For Acute Myeloid Leukemia

Published OnlineFirst September 21, 2010; DOI: 10.1158/1078-0432.CCR-10-0382 Published OnlineFirst on September 21, 2010 as 10.1158/1078-0432.CCR-10-0382

Clinical Therapy Clinical Cancer Research Sequential Cytarabine and α-Particle Immunotherapy with Bismuth-213–Lintuzumab (HuM195) for Acute Myeloid Leukemia

Todd L. Rosenblat1, Michael R. McDevitt1, Deborah A. Mulford1, Neeta Pandit-Taskar1, Chaitanya R. Divgi1, Katherine S. Panageas1, Mark L. Heaney1, Suzanne Chanel1, Alfred Morgenstern2, George Sgouros1, Steven M. Larson1, David A. Scheinberg1, and Joseph G. Jurcic1

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 potency of the antibody 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/d) 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 five patients (16%). Treatment- related deaths occurred in 2 of 21 (10%) patients 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. Clin Cancer Res; 16(21); OF1–9. ©2010 AACR.

Although standard induction therapy with cytarabine patients ≥65 years of age (3). Therefore, new therapies are and an anthracycline produces complete remissions needed to improve overall survival and reduce therapy- (CR) in 50% to 70% of patients with acute myeloid leu- related toxicity. kemia (AML), long-term survival is seen in only 20% to Early studies showed that β-particle–emitting anti-CD33 40% of patients (1). Following relapse, additional chemo- constructs containing iodine-131 or yttrium-90 could therapy produces remissions in only 20% to 25% of pa- eliminate large leukemic burdens but produced prolonged tients. Although allogeneic hematopoietic stem cell myelosuppression requiring HSCT (4, 5). The unique transplantation (HSCT) can produce long-term remissions physical and radiobiological properties of α-particles, in ∼30% of patients with relapsed AML, most patients are however, may provide more efficient tumor cell killing not appropriate candidates due to age, comorbidities, or and reduce the nonspecific cytotoxic effects seen with lack of a suitable donor (2). The prognosis for older pa- β-emitters. Compared with β-particles, α-particles have a tients is even worse, with a 5-year survival rate of 5% for shorter range (50-80 versus 800-10,000 μm) and a higher linear energy transfer (100 versus 0.2 keV/μm; ref. 6). As few as one or two α-particles can kill a target cell. There- Authors' Affiliations: 1Departments of Medicine, Radiology, α Epidemiology and Biostatistics, and Medical Physics and the Molecular fore, the potential for specific antitumor effects makes - Pharmacology and Chemistry Program, Memorial Sloan-Kettering particle immunotherapy an attractive approach for the Cancer Center, New York, New York and 2European Commission treatment of cytoreduced or minimal disease. Joint Research Center, Institute for Transuranium Elements, Karlsruhe, Germany Lintuzumab (HuM195) is a humanized monoclonal Corresponding Author: Joseph G. Jurcic, Memorial Sloan-Kettering antibody that targets CD33, a 67-kDa cell surface glycopro- Cancer Center, Box 458, 1275 York Avenue, New York, NY 10065. tein expressed on most myeloid leukemia cells. It is also Phone: 212-639-2955; Fax: 212-772-8441; E-mail: [email protected]. found on committed myelomonocytic and erythroid pro- doi: 10.1158/1078-0432.CCR-10-0382 genitors but not on pluripotent stem cells, granulocytes, or ©2010 American Association for Cancer Research. nonhematopoietic tissues (7, 8). Lintuzumab induces

