Radioimmunotherapy for Treatment of Acute Leukemia

Radioimmunotherapy for Treatment of Acute Leukemia Caroline Bodet-Milin, MD,*,† Françoise Kraeber-Bodéré, MD, PhD,*,†,‡ Thomas Eugène,† François Guérard,* Joëlle Gaschet, Clément Bailly,† Marie Mougin,*,† Mickaël Bourgeois,*,† Alain Faivre-Chauvet,*,† Michel Chérel,*,‡ and Patrice Chevallier*,§ Acute leukemias are characterized by accumulation of immature cells (blasts) and reduced production of healthy hematopoietic elements. According to the lineage origin, two major leukemias can be distinguished: acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Although the survival rate for pediatric ALL is close to 90%, half of the young adults with AML or ALL and approximately 90% of older patients with AML or ALL still die of their disease, raising the need for innovative therapeutic approaches. As almost all leukemic blasts express specific surface antigens, targeted immunotherapy appears to be particularly promising. However, published results of immunotherapy alone are generally modest. Radioimmunotherapy (RIT) brings additional therapeutic mechanisms using radiolabeled monoclonal antibodies (mAbs) directed to tumor antigens, thus adding radiobiological cytotoxicity to immunologic cytotoxicity. Because of the high radiosensitivity of tumor cells and the diffuse widespread nature of the disease, making it rapidly accessible to circulating radiolabeled mAbs, acute leukemias represent relevant indications for RIT. With the development of recombinant and humanized mAbs, innovative radionuclides, and more efficient radiolabeling and pretargeting techniques, RIT has significantly improved over the last 10 years. Different approaches of α and β RIT targeting CD22, CD33, CD45, or CD66 antigens have already been evaluated or are currently being developed in the treatment of acute leukemia. This review summarizes the preclinical and clinical studies demonstrating the potential of RIT in treatment of AML and ALL. Semin Nucl Med 46:135-146 C 2016 Elsevier Inc. All rights reserved.

Introduction

R

adioimmunotherapy (RIT) is a targeted molecular therapy whereby irradiation from radionuclides is delivered to tumor cells using monoclonal antibodies (mAbs) directed to tumor antigens.1 Delivering a heterogeneous low-dose-rate

*CRCNA, INSERM U892, Nantes University, France. †Department of Nuclear Medicine, Nantes University Hospital, France. ‡Department of Nuclear Medicine, ICO-René Gauducheau, Saint-Herblain, France. §Hematology Department, CHU, Nantes, France. This work has been supported in part by a grant from the French National Agency for Research titled “Investissements d’Avenir” No. ANR-11-LABX0018-01 and the ArronaxPlus Equipex No. ANR-11-EQPX-0004. Address reprint requests to Françoise Kraeber-Bodéré, MD, PhD, Nuclear Medicine Department, Hôtel Dieu University Hospital, 1 Place Ricordeau, Nantes 44093, France. E-mail: [email protected]

http://dx.doi.org/10.1053/j.semnuclmed.2015.10.007 0001-2998/& 2016 Elsevier Inc. All rights reserved.

irradiation to the targeted tumor, RIT combines therapeutic mechanisms including immunologic and radiobiological cytotoxicity, as well as bystander and abscopal effects.2 RIT has significantly improved over the 10 last years, due to the development of recombinant and humanized mAbs, access to innovative therapeutic radionuclides, synthesis of more stable chelates for radiolabeling, and pretargeting techniques that increase its therapeutic index.3,4 Hematological diseases are relevant indications for RIT because of the high radiosensitivity of these tumor cells, the diffuse widespread nature of the disease, which is rapidly accessible to circulating radiolabeled mAbs, the high expression of specific antigens not expressed in normal tissues, and the availability of several mAbs.3-5 Moreover, the immunization rate is lower than that for patients with solid tumors, thus allowing multiple injections. The availability of stem cell transplantation allows high-dose protocols, thus increasing RIT efficacy.3 135

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normal cells with a significant toxicity. Despite the fact that βRIT seems not relevant in the context of MRD, promising results have been reported using 90Y-RIT in the consolidation setting in responding patients with NHL after induction therapy8; β
emitters such as 131I, 177Lu, or 67Cu with shorterrange energies should be more favorable for microscopic disease, with 131I exhibiting high-energy gamma emissions requiring isolation. In contrast, α particle emitters offer the theoretical possibility of killing residual tumor cells.17-19 Owing to their high linear energy transfer (LET), α particles deliver a high fraction of their energy inside the targeted cells, leading to highly efficient killing, and could therefore be particularly adapted to target isolated tumor cells and MRD. Moreover, α-particle cytotoxicity is considered to be independent of the dose rate and oxygenation.18 For α emitters, 213 Bi is available through a 225Ac-213Bi generator but its short half-life (T1/2) of 45.6 minute makes it difficult to use. 225Ac, with a 10-day half-life, appears easier to transfer into the clinic. However, its decay produces a series of alpha-emitting daughter nucleons released from the chelating agent, and increases irradiation of normal tissues. Despite its complex chemistry and limited availability, 211At (T1/2: 7.2 h) may be a better candidate. Different approaches of α and β RIT targeting CD22, CD33, CD45 or CD66 antigens have been evaluated or are already developed in the treatment of acute leukemia. This review summarizes the preclinical and clinical studies demonstrating the potential of RIT in treatment of AML and ALL.

