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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Cancer Res. Author manuscript; available in PMC 2011 November 1.
Published in final edited form as:
PMCID: PMC2970691
NIHMSID: NIHMS223706

Sequential Cytarabine and Alpha-Particle Immunotherapy with Bismuth-213-Lintuzumab (HuM195) for Acute Myeloid Leukemia

Abstract

Purpose

Lintuzumab (HuM195), a humanized anti-CD33 antibody, targets myeloid leukemia cells and has modest single-agent activity against acute myeloid leukemia (AML). To increase the antibody’s potency without the nonspecific cytotoxicity associated with β-emitters, the α particle-emitting radionuclide bismuth-213 (213Bi) was conjugated to lintuzumab. This phase I/II trial was conducted to determine the maximum tolerated dose (MTD) and antileukemic effects of 213Bi-lintuzumab, the first targeted α-emitter, after partially cytoreductive chemotherapy.

Experimental Design

Thirty-one patients with newly diagnosed (n = 13) or relapsed/refractory (n = 18) AML (median age, 67 years; range, 37–80) were treated with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-lintuzumab 18.5–46.25 MBq/kg.

Results

The MTD of 213Bi-lintuzumab was 37 MB/kg; myelosuppression lasting > 35 days was dose-limiting. Extramedullary toxicities were primarily limited to ≤ grade 2 events, including infusion-related reactions. Transient grade 3/4 liver function abnormalities were seen in 5 patients (16%). Treatment-related deaths occurred in 2 of 21 patients (10%) who received the MTD. Significant reductions in marrow blasts were seen at all dose levels. The median response duration was 6 months (range, 2–12). Biodistribution and pharmacokinetic studies suggested that saturation of available CD33 sites by 213Bi-lintuzumab was achieved after partial cytoreduction with cytarabine.

Conclusions

Sequential administration of cytarabine and 213Bi-lintuzumab is tolerable and can produce remissions in patients with AML.

Keywords: Radioimmunotherapy, Alpha-particles, Acute myeloid leukemia, Monoclonal Antibodies, Bismuth-213

Translational Relevance

This report offers proof-of-principle that targeted α-particle immunotherapy can produce remissions in patients with advanced myeloid leukemia. Because of the short-range and high energy of α emissions, this approach may be beneficial in the setting of small-volume or micrometastatic disease in a variety of tumors. This study provides the rationale for the further development of α-particle immunotherapy using 213Bi and alternative radioisotopes in leukemia as well as other cancers, such as breast and prostate carcinoma, where micrometastatic disease is present.

INTRODUCTION

Although standard induction therapy with cytarabine and an anthracycline produces complete remissions (CR) in 50–70% of patients with acute myeloid leukemia (AML), long-term survival is seen in only 20–40% of patients (1). Following relapse, additional chemotherapy produces remissions in only 20–25% of patients. While allogeneic hematopoietic stem cell transplantation (HSCT) can produce long-term remissions in approximately 30% of patients with relapsed AML, most patients are not appropriate candidates due to age, co-morbidities, or lack of a suitable donor (2). The prognosis for older patients is even worse, with a 5-year survival rate of 5% for patients ≥ 65 years of age (3). Therefore, new therapies are needed to improve overall survival and reduce therapy-related toxicity.

Early studies showed that β particle-emitting anti-CD33 constructs containing iodine-131 or yttrium-90 could eliminate large leukemic burdens but produced prolonged myelosuppression requiring HSCT (4, 5). The unique physical and radiobiological properties of α-particles, however, may provide more efficient tumor cell killing and reduce the nonspecific cytotoxic effects seen with β-emitters. Compared with β-particles, α-particles have a shorter range (50–80 v 800–10,000 μm) and a higher linear energy transfer (LET) (100 v 0.2 keV/μm) (6). As few as 1 or 2 α-particles can kill a target cell. Therefore, the potential for specific antitumor effects makes α-particle immunotherapy an attractive approach for the treatment of cytoreduced or minimal disease.