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(17, 20–23). Unconjugated antibody was added to Translational Relevance adjust the specific activity to 555 to 740 MBq/mg to pre- serve the immunoreactivity of the radioconjugate. The This report offers proof of principle that targeted final product was administered as an injection over α -particle immunotherapy can produce remissions 5 minutes. in patients with advanced myeloid leukemia. Because α of the short range and high energy of -emissions, Patient eligibility this approach may be beneficial in the setting of Patients with previously untreated AML age >60 years or small-volume or micrometastatic disease in a variety those who were unable to receive intensive chemotherapy of tumors. This study provides the rationale for the due to comorbid conditions, such as cardiovascular dis- further development of α-particle immunotherapy 213 ease, were eligible. Patients with relapsed or primary re- using Bi and alternative radioisotopes in leukemia fractory AML were also included. More than 25% of the as well as other cancers, such as breast and prostate patients' bone marrow blasts were required to express carcinoma, where micrometastatic disease is present. CD33. No antileukemic therapy was administered for 3 weeks before study entry except for hydroxyurea, which was discontinued before treatment. Concurrent use of ei- antibody-dependent cell-mediated cytotoxicity and can ther oral or intravenous antibiotics was allowed. Entry cri- fix human complement in vitro (9). Previous studies teria included creatinine <2 mg/dL or creatinine clearance ≤ showed that lintuzumab can target leukemia cells in pa- >60 mL/min, bilirubin 2 mg/dL, and alkaline phospha- ≤ tients without immunogenicity (10), eliminate minimal tase and aspartate aminotransferase 2.5 times normal. residual disease in acute promyelocytic leukemia (11), Patients could not have detectable antibodies to lintuzu- and produce occasional remissions in AML (12–14). mab or active central nervous system involvement by leu- 213 kemia. Patients were treated from April 2001 to June 2006 Bismuth-213 ( Bi; t1/2 = 45.6 minutes) is a radiometal that emits an α-particle of 8 MeV and is prepared for at Memorial Sloan-Kettering Cancer Center on a protocol clinical use from an actinium-225 (225Ac)/213Bi genera- approved by the Center's institutional review board. All tor. Up to 37 MBq/kg of 213Bi-lintuzumab were safely subjects gave written informed consent according to the administered to patients with relapsed or refractory Declaration of Helsinki. AML in a phase I trial (15). γ-Camera imaging showed rapid uptake of 213Bi in the bone marrow, liver, and Treatment Patients were hospitalized and received cytarabine at a spleen, with tumor-to-whole body absorbed dose ratios 2 1,000-fold greater than β-emitting anti-CD33 constructs dose of 200 mg/m daily by i.v. continuous infusion for 5 days. Within 8 days after completion of cytarabine, two in a similar patient population. Although 14 of 18 pa- 213 tients had reductions in marrow blasts, none achieved to four injections of Bi-lintuzumab (518-1,262 MBq each) were given over 1 to 2 days. Because 213Bi yields CR. This was likely due to large tumor burdens in heavi- 225 213 ly pretreated patients and to the relatively low specific were limited by the activity of each Ac/ Bi generator, 213 we escalated radioactivity doses by increasing the num- activities of Bi-lintuzumab. We hypothesized that a 213 one- to two-log reduction in tumor burden could in- ber of injections. Four dose levels of Bi-lintuzumab crease the number of 213Bi atoms delivered to leukemia were administered in the phase I portion of the trial: cells and produce remissions. To determine the effects of 18.5, 27.75, 37, and 46.25 MBq/kg. Additional patients 213Bi-lintuzumab against cytoreduced disease, we con- were treated at the maximum tolerated dose (MTD) of ducted a phase I/II trial in which patients first received 37 MBq/kg in the phase II portion of the trial. Total ad- a nonremittive dose of cytarabine to decrease the leuke- ministered activities ranged from 1,195 to 4,755 MBq, mic burden. and total antibody doses ranged from 2 to 6.3 mg. Given the low level of γ-emissions from 213Bi, radiation isola- tion for patients and precautions for staff were not Materials and Methods required. Hematopoietic growth factor support was allowed if clinically indicated according to American Soci- 213Bi-lintuzumab preparation ety of Clinical Oncology guidelines (24). Prophylactic The bifunctional chelate 2-(4-isothiocyanatobenzyl) antibiotic and antifungal therapy was not given routine- diethylenetriamine pentaacetic acid (SCN-CHX-A-DTPA) ly. Toxicity was assessed according to the Common Tox- was conjugated to lintuzumab (Protein Design Labs, Inc.) icityCriteriaversion2.0establishedbytheNational by TSI Washington, with a ligand-to-protein ratio of 4.5 Cancer Institute. To measure antileukemic effects, we (16–19). 225Ac, supplied by Actinium Pharmaceuticals, did bone marrow aspirations at baseline, before admin- Inc., was obtained from Oak Ridge National Laboratory istration of 213Bi-lintuzumab, and then 4 and 8 weeks or the Institute for Transuranium Elements. Following after the start of treatment. Four of the six responding construction of 225Ac/213Bi generators, 213Bi was eluted patients received consolidation therapy, generally with every 3 to 4 hours and conjugated to lintuzumab– single-agent cytarabine, at the discretion of the treating SCN-CHX-A-DTPA using previously described methods physician.