The benefits of RIT for patients with hematological diseases have been demonstrated by several clinical trials, especially in patients with non–Hodgkin’s B-cell lymphoma (NHL), using anti-CD20 or CD22 radiolabeled mAbs.5-11 However, only one approved product targeting the CD20 antigen is available today in clinical practice: the intact murine immunoglobulin 90 Y-ibritumomab tiuxetan (Zevalin, Spectrum Pharmaceuticals, Henderson, NV) for treatment of patients with follicular lymphoma that is relapsed or refractory using nonablative activities or as consolidation after induction chemotherapy in front-line treatment.6-8 A number of publications have also reported promising efficacy in patients with other aggressive NHLs using high-dose myeloablative protocols, RIT consolidation, and RIT in first-line therapy or delivered using fractionation to increase tumor-absorbed dose without raising hematological toxicity.4,5 Despite this proven efficacy and the durable responses observed with nonablative activities delivered in outpatient procedures, RIT remains underused for patients with NHL, in part because of the development of multiple competing targeted therapies such as ibrutinib and brentuximab.3,11 Preclinical data and pilot clinical studies have also suggested potential efficacy of RIT in other hemopathies, such as multiple myeloma or acute leukemia.12-16 Leukemias are known to be highly sensitive to radiation, and total-body irradiation (TBI) has been used as a transplant conditioning regimen to maximize the antitumor effect before bone marrow transplantation (BMT). In these poor-prognosis diseases, RIT represents another promising therapeutic option adding complementary cytotoxic mechanisms to other therapeutic modalities, especially by using alpha emitters, which are more adapted to target isolated tumor cells and minimal residual disease (MRD). However, the choice of the radionuclide is critical (Table 1).17,18 The path length of penetration of the radioactive emissions should match the size of the targeted tumor. Because of their long range (a few mm), β
particles produce a crossfire effect leading to the destruction of targeted cells and surrounding tumor cells. Indeed, RIT with long-range β emitters can potentially overcome heterogeneous antigen expression, and it offers advantages for treatment of bulky disease. The cross-fire effect can also result in the killing of

Leukemia is a Relevant Indication for RIT Acute leukemias are clonal disorders of hematopoietic stem cells characterized by the accumulation of immature cells (blasts) and the reduced production of healthy hematopoietic elements, resulting in neutropenia, anemia, and thrombocytopenia. Diagnosis is obtained based on the morphologic and immunophenotypic analyses of bone marrow cells. This allows two major entities according to the lineage origin to be distinguished: acute myeloid leukemia (AML) and acute

Table 1 Principal Radionuclides Used for Antibody-Targeted Therapy Radionuclide

T1/2 (Hours)

Emissions

Emax (keV)

Maximum Range in Soft Tissue (mm)

Iodine-131

193 64 162

Copper-67

62

Bismuth-213

0.76

Astatine-211

7.2

Actinium-225

10 days

610 362 2250 498 208 392-577 184 8400 440 5870 and 7450 77-92 6000-8000 198-659 218-444