Lintuzumab (HuM195) is a humanized monoclonal antibody that targets CD33, a 67-kDa cell surface glycoprotein expressed on most myeloid leukemia cells. It is also found on committed myelomonocytic and erythroid progenitors but not on pluripotent stem cells, granulocytes, or non-hematopoietic tissues (7, 8). Lintuzumab induces antibody-dependent cell-mediated cytotoxicity and can fix human complement in vitro (9). Previous studies demonstrated that lintuzumab can target leukemia cells in patients without immunogenicity (10), eliminate minimal residual disease in acute promyelocytic leukemia (11), and produce occasional remissions in AML (1214).

Bismuth-213 (213Bi) (t1/2 = 45.6 minutes) is a radiometal that emits an α-particle of 8 MeV and is prepared for clinical use from an actinium-225 (225Ac)/213Bi generator. Up to 37 MBq/kg of 213Bi-lintuzumab were safely administered to patients with relapsed or refractory AML in a phase I trial (15). Gamma camera imaging showed rapid uptake of 213Bi in the bone marrow, liver, and spleen, with tumor-to-whole body absorbed dose ratios 1,000-fold greater than β-emitting anti-CD33 constructs in a similar patient population. Although 14 of 18 patients had reductions in marrow blasts, none achieved CR. This was likely due to large tumor burdens in heavily pre-treated patients and to the relatively low specific activities of 213Bi-lintuzumab. We hypothesized that a 1–2 log reduction in tumor burden could increase the number of 213Bi atoms delivered to leukemia cells and produce remissions. To determine the effects of 213Bi-lintuzumab against cytoreduced disease, we conducted a phase I/II trial in which patients first received a non-remittive dose of cytarabine to decrease the leukemic burden.

PATIENTS AND METHODS

213Bi-Lintuzumab Preparation

The bifunctional chelate 2-(4-isothiocyanatobenzyl) diethylenetriamine pentaacetic acid (SCN-CHX-A-DTPA) was conjugated to lintuzumab (Protein Design Labs, Inc.; Fremont, CA) by TSI Washington (Rockville, MD), with a ligand-to-protein ratio of 4.5 (1619). 225Ac, supplied by Actinium Pharmaceuticals, Inc. (Florham Park, NJ), was obtained from Oak Ridge National Laboratory (Oak Ridge, TN) or the Institute for Transuranium Elements (Karlsruhe, Germany). Following construction of 225Ac/213Bi generators, 213Bi was eluted every 3–4 hours and conjugated to lintuzumab-SCN-CHXA-DTPA using previously described methods (17, 2023). Unconjugated antibody was added to adjust the specific activity to 555–740 MBq/mg to preserve the immunoreactivity of the radioconjugate. The final product was administered as an injection over 5 minutes.

Patient Eligibility

Patients with previously untreated AML > age 60 years or those who were unable to receive intensive chemotherapy due to co-morbid conditions, such as cardiovascular disease, were eligible. Patients with relapsed or primary refractory AML were also included. More than 25% of the patients’ bone marrow blasts were required to express CD33. No antileukemic therapy was administered for 3 weeks before study entry except for hydroxyurea, which was discontinued prior to treatment. Concurrent use of either oral or intravenous antibiotics was allowed. Entry criteria included creatinine < 2 mg/dL or creatinine clearance > 60 mL/min, bilirubin ≤ 2 mg/dL, and alkaline phosphatase and aspartate aminotransferase (AST) ≤ 2.5 times normal. Patients could not have detectable antibodies to lintuzumab or active central nervous system involvement by leukemia. Patients were treated from April, 2001-June, 2006 at Memorial Sloan-Kettering Cancer Center on a protocol approved by the Center’s institutional review board. All subjects gave written informed consent according to the Declaration of Helsinki.