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Biodistribution and pharmacokinetics pixel/minute over the 60-minute imaging period as The 440-keV γ-emissions of 213Bi allowed biodistribu- previously described (26). tion studies to be done as previously described (15, 25). Patients underwent continuous γ-camera imaging for Response definitions 60 minutes beginning immediately after the first and last CR was defined as <5% bone marrow blasts with a injections of 213Bi-lintuzumab using a dual-head Vertex neutrophil count of >1,000/μL, a platelet count of γ-camera (ADAC Laboratories). We calculated activity >100,000/μL, and no extramedullary disease. CR with in- in the liver as the geometric mean of the counts/minutes complete platelet recovery (CRp) was defined similarly, in the anterior and posterior images. Activity in the spine except that the platelet count was <100,000/μL in the set- was estimated from the posterior view. Kinetic curves ting of transfusion independence. Partial remission (PR) were generated, corrected for decay, and converted to per- was defined as a >50% decrease in bone marrow blasts centage injected dose (%ID) for each region. The %ID for with all of the hematologic values for CR (27). the spine was converted to marrow %ID by scaling a nominal estimate of the red marrow mass in the verte- Statistical analysis brae according to body weight (25). Additional pharma- Three to six patients were treated at each dose level in the cokinetic data were obtained through parametric rate phase I portion of the study. The MTD was defined as the images by fitting a linear expression to the counts in each highest dose for which the incidence of dose-limiting toxicity (DLT) is <33%. DLTs included (a) any grade 4 non- hematologic toxicity, (b) grade 3 abnormalities of liver function or serum creatinine, and (c) grade 4 leukopenia lasting ≥35 days in patients with baseline leukocyte counts Table 1. Patient characteristics of >1,000/μL. The primary end point in the phase II portion of the trial was response (CR + CRp + PR). Secondary Characteristic No. patients % end points included disease-free survival and overall sur- N ( = 31) vival. A two-stage design was used in which the probabili- ties of type I and II errors were 0.05 and 0.2, respectively. Median age (range), y 67 (37-80) This design yielded at least 80% probability of a positive Sex result if the true response rate was at least 20%. Male 21 68 We correlated toxicity and antileukemic effects with Female 10 32 various clinical parameters. Reductions in marrow blasts Disease status following cytarabine alone and in combination with Untreated 13 42 213Bi-lintuzumab were compared using the two-sided De novo 516 t test. We compared clinical parameters, such as number Secondary 8 26 of prior treatment regimens, baseline percentage of mar- Median age (range), y 74 (51-80) row blasts, and level of CD33 expression, between those Charlson comorbidity index patients with untreated AML/relapsed AML receiving first 0431 salvage treatment and those patients with primary refrac- 1323 tory AML/multiply treated relapsed disease using the 2323 two-sided t test. We estimated the probability of overall 300 survival using the Kaplan-Meier method. 4215 518 Results Primary refractory 3 10 Median age (range), y 60 (37-66) Patient characteristics Relapsed 15 48 Thirty-one patients (median age, 67 years; range, 37-80) Untreated 8 25 were treated with sequential cytarabine and 213Bi-lintuzumab Previously treated 7 (1-4) 23 (18.5-46.25 MBq/kg; Table 1). Thirteen patients had un- (no. prior regimens)* treated AML (5 with de novo AML; 8 with secondary Median CR1 duration 4 (3-39) AML). Among these previously untreated patients, six (range), mo (46%) had Charlson comorbidity scores of >1, an estab- Median age (range), y 65 (49-75) lished adverse prognostic factor for response to standard Cytogenetics induction chemotherapy (28, 29). Fifteen patients had re- Intermediate-risk 22 71 lapsed AML (7 of whom received prior salvage treatment), Poor-risk 9 29 and 3 patients had primary refractory AML. According to Median CD33 expression 88 (36-100) the Cancer and Leukemia Group B risk classification (range), % system (30), 22 patients (71%) had intermediate-risk kar- *Includes two patients who underwent allogeneic HSCT. yotypes, whereas 9 (29%) had poor-risk cytogenetic abnormalities. Fifteen patients were treated in the phase I