2.9

Yttrium-90 Lutetium-177

β
γ β
βγ β
γ α γ α X 4α 2 β
γ

11 2.0 1.8 0.1 0.055-0.080 6 Successive disintegrations

RIT for acute leukemia lymphoid leukemia (ALL).20-22 Within the lymphoid category, ALLs are categorized, essentially for therapeutic reasons, as either T- or B-cell lineage specific. The B-ALL pathologies are then further phenotyped as being positive or negative for the Philadelphia chromosome (or its molecular equivalent—the bcr-abl gene). Indeed, new drugs are particularly indicated for B-ALL, such as for example clofarabine, whereas efficacy of nelarabine has been demonstrated for T-ALL. Moreover, antibcr-abl tyrosine kinase inhibitors have become a standard of care in combination with chemotherapy for Philadelphia positive B-ALL.21 AML can occur at all ages but particularly affects patients older than 65 years with an incidence of 2.4 per 100,000. On the contrary, patients with ALL are predominantly children with approximately 60% of them younger than 20 years, and approximately 6000 new cases per year are diagnosed in the United States. The specific causes of these diseases remain hypothetical including exogenous or endogenous exposures, genetic susceptibility, and chance.20,21 However, progress in the understanding of leukemogenesis has paved the way for better classification of patients to distinguish cases at high risk of relapse and those who benefit from allogeneic stem cell transplantation (allo-SCT). Indeed, nowadays, cytogenetics and molecular analyses at diagnosis provide key information regarding predicting factors in AML as well as ALL. Evaluation of these markers during the course of the disease and defining MRD has been integrated (especially in ALL) within the therapeutic strategy.20,23 Induction chemotherapy followed by various consolidations with or without maintenance therapy is currently the standard of care for patients with both AML and ALL as first-line therapy, although allo-SCT also remains indicated as consolidation in most patients (classified as high-risk patients) because it provides lower relapse compared with chemotherapy alone. Currently, half of the young adults with AML or ALL, and approximately 90% of older patients with AML or ALL still die of their disease.20,21 On the contrary, the survival rate for pediatric ALL is close to 90%. The prognosis for relapsed or refractory patients remains very poor, and overall, the main objective in these cases is to obtain cytologic remission as a bridge to transplant, the sole curative treatment at this stage. In summary, novel innovative approaches are needed for patients with acute leukemia, especially adults.20,21 Acute leukemias represent an appropriate model for immunotherapy and therefore RIT. Indeed, almost all leukemic blasts express surface antigens that can be targeted by therapeutic mAbs. These antibodies have been developed specifically for hematological disorders or other malignancies. Targets include the surface antigen CD45, a pan-leukocyte antigen for both AML and ALL, CD33 and CD66 for AML, and CD19, CD20, CD22, CD52, and HER2 for B-ALL.22,24 The success of immunotherapy alone is generally modest due to the high leukemic burden present in patients (mainly relapsed or refractory cases) at the time of testing. This may also be due to indirect and minimal cytotoxic effects of antibodies that include antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, or antibody-dependent cellular phagocytosis (ADCP).

137 One possible way to improve the efficacy of therapeutic antibodies is to combine several of them,24 or to combine them with either chemotherapy25 or a radionuclide (ie, RIT). As examples, anti-CD33, anti-CD45, and anti-CD66 RIT have been tested in the setting of AML, whereas anti-CD22 RIT is the only specific approach developed recently for ALL. RIT could be used in various situations with different objectives: obtaining cytologic complete response (CR) in relapsed or refractory patients, consolidating patients after achieving CR, and/or preventing relapse by maintenance RIT therapy after consolidation, decreasing MRD before allo-SCT, or intensifying the antileukemic activity of the conditioning regimen before allo-SCT. To date, obtaining cytologic CR in relapsed or refractory patients, and intensifying the antileukemic activity of the conditioning regimen before allo-SCT have been tested and are discussed further later.

Anti-CD33 RIT for Treatment of Acute Myeloid Leukemia The CD33 antigen is a 67-kD glycoprotein expressed on most myeloid leukemias and clonogenic leukemia progenitors but not on normal stem cells. Anti-CD33 RIT has been developed using the murine M195 and the HuM195 (lintuzumab) humanized antibodies, both developed by the Scheinberg group at the Memorial Sloan-Kettering Institute. In 1991, Scheinberg et al26 published the first pilot phase I clinical trial results assessing β-RIT using 131I-M195 in 10 patients with AML. The study demonstrated rapid tumor targeting starting within hours after injection, with saturation of CD33 antigens, followed by internalization of 131I-M195 by the blasts. Although no tumor responses were observed the dosimetric estimations suggested that whole bone marrow ablative doses could be expected using 131I-M195. However, it was also estimated that the liver might be the initial nonhematopoietic dose-limiting organ and could receive absorbed doses of up to 4 cGy/mCi, with an activity escalated as high as 600 mCi (22 GBq). A subsequent dose-escalation trial assessed 131 I-M195 in 24 patients with relapsed or refractory AML.27 In all, seven activity levels were evaluated ranging from 50-210 mCi/m2 (1.85-7.7 GBq/m2), with at least three patients at each level and patients being treated with 160 mCi/m2 or greater only if they were eligible for allogeneic or autologous BMT. Post-RIT scintigraphies in 22 patients showed positive uptake of the radiolabeled mAb in the principal areas of leukemic involvement, bone marrow, spleen, and liver. In all, 23 patients (96%) demonstrated decreases in peripheral blood cells with decreased percentage of bone marrow blasts seen in 83% of cases. Leukemia was sufficiently depleted in eight patients to allow BMT, with achievement of a marrow remission in three cases. Significant hepatic toxicity was seen in one patient with pre-existing liver dysfunction but the maximum-tolerated dose (MTD) was not reached. Overall, 37% of assessable patients developed human antimouse antibody responses, limiting retreatment. Importantly, a rapid clearance of reinjected anti-CD33 mAbs was observed in two human antimouse antibody-positive patients, resulting in