Treatment

Patients were hospitalized and received cytarabine at a dose of 200 mg/m2 daily by IV continuous infusion for 5 days. Within 8 days after completion of cytarabine, 2–4 injections of 213Bi-lintuzumab (518–1,262 MBq each) were given over 1–2 days. Because 213Bi yields were limited by the activity of each 225Ac/213Bi generator, we escalated radioactivity doses by increasing the number of injections. Four dose levels of 213Bi-lintuzumab were administered in the phase I portion of the trial: 18.5, 27.75, 37 and 46.25 MBq/kg. Additional patients were treated at the maximum tolerated dose (MTD) of 37 MBq/kg in the phase II portion of the trial. Total administered activities ranged from 1,195–4,755 MBq, and total antibody doses ranged from 2–6.3 mg. Given the low level of gamma emissions from 213Bi, radiation isolation for patients and precautions for staff were not required. Hematopoietic growth factor support was allowed if clinically indicated according to ASCO guidelines (24). Prophylactic antibiotic and antifungal therapy was not given routinely. Toxicity was assessed according to the common toxicity criteria established by the National Cancer Institute, version 2.0. To measure antileukemic effects, we performed bone marrow aspirations at baseline, before administration of 213Bi-lintuzumab, then 4 and 8 weeks after the start of treatment. Four of the six responding patients received consolidation therapy, generally with single-agent cytarabine, at the discretion of the treating physician.

Biodistribution and Pharmacokinetics

The 440-keV γ emissions of 213Bi allowed biodistribution studies to be performed as previously described (15, 25). Patients underwent continuous gamma camera imaging for 60 minutes beginning immediately after the first and last injections of 213Bi-lintuzumab using a dual-head Vertex gamma camera (ADAC Laboratories, Milpitas, CA). We calculated activity in the liver as the geometric mean of the counts/minutes in the anterior and posterior images. Activity in the spine was estimated from the posterior view. Kinetic curves were generated, corrected for decay, and converted to percentage injected dose (%ID) for each region. The %ID for the spine was converted to marrow %ID by scaling a nominal estimate of the red marrow mass in the vertebrae according to body weight (25). Additional pharmacokinetic data was obtained through parametric rate images by fitting a linear expression to the counts in each pixel/minute over the 60-minute imaging period as previously described (26).

Response Definitions

CR was defined as < 5% bone marrow blasts with a neutrophil count of > 1000/μL, a platelet count of > 100,000/μL, and no extramedullary disease. CR with incomplete platelet recovery (CRp) was defined similarly, except that the platelet count was <100,000/μL in the setting of transfusion independence. Partial remission (PR) was defined as a > 50% decrease in bone marrow blasts with all of the hematologic values for CR (27).

Statistical Analysis

Three to 6 patients were treated at each dose level in the phase I portion of the study. The MTD was defined as the highest dose for which the incidence of dose-limiting toxicity (DLT) is < 33%. DLT’s included: (1) any grade 4 non-hematologic toxicity, (2) grade 3 abnormalities of liver function or serum creatinine, and (3) grade 4 leukopenia lasting ≥35 days in patients with baseline leukocyte counts of >1,000/μL. The primary endpoint in the phase II portion of the trial was response (CR + CRp + PR). Secondary endpoints included disease-free survival (DFS) and overall survival. A two-stage design was used in which the probabilities of a type I error and type II error were 0.05 and 0.2, respectively. This design yielded at least 80% probability of a positive result if the true response rate was at least 20%.

We correlated toxicity and antileukemic effects with various clinical parameters. Reductions in marrow blasts following cytarabine alone and in combination with 213Bi-lintuzumab were compared using the two-sided t-test. We compared clinical parameters, such as number of prior treatment regimens, baseline percentage of marrow blasts, and level of CD33 expression, between those patients with untreated AML/relapsed AML receiving first salvage treatment and those patients with primary refractory AML/multiply treated relapsed disease using the two-sided t test. We estimated the probability of overall survival using the Kaplan-Meier method.