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Table 2. Extramedullary toxicity

Toxicity Dose level 18.5 MBq/kg 27.75 MBq/kg 37 MBq/kg 46.25 MBq/kg (n =3) (n =3) (n = 21) (n =4)

Bilirubin Grade 3 0 0 3 1 Grade 4 0 0 1 0 Alkaline phosphatase (grade 3) 0 0 1 0 AST/ALT (grade 3) 0 0 1 0 Creatinine (grade 3) 0 0 1 0 Infusion-related reaction (grade 3) 0 0 1 0 GI bleeding (grade 4) 0 0 1 0

Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; GI, gastrointestinal.

portion of the trial: 3 patients each received 18.5 and irubin, alkaline phosphatase, or transaminases; however, 27.75 MBq/kg, 5 received 37 MBq/kg, and 4 were treated grade 3 or 4 abnormalities were seen in only 5 patients with 46.25 MBq/kg. An additional 16 patients were (16%). No patients had evidence of sinusoidal obstructive enrolled in the phase II portion of the study. syndrome. The median time to the onset of liver function abnormalities was 7 days (range, 3-30), and the median Adverse effects duration was 6 days (range, 1-27). Increases in serum cre- As expected, myelosuppression was the most common atinine occurred in 11 patients (35%), but only 1 devel- toxicity. Among 10 patients who began therapy with an oped a grade 3 elevation while receiving concomitant absolute neutrophil count of ≥1,000/μL, 9 (90%) had liposomal amphotericin and aminoglycosides. Nine pa- grade 3 (n =1)orgrade4(n = 8) neutropenia. Among tients (29%) had infusion-related reactions following ad- 15 patients who were platelet transfusion independent ministration of 213Bi-lintuzumab, typically characterized before treatment, all developed grade 3 (n = 4) or grade by fever, chills, and rigors, including 1 patient with grade 4(n = 11) thrombocytopenia. The median time from 3 bronchospasm. Additionally, 1 patient developed a the start of cytarabine until recovery from grade 4 leuko- grade 4 gastrointestinal hemorrhage. penia was 29 days (range, 2-59). In the six patients who responded, the median time to neutrophil recovery Antileukemic activity and responses ≥1,000/μL was 30 days (range, 25-57), and the median Patients underwent bone marrow aspirations at base- time to platelet transfusion independence was 34 days line, after receiving cytarabine but before administration (range, 23-87). The duration of myelosuppression was un- of 213Bi-lintuzumab, and finally 4 or 8 weeks after the start related to the administered activity (P = 0.221) or the level of therapy. Not all patients had evaluable specimens at of CD33 expression (P = 0.084) due in part to the effect of each time point. Reductions in bone marrow blasts were cytarabine. During the phase I portion of the trial, dose- seen at all dose levels (Fig. 1). Of the 31 patients, 26 limiting myelosuppression (defined as grade 4 leukopenia had interpretable bone marrow aspirations 4 or 8 weeks lasting ≥35 days) was seen in two of four patients treated after the start of therapy. Among these 26 patients, 20 with 46.25 MBq/kg. Therefore, we determined the MTD of (77%) had a >20% decrease in marrow blasts. The mean 213Bi-lintuzumab following cytarabine to be 37 MBq/kg. reduction ± SD was 47 ± 71% (range, −223 to 99). The Myelosuppression-associated febrile episodes occurred reduction of marrow blasts was not related to level of in most patients. Twenty (65%) had documented bacte- CD33 expression (P = 0.083) or the administered activity rial infections, predominantly catheter-related coagulase- (P = 0.439), likely due to variability in disease burden and negative Staphylococcal bacteremia. Nineteen patients leukemia subtypes, and in resistance to chemotherapy or (61%) developed presumed fungal pneumonia, and 1 radiation. had candidemia. Eight patients (26%) had neutropenic A bone marrow aspiration done following the com- fever without an identifiable source of infection. Peri- pletion of cytarabine but before administration of induction mortality related to infectious complications 213Bi-lintuzumab served to evaluate the relative contri- occurred in 2 of 21 patients (10%) receiving 37 MBq/kg bution of each agent to the overall activity of the regi- and in 1 of 4 patients (25%) treated with 46.25 MBq/kg. men. Typically, the marrow was assessed on day 6 or 7 Liver function abnormalities were the most common ex- of treatment before the full antileukemic effects of cytar- tramedullary toxicity (Table 2). Across all dose levels, 21 abine may be apparent. Nevertheless, among 25 patients of 31 patients (68%) developed transient elevations of bil- whose bone marrow was evaluable at this time point, all