138 immediate suppression of serum M195 levels and bone marrow targeting. Overall, this study suggested that leukemic cytoreduction can be achieved using 131I-M195, without nonhematologic toxicity, in patients with relapsed or chemotherapy-refractory AML, and that RIT can be considered as part of a preparative regimen for BMT. 131I-RIT against CD33 was also safely combined with busulfan and cyclophosphamide (BU-CY) as conditioning for BMT in patients with AML.28 Because of their high LET, short range, and high cytotoxic potential, α-RIT appears more suitable for leukemia treatment than β-RIT. 213Bi emits an α particle of 8 MeV and a photon of 440 keV, allowing biodistribution and dosimetry studies. 213Bi produced from an 225Ac-213Bi generator has been conjugated to the murine and the humanized forms of anti-CD33 mAbs, M195 and HuM195, using the bifunctional 2-(4-isothiocyanatobenzyl) diethylenetriamine pentaacetic acid (SCN-CHXA-DTPA) chelate.29 Preclinical studies in mice showed that there was no uptake of 213Bi in tissues not expressing CD33, or in the kidney, which has avidity for free bismuth,30 and intravenous doses up to 370 MBq/kg of 213Bi-lintuzumab were shown to be safe. Leukemia cell death in vitro with 213Bilintuzumab showed dose and specific activity
dependent killing of CD33þ HL60 cells, with approximately 50% cell death observed when two bismuth atoms were bound onto the target cell surface. Despite the short half-life of the α emitter (45.6 minutes), a phase I dose-escalation study assessing 213Bi-lintuzumab was conducted in 18 patients with relapsed and refractory AML or chronic myelomonocytic leukemia treated with 10.3637.0 MBq/kg 213Bi-RIT.16 No significant nonhematologic toxicity was observed. All of the 17 evaluable patients developed myelosuppression, with a median time to recovery of 22 days. Dose-limiting toxicity (DLT), defined as grade 4 leukopenia for more than 35 days from the start of therapy, was seen in one patient treated at the 37 MBq/kg dose level following relapse after allogeneic BMT. As in the β-RIT study, 213 Bi-HuM195 was retained in areas of leukemic involvement (bone marrow, liver, and spleen). The estimated total absorbed dose to the marrow, and therefore to CD33þ target cells, ranged from 6.6-73 Sv, whereas the total dose to the liver, spleen, and blood ranged from 2.4-23.5 Sv, 2.9-36.8 Sv, and 1.1-11 Sv, respectively. Absorbed dose ratios of the bone marrow, liver, and spleen, to the whole body were approximately 1000 times higher for 213Bi-HuM195 than those for the β-emitting immunoconjugates because of the markedly decreased whole-body doses and the greater target organ doses for 213Bi compared with 131I.31 An antileukemic effect was observed: 15 of 18 patients had leukemic blasts in the peripheral blood before treatment with 213Bi-HuM195, and 14 of 15 patients (93%) had reductions in circulating blasts following therapy. Even at the lowest activity level, patients showed elimination of more than 99% of peripheral blasts. Up to three logs of circulating leukemia cells were killed and four patients (27%) had complete eradication of peripheral leukemia cells. Circulating blasts decreased rapidly, with nadirs occurring after a median of 10 days (range: 4-17 days). Of the 18 patients, 14 (78%) had reductions in percentages of bone

C. Bodet-Milin et al. marrow leukemia cells 7-10 days after α-RIT. Among the four patients with complete elimination of peripheral blood blasts, three had reductions in bone marrow blasts. The percentage reduction of marrow blasts and the level of CD33 expression were not related (r ¼ 0.074; P ¼ 0.769), and the reductions in bone marrow blasts occurred more consistently with higher injected activities. However, no complete remissions were achieved in this study. Jurcic et al16 proposed several reasons for the absence of CR: firstly, with approximately 1 in 2700 molecules of HuM195 carrying the radiolabel at the specific activities injected, it remains difficult to deliver one to two 213Bi atoms to every leukemia cell. Moreover, because of the selectivity of α particle irradiation, CD33
leukemic progenitors may escape its cytotoxic effects. Because of the short range and high LET, α-RIT is ideally suited to MRD treatment after partial cytoreduction with chemotherapeutic agents such as cytarabine. Thus, a phase III trial was designed to determine the MTD and antileukemic effects of 213Bi-lintuzumab after partially cytoreductive chemotherapy.32 In all, 31 patients with newly diagnosed (n ¼ 13) or relapsed or refractory (n¼18) AML were treated with cytarabine (200 mg/m2/day) for 5 days followed by 18.546.25 MBq/kg of 213Bi-lintuzumab. Myelosuppression lasting more than 35 days was the DLT, and the MTD of 213Bilintuzumab was determined at 37 MBq/kg. Of the 31 patients, 21 (68%) developed transient elevations of bilirubin, alkaline phosphatase, or transaminases but grade 3-4 liver toxicity was reported in only five patients (16%). Significant reductions in marrow blasts were seen at all dose levels, with a median response duration of 6 months, ranging from 2-12 months. A total of 21 patients had assessable bone marrow evaluations after cytarabine therapy but before 213Bi-lintuzumab treatment, and after recovery following 213Bi-RIT. Following cytarabine, 13 patients (62%) had reductions in marrow blasts with a mean decrease of 10 ⫾ 58%. From the postcytarabine time point to recovery following 213Bi-RIT, 16 patients (76%) had reductions in marrow blasts, including six (29%) who demonstrated progression with cytarabine alone. The mean decrease in blasts during this interval was 41 ⫾ 57%, suggesting an effective antileukemic effect of 213Bi-lintuzumab. Clinical responses were seen in 6 of the 25 patients (24%) who received doses of Z37 MBq/kg. Among the 11 patients with untreated AML who received doses ZMTD, two achieved CR (18%), one achieved CR with incomplete platelet recovery (CRp) (9%), and two achieved partial response (PR) (18%). Only one CRp (14%) was observed in the seven patients with AML in first relapse who had not received prior salvage therapy, and none of the seven patients with primary refractory AML or multiply treated relapsed disease responded, indicating the need for effective reduction in disease burden before administration of α-particle immunotherapy to achieve remission. Biodistribution and pharmacokinetic studies suggested that saturation of available CD33 sites by 213Bilintuzumab was achieved after partial cytoreduction with cytarabine. These studies assessing 213Bi-lintuzumab provide the rationale for the use of α-particle immunotherapy in the setting of small-volume leukemias or MRD. However, the major