RESULTS

Patient Characteristics

Thirty-one patients (median age, 67 years; range, 37–80) were treated with sequential cytarabine and 213Bi-lintuzumab (18.5–46.25 MBq/kg) (Table 1). Thirteen patients had untreated AML (5 with de novo AML; 8 with secondary AML). Among these previously untreated patients, 6 (46%) had Charlson comorbidity scores of > 1, an established adverse prognostic factor for response to standard induction chemotherapy (28, 29). Fifteen patients had relapsed AML (7 of whom received prior salvage treatment), and 3 patients had primary refractory AML. According to the Cancer and Leukemia Group B (CALGB) risk classification system (30), 22 patients (71%) had intermediate-risk karyotypes, while 9 (29%) had poor-risk cytogenetic abnormalities. Fifteen patients were treated in the phase I portion of the trial: 3 patients each received 18.5 MBq/kg and 27.75 MBq/kg; 5 received 37 MBq/kg, and 4 were treated with 46.25 MBq/kg. An additional 16 patients enrolled in the phase II portion of the study.

Table 1
Patient Characteristics

Adverse Effects

As expected, myelosuppression was the most common toxicity. Among 10 patients who began therapy with an absolute neutrophil count ≥ 1,000/μL, 9 (90%) had grade 3 (n = 1) or grade 4 (n = 8) neutropenia. Among 15 patients who were platelet-transfusion independent before treatment, all developed grade 3 (n = 4) or grade 4 (n = 11) thrombocytopenia. The median time from the start of cytarabine until recovery from grade 4 leukopenia was 29 days (range, 2–59). In the 6 patients who responded, the median time to neutrophil recovery ≥ 1,000/μL was 30 days (range, 25–57), and the median time to platelet-transfusion independence was 34 days (range, 23–87). The duration of myelosuppression was unrelated to the administered activity (P = 0.221) or the level of CD33 expression (P = 0.084), due in part to the effect of cytarabine. During the phase I portion of the trial, dose-limiting myelosuppression (defined as grade 4 leukopenia lasting ≥ 35 days) was seen in 2 of 4 patients treated with 46.25 MBq/kg. Therefore, we determined the MTD of 213Bi-lintuzumab following cytarabine to be 37 MBq/kg.

Myelosuppression-associated febrile episodes occurred in most patients. Twenty (65%) had documented bacterial infections, predominantly catheter-related coagulase-negative Staphylococcal bacteremia. Nineteen patients (61%) developed presumed fungal pneumonia, and 1 had candidemia. Eight patients (26%) had neutropenic fever without an identifiable source of infection. Peri-induction mortality related to infectious complications occurred in 2 of 21 patients (10%) receiving 37 MBq/kg and in 1 of 4 patients (25%) treated with 46.25 MBq/kg.

Liver function abnormalities were the most common extramedullary toxicity (Table 2). Across all dose levels, 21 of 31 patients (68%) developed transient elevations of bilirubin, alkaline phosphatase, or transaminases; however, grade 3 or 4 abnormalities were seen in only 5 patients (16%). No patients had evidence of sinusoidal obstructive syndrome. The median time to the onset of liver function abnormalities was 7 days (range, 3–30), and the median duration was 6 days (range, 1–27). Increases in serum creatinine occurred in 11 patients (35%), but only 1 developed a grade 3 elevation while receiving concomitant liposomal amphotericin and aminoglycosides. Nine patients (29%) had infusion-related reactions following administration of 213Bi-lintuzumab, typically characterized by fever, chills, and rigors, including 1 with grade 3 bronchospasm. Additionally, 1 patient developed a grade 4 gastrointestinal hemorrhage.