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Table 3. Characteristics of responding patients

Patient no. Age/sex Diagnosis Cytogenetics Prior therapy Dose level Response (MBq/kg) (duration, mo)

7 74/M 2° AML after MDS Complex 5-Aza (MDS) 37 PR (4) 10 79/M 2° AML, h/o APL der(7), t(1,7) ATO, SAHA (AML); 37 CR (9) ATRA, IDR/Ara-C × 3 (APL) 11 80/M AML, M2 de novo 8 None 37 CR (12) 14 74/M 2° AML after MDS Normal None 46.25 PR (7) 15 70/F AML, M2, relapsed Normal IDR/Ara-C × 3 46.25 CRp (5) 18 75/M 2° AML after MDS del(9q) Decitabine (MDS) 37 CRp (2)

Abbreviations: MDS, myelodysplastic syndrome; 5-Aza, azacitidine; ATO, arsenic trioxide; SAHA, vorinostat; ATRA, all-trans retinoic acid; IDR, idarubicin; Ara-C, cytarabine; APL, acute promyelocytic. had detectable disease with blast counts ranging from Clinical responses were seen in 6 of the 25 patients 7% to 96%. Only 11 (40%) had a >20% reduction in (24%; 95% confidence interval, 11-44) who received marrow blasts, and the mean decrease was 10 ± 55% doses of ≥37 MBq/kg (Table 3). All responders had (range, −116 to 88). poor-risk features, including age ≥70 years or secondary A total of 21 patients had assessable bone marrow eva- AML; however, none of the 6 patients receiving luations before treatment, after cytarabine but before 213Bi- <37 MBq had a clinical response. Among the 11 patients lintuzumab, and 4 or 8 weeks after the start of treatment. with untreated AML who received doses greater than or Following cytarabine alone, 13 patients (62%) had reduc- equaltotheMTD,2achievedCR(18%),1achieved tions in marrow blasts with a mean decrease of 10 ± 58% CRp (9%), and 2 achieved PR (18%). Among the seven (range, −116 to 88). From the postcytarabine time point to patients with AML in first relapse who had not received recovery following treatment with 213Bi-lintuzumab, 16 prior salvage therapy, one (14%) attained a CRp. None patients (76%) had reductions in marrow blasts, including of the seven patients with primary refractory AML or 6 (29%) who showed progression with cytarabine alone. multiply treated relapsed disease responded, indicating Five patients (24%) had increases in marrow blasts com- that effective cytoreduction was necessary to achieve pared with the postcytarabine evaluation. The mean de- remission after administration of 213Bi-lintuzumab. crease in blasts during this interval was 41 ± 57% (range, Except for the number of prior regimens (P < 0.005), −90 to 98). These data suggest that 213Bi-lintuzumab no clinical differences, such as baseline marrow blast enhanced the antileukemic effect of cytarabine in most percentage (P = 0.779) or level of CD33 expression patients. (P = 0.258), between the group with untreated AML

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

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The overall response rate (CR + CRp + PR) among the 21 patients receiving the MTD of 37 MBq/kg was 19% (95% confidence interval, 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 six 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 Fig. 2. Kaplan-Meier plot showing the probability of survival for patients for patients with previously treated AML (n =18),the with previously untreated AML and those with relapsed or refractory median survival was 3.1 months (range, 1-13; Fig. 2). disease. Biodistribution and pharmacokinetics or receiving first salvage therapy and the group with Four patients (one at each dose level) underwent detailed refractory or multiply treated relapsed AML were appar- biodistribution and pharmacokinetic studies. Posterior ent. The lack of responses in heavily pretreated patients γ-camera imaging showed rapid localization of isotope to indicates the need for effective reduction in disease bur- areas of leukemic involvement, including the bone marrow den before administration of α-particle immunotherapy of the vertebrae and pelvis, the liver, and the spleen in all to achieve remission. patients (Fig. 3). Despite avidity for free bismuth, the