RIT for acute leukemia obstacles to the widespread clinical use of 213Bi-lintuzumab are the short half-life of 213Bi and the requirement of an on-site 225 Ac-213Bi generator. To overcome this, second-generation immunoconjugates in which the isotope generator is directly conjugated to the mAb have been developed. 225Ac (half-life ¼ 10 days) can serve as an in vivo generator (atomic nanogenerator) of four α-33 particles. A phase I trial evaluating 225 Ac-lintuzumab was conducted on 18 patients with relapsed or refractory AML. Patients were treated with a single infusion of 0.5-4 μCi/kg (18.5-150 kBq/kg) of 225Ac-lintuzumab. The MTD was determined to be 3 μCi/kg (110 kBq/kg). Serious nonhematologic toxicity was observed in three patients (transient grade 3 liver-function abnormalities), but there was no evidence of radiation-induced nephrotoxicity. Peripheral blasts were eliminated in 10 of 16 evaluable patients (63%), but only at doses of 1 μCi/kg (37 kBq/kg) or more. Bone marrow blast reductions were observed in 10 of 15 evaluable patients (67%) at 4 weeks, and three patients receiving 1, 3, and 4 μCi /kg (37, 110, and 150 kBq/kg) achieved marrow blasts of 5% or less.34 Fractionated injection of 225Ac-lintuzumab in combination with low-dose cytarabine (LDAC) is now under investigation in untreated older patients with AML in a multicenter trial (NCT01756677).35 In this study, patients receive LDAC twice daily for 10 days every 4-6 weeks. During the first cycle, two doses of 225Ac-lintuzumab are given approximately 1 week apart following completion of LDAC. To prevent radiationinduced nephrotoxicity, patients receive furosemide during 225 Ac-RIT and spironolactone for 1 year afterward. This study is still in progress to define the MTD, with planned dose levels up to 2 μCi/kg/fraction (74 kBq kg/fraction). Additional patients would be treated at the MTD in the phase II portion of this trial to determine response rates and survival.

Anti-CD45 RIT for Treatment of Acute Leukemia and Myelodysplasia The group of Fred Hutchinson Cancer Center (Seattle, WA) developed RIT using radiolabeled anti-CD45 mAb. CD45 is a pan-leucocyte antigen tyrosine phosphatase expressed on virtually all leukocytes, including myeloid and lymphoid precursors in bone marrow and mature lymphocytes in lymph nodes.36 CD45 antigen is expressed on the surface of 85%95% of both B-cell lymphoma and leukemic cells at a relatively high level (100-300,000 molecules per cell) and remains stable on the cell surface with minimal internalization after ligand binding.37,38 The same groups have examined the effectiveness of RIT using radiolabeled anti-CD45 mAb. The initial phase I trial was conducted in 34 patients with refractory AML or ALL, or myelodysplastic syndrome (MDS). The study examined the biodistribution of anti-CD45 131I-BC8 mAb and determined the toxicity of escalating doses of targeted radiation combined with 120 mg/kg CY and 12 Gy TBI followed by HLA-matched related allogeneic or autologous BMT.39 In the initial study, the