Table 2
Extramedullary Toxicity

Antileukemic Activity and Responses

Patients underwent bone marrow aspirations at baseline, after receiving cytarabine but before administration of 213Bi-lintuzumab, and finally 4 or 8 weeks after the start of therapy. Not all patients had evaluable specimens at each time point. Reductions in bone marrow blasts were seen at all dose levels (Fig. 1). Of the 31 patients, 26 had interpretable bone marrow aspirations 4 or 8 weeks after the start of therapy. Among these 26 patients, 20 (77%) had a > 20% decrease in marrow blasts. The mean reduction ± standard deviation was 47 ± 71% (range, −223–99). The reduction of marrow blasts was not related to level of CD33 expression (P = 0.083) or the administered activity (P = 0.439), likely due to variability in disease burden and leukemia subtypes, and in resistence to chemotherapy or radiation.

Fig. 1
Percentage change in bone marrow blasts after treatment with sequential cytarabine and 213Bi-lintuzumab in 26 evaluable patients.

A bone marrow aspiration performed following the completion of cytarabine but before administration of 213Bi-lintuzumab served to evaluate the relative contribution of each agent to the overall activity of the regimen. Typically, the marrow was assessed on day 6 or 7 of treatment, before the full antileukemic effects of cytarabine may be apparent. Nevertheless, among 25 patients whose bone marrow was evaluable at this time point, all had detectable disease with blast counts ranging from 7–96%. Only 11 (40%) had a > 20% reduction in marrow blasts, and the mean decrease was 10 ± 55% (range, −116–88).

A total of 21 patients had assessable bone marrow evaluations before treatment, after cytarabine but before 213Bi-lintuzumab, and 4 or 8 weeks after the start of treatment. Following cytarabine alone, 13 patients (62%) had reductions in marrow blasts with a mean decrease of 10 ± 58% (range, −116–88). From the post-cytarabine time point to recovery following treatment with 213Bi-lintuzumab, 16 patients (76%) had reductions in marrow blasts, including six (29%) who demonstrated progression with cytarabine alone. Five patients (24%) had increases in marrow blasts, compared to the post-cytarabine evaluation. The mean decrease in blasts during this interval was 41 ± 57% (range, −90–98). These data suggest that 213Bi-lintuzumab enhanced the antileukemic effect of cytarabine in most patients.

Clinical responses were seen 6 of the 25 patients (24%; 95% CI, 11–44) who received doses of ≥ 37 MBq/kg (Table 3). All responders had poor-risk features, including age ≥ 70 years or secondary AML; however, none of the 6 patients receiving < 37 MBq had a clinical response. Among the 11 patients with untreated AML who received doses ≥ MTD, 2 achieved CR (18%), 1 achieved CRp (9%), and 2 achieved PR (18%). Among the 7 patients with AML in first relapse who had not received prior salvage therapy, 1 (14%) attained a CRp. None of the 7 patients with primary refractory AML or multiply treated relapsed disease responded, indicating that effective cytoreduction was necessary to achieve remission after administration of 213Bi-lintuzumab. Except for the number of prior regimens (P < 0.005), no clinical differences, such as baseline marrow blast percentage (P = 0.779) or level of CD33 expression (P = 0.258), between the group with untreated AML or receiving first salvage therapy and the group with refractory or multiply treated relapsed AML were apparent. The lack of responses in heavily pretreated patients indicates the need for effective reduction in disease burden prior to administration of α-particle immunotherapy to achieve remission.

Table 3
Characteristics of Responding Patients

The overall response rate (CR + CRp + PR) among the 21 patients receiving the MTD of 37 MBq/kg was 19% (95% CI, 7–41). Two patients achieved CR; one had a CRp, and one had a PR. This group included 9 patients with previously untreated AML (3 de novo AML; 6 secondary AML), 10 with relapsed AML (6 first salvage; 4 previously treated), and 2 with primary refractory AML. Although all four responding patients were > 70 years of age and three had secondary AML, none had received prior therapy for AML.