Fig. 3. γ-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 h after the first injection (C) and clearance after the last (D). Time-activity curves show localization and retention of 213Bi in the marrow (E) and liver (F). (-○-), first injection; (-♦-), fourth injection.

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kidneys were not visualized, confirming the stability of are feasible, only 1 in 2,700 lintuzumab molecules carries 213Bi-lintuzumab in vivo. In contrast to the results seen in the radiolabel. Therefore, it remains difficult to deliver one the initial phase I trial, in which 213Bi-lintuzumab was given to two 213Bi atoms to every leukemia cell, particularly be- without prior cytoreduction (15), cardiac blood pooling cause of its 46-minute physical half-life. In the setting of was seen after the last injection in one patient treated small-volume disease, however, the short path length and with 27.5 MBq/kg, indicating saturation of CD33 antigen high linear energy transfer of α-particles are ideal. In this sites within the bone marrow, liver, and spleen (Fig. 3A study, cytoreduction provided by cytarabine allowed fur- and B). Additional pharmacokinetic data were obtained ther reduction of residual leukemia by 213Bi-lintuzumab by parametric rate imaging, in which an increase in the rate to produce remissions. of isotope accumulation over the 1-hour imaging period is Although decreases in marrow blasts were seen at all depicted by red-orange, and clearance is shown in blue- dose levels, we observed a 213Bi dose-response relationship green. Reduced bone marrow uptake or clearance of 213Bi- with remission occurring only at doses ≥37 MBq/kg. This lintuzumab was seen after multiple injections in all four suggests that cytarabine was not likely the sole cause of patients who were studied, indicating saturation of antigen remissions. Moreover, serial bone marrow evaluations sites after partial cytoreduction with cytarabine (Fig. 3C and suggest that 213Bi-lintuzumab augmented the antileuke- D). This is consistent with a reduction in the total number mic activity of cytarabine alone. Although blasts were of target antigens by cytarabine and not due to loss of target seen on all marrow samples after administration of cytar- leukemia cells because imaging was done at the time of abine but before 213Bi-lintuzumab, these specimens were injection and α-particle–mediated cytoreduction of the obtained before day 14 of therapy. Because of this early leukemia would not be expected for several days. time point, it is impossible to determine whether any pa- Uptake of 213Bi by the marrow and liver, accounting for tient would have achieved a CR with cytarabine alone. 70% to 90% of the injected activity, occurred within 5 to The ability of sequential cytarabine and 213Bi-lintuzumab 10 minutes after injection and was maintained throughout to induce remissions was seen only in patients where the 1-hour period of image collection. Marrow activity af- effective cytoreduction with cytarabine was possible. Six ter the first and last injections of 213Bi-lintuzumab was of 18 patients (33%) with untreated AML or untreated first constant in three patients and decreased in one patient relapse who received doses of ≥37 MBq/kg responded, after multiple injections (Fig. 3E). All four patients had de- whereas none of the 7 patients with primary refractory creases in liver uptake following multiple injections, indi- AML or multiply treated relapsed disease benefited. This cating a “first-pass” binding effect with CD33 saturation of group of patients would not be expected to have a signifi- target cells (including leukemia cells and Kupffer cells) cant reduction in leukemic burden after single-agent cytar- within the imaged liver space after several milligrams of abine, and, as noted in an initial phase I study, treatment antibody, as previously observed (Fig. 3F; ref. 15). with 213Bi-lintuzumab alone at similar activities did not produce CRs in patients with large disease volumes. Discussion The response rate in this trial was higher than expected from a single course of standard-dose cytarabine alone In this study, we show that sequential administration (31). Bodey et al. (32) reported a 3% response rate after a of cytarabine and 213Bi-lintuzumab is tolerable and can single course of cytarabine at the dose and schedule used in produce remissions in some patients with AML. Al- this study. Similarly, in a report by Bickers et al. (33), no though a relatively small group of heterogeneous patients responses were seen after one course of cytarabine at doses was included in this trial, it provides proof of principle of 200 mg/m2/d for 5 days. The response rate reported in that targeted α-particle immunotherapy may be effective these early trials may be lower than expected today because at reducing low-volume disease. These results suggest of significant improvements in supportive care over the past that further investigation of radioimmunotherapy with 30 years. There are numerous nonrandomized trials in the α-emitters after cytoreduction and in the postremission literature that report a response rate of 10% to 20% with or adjuvant setting is warranted. low-dose cytarabine in older patients with AML, but, in gen- Except as conditioning for HSCT, radioimmunotherapy eral, multiple cycles of therapy are necessary. The largest with long-range, low-energy β-emitters is useful only in randomized trial of low-dose cytarabine compared with hy- treating bulky, radiosensitive cancers such as lymphoma droxyurea confirmed a response rate of 18% but only after a because DLT usually results from nonspecific irradiation median of three courses. One of 103 patients (1%) achieved of normal tissues. Conversely, therapy with high-energy, CR after the first cycle of therapy (34). short-range α-particles can provide far more potent and The current study shows the effect of disease burden on selective delivery of radiation to individual tumor cells, antibody biodistribution. In the initial phase I study of yielding enhanced antitumor activity with decreased toxic- single-agent 213Bi-lintuzumab, in which similar total anti- ity. Despite these advantages, CRs with 213Bi-lintuzumab body doses were used, the percentage of injected activity alone would require extraordinarily high injected activi- reaching the marrow after multiple doses increased in ties. If we assume tumor burdens of 1012 cells with an av- 38% of patients, whereas activity in the liver and spleen erage CD33 density of 10,000/cell, ∼1016 binding sites are decreased in 75% and 56% of patients, respectively (15). available to lintuzumab. With the specific activities that This suggested that CD33 sites in the liver and spleen can