139 effect of a preclearing treatment of cold mAbs on liver uptake of labeled mAbs was assessed, demonstrating that the precleared dose did not modify estimated absorbed doses to liver or marrow. Therefore, the two groups (with or without precleared dose) were combined in the analysis. In all, 37 patients (84%) had favorable biodistribution, with a higher estimated radiation absorbed dose to marrow and spleen than to normal organs. The reported MTD delivered by 131I-BC8 was 10.5 Gy to the normal organ receiving the highest dose. Although the liver was the normal organ receiving the highest dose, the DLT was mucositis. Based on the average estimates of absorbed radiation doses, this dose level would deliver an average of 24 Gy to bone marrow and 50 Gy to the spleen, combined with CY and 12 Gy of TBI. Of the 25 patients with AML or MDS, seven survived disease free from 15-89 months (median ¼ 65 months) after BMT. Of the nine patients with ALL, three survived disease free for 19, 54, and 66 months after BMT. A phase 1-2 study was also conducted, assessing anti-CD45 131 I-BC8 combined with BU or CY before allogeneic BMT in patients with AML.40 In all, 59 patients with AML in first remission received a biodistribution infusion of 131I-BC8 and 46 patients with a favorable biodistribution received a therapeutic dose of 131I-BC8, estimated to deliver 3.5 Gy (first four patients) to 5.25 Gy (all subsequent patients) to the normal organ receiving the highest dose. Intensified conditioning with 131 I-BC8 mAb resulted in low toxicities compared with that expected from BU or CY alone. Engraftment was not delayed and the most frequent severe toxicity was grade 3 mucositis observed in two patients. Despite the planned delivery of 5.25 Gy to the liver, severe hepatic toxicity did not appear to be higher than those reported with BU or CY alone. These data were compared retrospectively to registry data from the International Bone Marrow Transplant Registry on firstremission patients with AML conditioned with BU or CY alone. After adjusting for differences in age and cytogenetic risk, the hazard of mortality among 131I-BC8 patients was 0.65 times that of the registry patients, suggesting potential efficacy of 131I-BC8 as a conditioning regimen, even if such retrospective nonrandomized comparisons may have many weaknesses. The encouraging results achieved with 131I-BC8 and myeloablative conditioning regimens in younger patients conducted by the Seattle group led them to reflect on how this approach might be applied to older patients with AML or MDS who would not be considered as candidates for high-dose conventional therapies. Therefore, a study was conducted to estimate the MTD of 131I-BC8 that can be combined with a standard reduced-intensity conditioning regimen before allogeneic BMT in 58 patients older than 50 years with advanced AML or high-risk MDS.41 The patients received RIT and fludarabine along with 2-Gy TBI. All patients showed a CR and had 100% donor-derived CD3þ and CD33þ cells in the blood by day 28 after the BMT. The MTD determined in this study was 24 Gy, considerably higher than the 10.5-Gy MTD estimated in the previous myeloablative study combining 131 I-BC8-CY-TBI.42 Based on the dosimetric estimation, the 24-Gy dose level delivered an average of 36 Gy to the bone marrow and 102 Gy to the spleen, combined with FLU and

140 2-Gy TBI. The estimated probability of recurrent malignancy at 1 year was 40% and the 1-year survival rate 41%, obviously quite encouraging results for these older, high-risk patients with AML or MDS. The same protocol was also assessed in 15 patients younger than 50 years with advanced AML or high-risk MDS.43 High activities from 332-1561 mCi (12-57 GBq) of 131I-BC8 were administered, delivering an average of 27 Gy to the bone marrow, 84 Gy to the spleen, and 21 Gy to the liver. Although a maximum dose of 28 Gy was delivered to the liver, no DLT was observed, suggesting that patients may tolerate higher myeloablative doses. Marrow doses were arbitrarily capped at 43 Gy to avoid radiation-induced stromal damage. However, no graft failure or evidence of stromal damage was observed, suggesting that this arbitrary limit of marrow irradiation may be unnecessarily conservative. Anti-CD45 RIT has also been evaluated using other α and β emitters, with the high-energy gamma emission of 131I requiring important radioprotection constraints and patient isolation. Unlike 131I, 177Lu has a lower and safer gamma energy emission facilitating imaging for dosimetry, and 90Y is a pure β emitter. The efficacy and toxicity of β anti-CD45 RIT using 90Y and 177Lu have been compared in a syngeneic murine myeloid leukemia model.44 Whereas 177Lu-RIT yielded no long-term survivors, 90Y-RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300-mCi (11-MBq) 90Y-RIT survived for more than 180 days after therapy, and mice treated with 100-mCi (3.7-MBq) 90YRIT had a median survival of 66 days. Biodistribution studies showed both 90Y- and 177Lu-mAbs localized similarly to target sites with the highest disease burden, bone marrow and spleen. Thus, the lack of efficacy of 177Lu-RIT was not explained by an inability to deliver 177Lu to sites of disease, but may be explained by the differences in physical energy properties between 90Y and 177Lu inducing different doses and dose rates. Indeed, the mean beta-particle energy per decay emitted by 90Y appears to be seven-fold higher than with 177Lu, resulting in higher effective absorbed doses using 90Y, as suggested by dosimetry calculations. Absorbed doses were estimated to be lower in 177Lu-RIT-treated mice compared with 90Y-RITtreated mice when each group received the same activity of 300 mCi (11 MBq). However, the absorbed dose alone may not explain the differential efficacy, RIT effects being different between mice treated with 665 mCi (24.6 MBq) of 177Lu-RIT receiving 212 Gy to the spleen compared with a similar dose of 248 Gy delivered to the spleen when treated with 300 mCi (11 MBq) 90Y-RIT. A correlate explanation for the differences may lie in dose-rate differences, 248 Gy from 300 mCi (11 MBq) of 90Y-DOTA-RIT being delivered at a higher dose rate over a shorter time frame given the shorter half-life of 90 Y, compared with the 212 Gy from 665 mCi (24.6 MBq) 177 Lu-RIT at a lower dose rate over its longer half-life. In summary, these preclinical studies confirm the therapeutic efficacy of 90Y-anti-CD45 RIT for leukemia but do not support the use of 177Lu. Because of the advantages of α emitters to target MRD, anti-CD45 RIT has also been developed using 211At, with its reasonable half-life (t1/2¼7.2 hours) appearing suitable for