The median response duration was 6 months (range, 2–12). The median overall survival duration for all patients was 4.6 months (range, 1–30). Among the 6 responders, the median survival was 13.7 months (range, 5–30). The median survival for patients with untreated AML (n = 13) was 7.7 months (range 1–30), whereas for patients with previously treated AML (n = 18), the median survival was 3.1 months (range, 1–13) (Fig. 2).

Fig. 2
Kaplan-Meier plot showing the probability of survival for patients with previously untreated AML and those with relapsed or refractory disease.

Biodistribution and Pharmacokinetics

Four patients (one at each dose level) underwent detailed biodistribution and pharmacokinetic studies. Posterior gamma camera imaging showed rapid localization of isotope to areas of leukemic involvement, including the bone marrow of the vertebrae and pelvis, the liver, and the spleen in all patients (Fig. 3). Despite avidity for free bismuth, the kidneys were not visualized, confirming the stability of 213Bi-lintuzumab in vivo. In contrast to the results seen in the initial phase I trial, in which 213Bi-lintuzumab was given without prior cytoreduction (15), cardiac blood pooling was seen after the last injection in 1 patient treated with 27.5 MBq/kg, indicating saturation of CD33 antigen sites within the bone marrow, liver, and spleen (Fig. 3A and B). Additional pharmacokinetic data was obtained by parametric rate imaging, in which an increase in the rate of isotope accumulation over the 1-hour imaging period is depicted by red-orange, and clearance is shown in blue-green. Reduced bone marrow uptake or clearance of 213Bi-lintuzumab was seen after multiple injections in all 4 patients who were studied, indicating saturation of antigen sites after partial cytoreduction with cytarabine (Fig. 3C and D). This is consistent with a reduction in the total number of target antigens by cytarabine and not due to loss of target leukemia cells, since imaging was done at the time of injection and α particle-mediated cytotoreduction of the leukemia would not be expected for several days.

Fig. 3
Gamma camera images show targeting of isotope to marrow, liver, and spleen after the first injection (A) and blood pooling after the last (B). Rate images show uptake of 213Bi by target organs over 1 hour after the first injection (C) and clearance after ...

Uptake of 213Bi by the marrow and liver, accounting for 70–90% of the injected activity, occurred within 5–10 minutes after injection and was maintained throughout the 1-hour period of image collection. Marrow activity after the first and last injections of 213Bi-lintuzumab was constant in 3 patients and decreased in 1 patient after multiple injections (Fig. 3E). All 4 patients had decreases in liver uptake following multiple injections, indicating a “first pass” binding effect with CD33 saturation of target cells (including leukemia cells and Kupffer cells) within the imaged liver space after several milligrams of antibody, as previously observed (Fig. 3F) (15).

DISCUSSION

In this study, we demonstrate that sequential administration of cytarabine and 213Bi-lintuzumab is tolerable and can produce remissions in some patients with AML. Although a relatively small group of heterogenous patients were included in this trial, it provides proof-of-principle that targeted α-particle immunotherapy may be effective at reducing low-volume disease. These results suggest that further investigation of radioimmunotherapy with α-emitters after cytoreduction and in the postremission or adjuvant setting is warranted.

Except as conditioning for HSCT, radioimmunotherapy with long-range, low energy β-emitters is useful only in treating bulky, radiosensitive cancers such as lymphoma, since DLT usually results from nonspecific irradiation of normal tissues. Conversely, therapy with high-energy, short-range α-particles can provide far more potent and selective delivery of radiation to individual tumor cells, yielding enhanced antitumor activity with decreased toxicity. Despite these advantages, CRs with 213Bi-lintuzumab alone would require extraordinarily high injected activities. If we assume tumor burdens of 1012 cells with an average CD33 density of 10,000/cell, approximately 1016 binding sites are available to lintuzumab. With the specific activities that are feasible, only 1 in 2,700 lintuzumab molecules carry the radiolabel. Therefore, it remains difficult to deliver 1–2 213Bi atoms to every leukemia cell, particularly because of its 46-minute physical half-life. In the setting of small-volume disease, however, the short path length and high LET of α-particles are ideal. In this study, cytoreduction provided by cytarabine allowed further reduction of residual leukemia by 213Bi-lintuzumab to produce remissions.