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act as antigen sinks and that, as they become saturated, sequential therapy with cytarabine and 213Bi-lintuzumab, more drug reaches the marrow with repeated injections. responding patients in the current trial had neutrophil re- Similarly, high numbers of circulating CD33-positive covery after a median of 30 days. Treatment with GO is also blasts or cell-free CD33 can adversely affect biodistribu- associated with significant liver function abnormalities. tion of the drug by rapidly binding antibody and prevent- Grade 3 or 4 hyperbilirubinemia and transaminase eleva- ing it from reaching target sites within the marrow. In tions were reported in 29% and 18% of patients, respective- contrast, after partial cytoreduction in this trial, marrow ly. Sinusoidal obstructive syndrome was seen in 5% of activity remained constant or decreased in all four patients patients (36). Grade 3 or 4 liver function abnormalities, who were studied. γ-Camera imaging revealed cardiac however, did not occur with single-agent 213Bi-lintuzumab blood pooling after the last injection of 213Bi-lintuzumab (15). When given after cytarabine in the current study, only in one patient, suggesting that saturation of all antigen 16% of patients developed significant hyperbilirubinemia, sites was possible in patients with smaller disease burdens. and 3% had grade 3/4 transaminase elevations. Sinusoidal Additionally, parametric rate imaging following the last obstructive syndrome was not observed. The more favor- injection of 213Bi-lintuzumab showed decreased uptake able toxicity profile of 213Bi-lintuzumab suggests that or clearance of drug when compared with the first injec- integration of targeted α-particle immunotherapy into tion in all patients. Taken together, these data indicate treatment strategies with standard chemotherapy may be greater saturation of antigen sites by 213Bi-lintuzumab more feasible than chemotherapy-GO combinations. in target organs after partial cytoreduction than with The ability of 213Bi-lintuzumab to produce remissions 213Bi-lintuzumab alone. in some patients with poor-risk AML in this trial provides The strategy of arming lintuzumab with an α-particle– the rationale for the use of α-particle immunotherapy in emitting radionuclide was originally proposed to increase the setting of small-volume leukemias and cancers, or mi- the modest immunologically mediated antileukemic ef- crometastatic disease. The major obstacles to the wide- fects of the antibody itself. Based on a pilot study in which spread use of radioimmunotherapy with 213Bi, however, 1 of 10 patients with relapsed or refractory AML achieved a are its short half-life and the requirement of an on-site CR lasting over 5 years (12) and a phase II study that con- 225Ac/213Bi generator. Therefore, we developed a second- firmed a 6% response rate (13), the role of lintuzumab in generation construct in which the isotope generator is cytoreduced disease was examined in a randomized phase directly conjugated to a tumor-specific antibody. In this 225 III trial (35). Patients with relapsed or refractory AML re- strategy, Ac (t1/2 = 10 days) can serve as an in vivo gen- ceived mitoxantrone, etoposide, and cytarabine alone or erator of four α-particles at or within a cancer cell. Based on with lintuzumab. Although an improvement in response the activity of 225Ac-containing radioimmunoconjugates in rate attributable to