C. Bodet-Milin et al. clinical use. Its chemistry is complex, and astatinated mAbs exhibit in vivo dehalogenation when labeling is done via conventional approaches. Therefore, Orozco et al45 from the Seattle group developed a novel approach of 211At-labeling and assessed anti-CD45 α-RIT in a preclinical mouse model of syngeneic-disseminated AML. 211At was linked to the antiCD45 B10 mAb via a lysine amine reaction with B10-NCS. RIT was delivered 2 days after the tumor graft, using escalated activities of 211At-B10 (from 12-24 mCi; 444888 kBq). Some groups of mice underwent a syngeneic BMT 2 days after the RIT, at a time when 99% of 211At had decayed. However, 3 hours after RIT, the α-camera imaging revealed that 211At was distributed throughout the spleen and bone marrow, with some variability depending on suborgan architecture. Studies conducted without BMT demonstrated a survival benefit for the mice treated with 12-mCi (444-kBq) 211At-RIT with a median survival of 69 days compared with a median survival of 36 days for untreated control mice (P o 0.0001). In syngeneic BMT studies, 211At-RIT improved the median survival of leukemic mice in a dose-dependent fashion (123, 101, 61, and 37 days given 24, 20, 12, and 0 mCi, respectively). This approach had minimal toxicity with a white blood cell nadir 2 weeks after BMT and recovery by 4 weeks. Renal and hepatic function in mice treated with 24 mCi (888 kBq) did not deviate from the reference ranges for at least 180 days after BMT. These preclinical data suggest that 211At-anti-CD45 RIT may be a relevant conditioning regimen option for patients with AML before BMT. The same group also developed pretargeted RIT (PRIT) to enhance RIT efficacy by increasing the tumor-to-normal tissue uptake ratio.46 Indeed, the efficacy of RIT using directly labeled mAbs is limited by nonspecific irradiation of normal tissues because of the long blood-circulating halflife of radiolabeled mAbs. Multistep pretargeting approaches dissociate the injection of mAbs showing a low distribution of the molecule, and the injection of a radionuclide coupled with a small molecule showing a rapid distribution.4,47 Several PRIT strategies have been proposed, such as the method of using a radiolabeled bivalent hapten pretargeted by bispecific antibodies developed by Goldenberg et al,47 or the strategy exploiting the high affinity of avidin or strepatavidin (SA) for biotin used by the Seattle group. The second method requires a clearing step between mAb-SA and the radiolabeled biotin administrations. Pagel et al46 evaluated PRIT using an antimurine CD45 mAb-SA immunoconjugate followed by radiolabeled biotin in a murine syngeneic myeloid leukemia system in which the target antigen is present on both leukemic cells as well as on normal myeloid, lymphoid, and reticuloendothelial tissues. A synthetic biotinylated clearing agent (CA) was used to eliminate excess mAb-SA molecules from the circulation before the administration of radiolabeled biotin. The CA increased the tumorto-normal tissue ratio with a marrow-to-blood ratio after 24 hours exceeding 220:1 when using the CA and approximately 90:1 without the CA. However, it is important to note that the pretargeting approach, even without the clearing step, generated superior marrow-to-normal organ ratios of

