molinolo A, Simpson JF, et al. Advantage of dose fractionation in monoclonal antibody-targeted radioimmunotherapy.
J Natl Cancer Inst
1990
;
82
:
763
–71.
13
DeNardo GL, DeNardo SJ, Lamborn KR, et al. Low-dose, fractionated radioimmunotherapy for B-cell malignancies using 131I-Lym-1 antibody.
Cancer Biother Radiopharm
1998
;
13
:
239
–54.
14
Vose JM, Colcher D, Gobar L, et al. Phase I/II trial of multiple dose 131Iodine-MAb LL2 (CD22) in patients with recurrent non-Hodgkin's lymphoma.
Leuk Lymphoma
2000
;
38
:
91
–101.
15
Leung SO, Goldenberg DM, Dion AS, et al. Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL2.
Mol Immunol
1995
;
32
:
1413
–27.
16
Stein R, Belisle E, Hansen HJ, Goldenberg DM. Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody, LL2.
Cancer Immunol Immunother
1993
;
37
:
293
–8.
17
Shih LB, Lu HH, Xuan H, Goldenberg DM. Internalization and intracellular processing of an anti-B-cell lymphoma monoclonal antibody, LL2.
Int J Cancer
1994
;
56
:
538
–45.
18
Cyster JG, Goodnow CC. Tuning antigen receptor signaling by CD22: integrating cues from antigens and the microenvironment.
Immunity
1997
;
6
:
509
–17.
19
Carnahan J, Wang P, Kendall R, et al. Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties.
Clin Cancer Res
2003
;
9
:
3982
–90S.
20
Leonard JP, Coleman M, Ketas JC, et al. Phase I/II trial of epratuzumab (humanized anti-CD22 antibody) in indolent non-Hodgkin's lymphoma.
J Clin Oncol
2003
;
21
:
3051
–9.
21
Leonard JP, Coleman M, Ketas JC, et al. Epratuzumab, a humanized anti-CD22 antibody, in aggressive non-Hodgkin's lymphoma: phase I/II clinical trial results.
Clin Cancer Res
2004
;
10
:
5327
–34.
22
Siegel AB, Goldenberg DM, Cesano A, Coleman M, Leonard JP. CD22-directed monoclonal antibody therapy for lymphoma.
Semin Oncol
2003
;
30
:
457
–64.
23
Emmanoulides C, Leonard JP, Schuster SJ, et al. Multi-center, phase 2 study of combination antibody therapy with epratuzumab plus rituximab in recurring low-grade NHL.
Blood
2003
;
102/11
:
69a
.
24
Linden O, Tennvall J, Cavallin-Stahl E, et al. Radioimmunotherapy using 131I-labeled anti-CD22 monoclonal antibody (LL2) in patients with previously treated B-cell lymphomas.
Clin Cancer Res
1999
;
5
:
3287
–91s.
25
Sharkey RM, Behr TM, Mattes MJ, et al. Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22.
Cancer Immunol Immunother
1997
;
44
:
179
–88.
26
Griffiths GL, Govindan SV, Sharkey RM, Fisher DR, Goldenberg DM. 90Y-DOTA-hLL2: an agent for radioimmunotherapy of non-Hodgkin's lymphoma.
J Nucl Med
2003
;
44
:
77
–84.
27
Sharkey RM, Brenner A, Burton J, et al. Radioimmunotherapy of non-Hodgkin's lymphoma with 90Y-DOTA humanized anti-CD22 IgG (90Y-Epratuzumab): do tumor targeting and dosimetry predict therapeutic response?
J Nucl Med
2003
;
44
:
2000
–18.
28
Linden O, Hindorf C, Cavallin-Stahl E, et al. Outcome and absorbed dose following 90-Yttrium-epratuzumab in B-cell lymphoma, using a dose-fractionation schedule. In: American Society of Hematology 45th annual meeting 2003. Blood 2003;102:407a.
29
Sgouros G. Bone marrow dosimetry for radioimmunotherapy: theoretical considerations.
J Nucl Med
1993
;
34
:
689
–94.
30
Fleming A. technique for the measurement of activity using a gamma camera and computer.
Phys Med Biol
1979
;
24
:
176
–80.
31
Loevinger R, Budinger TF, Watson E. MIRD primer for absorbed dose calculations. Revised edition. New York: The Society of Nuclear Medicine; 1991.
32
Cheson BD, Horning SJ, Coiffier B, et al. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group.
J Clin Oncol
1999
;
17
:
1244
–53.
33
Kaminski MS, Tuck M, Estes J, et al. 131I-tositumomab therapy as initial treatment for follicular lymphoma.
N Engl J Med
2005
;
352
:
441
–9.
34
Sawyer EJ, Timothy AR. Low dose palliative radiotherapy in low grade non-Hodgkin's lymphoma.
Radiother Oncol
1997
;
42
:
49
–51.
35
Britton KE. Radioimmunotherapy of Non-Hodgkin's lymphoma.
J Nucl Med
2004
;
45
:
924
–5.
36
Goldenberg DM, Sharkey RM. Radioimmunotherapy of non-Hodgkin's lymphoma revisited.
J Nucl Med
2005
;
46
:
383
–4.
37
Davis TA, Kaminski MS, Leonard JP, et al. The radioisotope contributes significantly to the activity of radioimmunotherapy.
Clin Cancer Res
2004
;
10
:
7792
–8.
38
Hindorf C, Linden O, Stenberg L, Tennvall J, Strand SE. Change in tumor-absorbed dose due to decrease in mass during fractionated radioimmunotherapy in lymphoma patients.
Clin Cancer Res
2003
;
9
:
4003
–6S.
39
Hartmann Siantar CL, DeNardo GL, DeNardo SJ. Impact of nodal regression on radiation dose for lymphoma patients after radioimmunotherapy.
J Nucl Med
2003
;
44
:
1322
–9.
40
Press OW, Eary JF, Appelbaum FR, et al. Radiolabeled-antibody therapy of B-cell lymphoma with autologous bone marrow support.
N Engl J Med
1993
;
329
:
1219
–24.