Although decreases in marrow blasts were seen at all dose levels, we observed a 213Bi dose-response relationship with remission occurring only at doses ≥ 37 MBq/kg. This suggests that cytarabine was not likely the sole cause of remissions. Moreover, serial bone marrow evaluations suggest that 213Bi-lintuzumab augmented the antileukemic activity of cytarabine alone. Although blasts were seen on all marrow samples after administration of cytarabine but before 213Bi-lintuzumab, these specimens were obtained before day 14 of therapy. Because of this early time point, it is impossible to determine whether any patient would have achieved a CR with cytarabine alone. The ability of sequential cytarabine and 213Bi-lintuzumab to induce remissions was seen only in patients where effective cytoreduction with cytarabine was possible. Six of 18 patients (33%) with untreated AML or untreated first relapse who received doses of ≥ 37 MBq/kg responded, while none of the 7 patients with primary refractory AML or multiply treated relapsed disease benefitted. This group of patients would not be expected to have a significant reduction in leukemic burden after single-agent cytarabine, and, as noted in an initial phase I study, treatment with 213Bi-lintuzumab alone at similar activities did not produce CRs in patients with large disease volumes.

The response rate in this trial was higher then expected from a single course of standard-dose cytarabine alone (31). Bodey et al. reported a 3% response rate after a single course of cytarabine at the dose and schedule used in this study (32). Similarly, in a report by Bickers et al., no responses were seen after one course of cytarabine at doses of 200 mg/m2/day for 5 days (33). The response rate reported in these early trials may be lower than expected today because of significant improvements in supportive care over the past 30 years. There are numerous nonrandomized trials in the literature that report a response rate of 10–20% with low-dose cytarabine in older patients with AML, but, in general, multiple cycles of therapy are necessary. The largest randomized trial of low-dose cytarabine compared with hydroxyurea confirmed a response rate of 18%, but only after a median of three courses. One of 103 patients (1%) achieved CR after the first cycle of therapy (34).

The current study demonstrates the impact of disease burden on antibody biodistribution. In the initial phase I study of single-agent 213Bi-lintuzumab, in which similar total antibody doses were used, the percentage of injected activity reaching the marrow after multiple doses increased in 38% of patients, whereas activity in the liver and spleen decreased in 75% and 56% of patients, respectively (15). This suggested that CD33 sites in the liver and spleen can act as antigen sinks and that as they become saturated, more drug reaches the marrow with repeated injections. Similarly, high numbers of circulating CD33-positive blasts or cell-free CD33 can adversely affect biodistribution of the drug by rapidly binding antibody and preventing it from reaching target sites within the marrow. In contrast, after partial cytoreduction in this trial, marrow activity remained constant or decreased in all 4 patients who were studied. Gamma camera imaging revealed cardiac blood pooling after the last injection of 213Bi-lintuzumab in one patient, suggesting that saturation of all antigen sites was possible in patients with smaller disease burdens. Additionally, parametric rate imaging following the last injection of 213Bi-lintuzumab showed decreased uptake or clearance of drug when compared to the first injection in all patients. Taken together, these data indicate greater saturation of antigen sites by 213Bi-lintuzumab in target organs after partial cytoreduction than with 213Bi-lintuzumab alone.