unconjugated antibody therapy did several xenograft models (37), we are currently conducting not reach statistical significance (36% versus 28%; P = aphaseItrialof225Ac-lintuzumab in advanced myeloid 0.28), no difference in adverse events or treatment-related leukemia. Additional studies combining 225Ac-lintuzumab mortality between the two groups was seen. A more recent with cytoreductive chemotherapy are planned. phase I trial was conducted to determine whether higher concentrations of lintuzumab sustained over prolonged Disclosure of Potential Conflicts of Interest periods could result in greater therapeutic efficacy (14). In this study, 7 of 17 patients with AML responded. These M.R. McDevitt, advisory board and commercial research support, results have led to an ongoing randomized phase II study Actinium Pharmaceuticals. J.G. Jurcic, commercial research grant and advisory board, Actinium Pharmaceuticals. D.A. Scheinberg, advisory board of low-dose cytarabine with or without lintuzumab in old- and commercial research support, Actinium Pharmaceuticals. er patients with untreated AML who are unable to tolerate standard induction chemotherapy. Acknowledgments Gemtuzumab ozogamicin (GO) represents an alternative antibody-based treatment to this radioimmunothera- We thank Renier Brentjens, Ellin Berman, Mark Frattini, Nicole peutic approach. GO agent is composed of a humanized Lamanna, Peter Maslak, and Mark Weiss for clinical care; Ronald Finn for anti-CD33 monoclonal antibody conjugated to a deriva- radiolabeling of the immunoconjugate; Michael Curcio, Jing Qiao, Eva Burnazi, Catalina Cabassa, and Yan Ma for laboratory assistance; and tive of the potent antitumor antibiotic calicheamicin. Dragan Cicic (Actinium Pharmaceuticals, Inc.) for supplying 225Ac. When released from the immunoconjugate within the cy- toplasm of a leukemic cell, calicheamicin induces DNA Grant Support damage and subsequent apoptotic cell death. In a series of trials conducted in adults with AML in first relapse, a NIH grants PO1 CA33049 and RO1 55349, Leroy Foundation, Experi- response rate (CR + CRp) of 26% was achieved (36). mental Therapeutics Center, Lauri Stauss Leukemia Foundation, Lymphoma Typically, significant myelosuppression is seen with GO, Foundation, and Actinium Pharmaceuticals, Inc. (Florham Park, NJ). The costs of publication of this article were defrayed in part by the even as a single agent. The median time to neutrophil recov- payment of page charges. This article must therefore be hereby marked ery ≥1,500/μL in responding patients was 48 days (36). In advertisement in accordance with 18 U.S.C. Section 1734 solely to contrast, resolution of grade 4 leukopenia occurred after a indicate this fact. median of 22 days following administration of single-agent Received 02/11/2010; revised 06/18/2010; accepted 06/28/2010; 213 Bi-lintuzumab in an earlier phase I study (15). Following published OnlineFirst 09/21/2010.

OF8 Clin Cancer Res; 16(21) November 1, 2010 Clinical Cancer Research

Cytarabine and α-Particle Immunotherapy for AML

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www.aacrjournals.org Clin Cancer Res; 16(21) November 1, 2010 OF9

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

Todd L. Rosenblat, Michael R. McDevitt, Deborah A. Mulford, et al.

Clin Cancer Res Published OnlineFirst September 21, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-0382

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