The strategy of arming lintuzumab with an α particle-emitting radionuclide was originally proposed to increase the modest immunologically-mediated antileukemic effects of the antibody itself. Based on a pilot study in which one of 10 patients with relapsed or refractory AML achieved a CR lasting over five years (12) and a phase II study that confirmed a 6% response rate (13), the role of lintuzumab in cytoreduced disease was examined in a randomized phase III trial (35). Patients with relapsed or refractory AML received mitoxantrone, etoposide, and cytarabine alone or with lintuzumab. While an improvement in response rate attributable to unconjugated antibody therapy did not reach statistical significance (36% v 28%; P = 0.28), no difference in adverse events or treatment-related mortality between the two groups was seen. A more recent phase I trial was conducted to determine whether higher concentrations of lintuzumab sustained over prolonged periods could result in greater therapeutic efficacy (14). In this study, 7 of 17 patients with AML responded. These results have led to an ongoing randomized phase II study of low-dose cytarabine with or without lintuzumab in older patients with untreated AML who are unable to tolerate standard induction chemotherapy.

Gemtuzumab ozogamicin (GO) represents an alternative antibody-based treatment to this radioimmunotherapeutic approach. GO agent is composed of a humanized anti-CD33 monoclonal antibody conjugated to a derivative of the potent antitumor antibiotic calicheamicin. When released from the immunoconjugate within the cytoplasm of a leukemic cell, calicheamicin induces DNA damage and subsequent apoptotic cell death. In a series of trials conducted in adults with AML in first relapse, a response rate (CR + CRp) of 26% was achieved (36).

Typically, significant myelosuppression is seen with GO, even as a single agent. The median time to neutrophil recovery ≥ 1500/μL in responding patients was 48 days (36). In contrast, resolution of grade 4 leukopenia occurred after a median of 22 days following administration of single-agent 213Bi-lintuzumab in an earlier phase I study (15). Following sequential therapy with cytarabine and 213Bi-lintuzumab, responding patients in the current trial had neutrophil recovery after a median of 30 days. Treatment with GO is also associated with significant liver function abnormalities. Grade 3 or 4 hyperbilirubinemia and transaminase elevations were reported in 29% and 18% of patients, respectively. Sinusoidal obstructive syndrome was seen in 5% of patients (36). Grade 3 or 4 liver function abnormalities, however, did not occur with single-agent 213Bi-lintuzumab (15). When given after cytarabine in the current study, only 16% of patients developed significant hyperbilirubinemia, and 3% had grade 3/4 transaminase elevations. Sinusoidal obstructive syndrome was not observed. The more favorable toxicity profile of 213Bi-lintuzumab suggests that integration of targeted α-particle immunotherapy into treatment strategies with standard chemotherapy may be more feasible than chemotherapy-GO combinations.

The ability of 213Bi-lintuzumab to produce remissions in some patients with poor-risk AML in this trial provide the rationale for the use of α-particle immunotherapy in the setting of small-volume leukemias and cancers, or micrometastatic disease. The major obstacles to the widespread use of radioimmunotherapy with 213Bi, however, are its short half-life and the requirement of an on-site 225Ac/213Bi generator. Therefore, we developed a second generation construct in which the isotope generator is directly conjugated to a tumor-specific antibody. In this strategy, 225Ac (t1/2 = 10 days) can serve as an in vivo generator of 4 α-particles at or within a cancer cell. Based on the activity of 225Ac-containing radioimmunoconjugates in several xenograft models (37), we are currently conducting a phase I trial of 225Ac-lintuzumab in advanced myeloid leukemia. Additional studies combining 225 Ac-lintuzumab with cytoreductive chemotherapy are planned.

Acknowledgments

We thank Renier Brentjens, Ellin Berman, Mark Frattini, Nicole Lamanna, Peter Maslak, and Mark Weiss for clinical care; Ronald Finn for radiolabeling of the immunoconjugate; Michael Curcio, Jing Qiao, Eva Burnazi, Catalina Cabassa, and Yan Ma for laboratory assistance, and Dragan Cicic (Actinium Pharmaceuticals, Inc.) for supplying 225Ac.

Supported by National Institutes of Health grants PO1 CA33049 and RO1 55349, the Leroy Foundation, the Experimental Therapeutics Center, the Lauri Stauss Leukemia Foundation, the Lymphoma Foundation, and Actinium Pharmaceuticals, Inc., Florham Park, NJ.

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