RIT for acute leukemia radioactivity with the anti-mCD45 mAb-SA immunoconjugate compared with results from conventional radiolabeling. For PRIT experiments, escalated activities from 100-800 μCi (3.7 MBq-29.6 MBq) 90Y-DOTA-biotin were administered 8 hours after anti-mCD45 mAb-SA immunoconjugate. The level at 800 μCi (29.6 MBq) was considered to be too toxic. Whereas all control mice required euthanasia by day 35 owing to progressive myeloid leukemia, mice treated with 100-200 μCi (3.7-7.4 MBq) of 90Y-DOTA-biotin exhibited prolonged survival but eventually died of leukemic progression. In comparison, four of five mice treated with 300 μCi (11.1 MBq) 90Y-DOTA-biotin survived leukemia free for the duration of the experiment (4100 days). These data suggest that anti-CD45 PRIT using an anti-CD45-SA immunoconjugate may be more effective and less toxic than directly labeled mAbs. The potential therapeutic advantage of anti-CD45 PRIT was evaluated also in nonhuman primates (Macaca fascicularis).48 In this study, radio-DOTA-biotin was administered 48 hours after the injection of the anti-CD45 tetrameric construct BC8 (scFv)(4)SA, resulting in markedly lower uptake in normal tissues compared with conventional RIT (target-to-normal organ ratios in the blood, lung, and liver of 10.3:1, 18.9:1, and 9.9:1 using PRIT, respectively, and 2.6:1, 6.4:1, and 2.9:1 using RIT, respectively). Moreover, the rapid blood clearance observed in primates using the tetrameric construct obviates the need for the clearing step. The rapid distribution of the PRIT reagents appears also relevant for labeling using very short half-life α-emitters such as 213Bi. Thus, to assess the potential of α- vs β-emitting CD45 PRIT for leukemia, Pagel et al49 compared biodistribution and therapeutic experiments in human leukemia xenografts implanted in athymic mice of PRIT using antiCD45 BC8 mAb-SA immunoconjugate and 90Y- or 213BiDOTA-biotin and a clearing step. Mice received 300 μg of BC8 and 50 μg of CA 21 hours later, 2 days after tumor injection. Groups of mice received 1 μg of DOTA-biotin labeled with increasing activities of either 90Y (112, 800, and 1200 μCi; 4.1, 29.6, and 44.4 MBq) or 213Bi (400 and 800 μCi; 14.8 and 29.6 MBq) 3 hours after the clearing step. An impressive localization of 213Bi-DOTA-biotin to tumors was obtained with minimal uptake into normal organs, with a uniform tumor radionuclide distribution detected by α-camera 45 minutes after 213Bi-biotin injection. Tissue dosimetry values for each radionuclide were normalized to an equivalent liver dose for comparative purposes and an equivalent liver dose was estimated for mice receiving 800 μCi (29.6 MBq) of 213Bi-biotin and 112 μCi (4.14 MBq) 90 Y-biotin. These respective activities were therefore used for anti-CD45 PRIT in the model of minimal residual leukemia, showing no benefit in terms of survival using 90Y (17 days survival) as compared to the control group (14 days survival). Mice treated with 213Bi had significant improvement of survival (P o 0.0001), with seven mice achieving long-term disease-free survival (DFS) of at least 120 days with minimal toxicity. These data suggest that anti-CD45 PRIT using a α-emitting radionuclide may be highly effective and minimally toxic for treatment of AML.

141

Anti-CD66 RIT as Treatment of Acute Myeloid Leukemia CD66, also known as the carcinoembryonic antigen, is a 180kDa glycoprotein expressed on granulocytes and epithelial cells. Efficacy of anti-CD66 RIT is indirect as selective irradiation of leukemic cells can occur from contact with normal granulocytes, which express CD66 on the cell surface and have bound the radioimmunoconjugate (“cross-fire effect”). Anti-CD66 RIT has been mainly tested in AML models as part of both myeloablative or reduced-intensity conditioning regimens before allo-SCT, using the murine BW250/183 antiCD66 antibody. It has been radioimmunoconjugated with 188 Rhenium or 90Yttrium, two beta-emitters with almost similar mean range of emission (2.4 and 2.7 mm).50 Only one study reported the conjugation with 99Tc.51 Overall, antiCD66 RIT combined with conventional or reduced-intensity conditioning regimens is safe, providing expected engraftment or graft vs host disease rates and tolerable toxicities. However, 90 Y-labeled anti-CD66 RIT seems to provide higher bone marrow radiation dose delivery 52 and may exhibit higher efficacy than that by 188Rh-anti-CD66 RIT. Schulz et al published the results of a single-center pediatric study in 2011 in which 90Y-labeled anti-CD66 RIT was combined with reduced-intensity or myeloablative conditioning regimens depending on the presence or absence of comorbidities in 30 patients with malignant (including leukemia) or nonmalignant diseases before allo-SCT. The most interesting result was the low nondisease-related mortality (NRM) rate of 13%, suggesting a minimal additional toxicity of RIT as part of the conditioning regimen. Survival rates were also interesting; malignant patients achieved 2-year overall survival (OS) and DFS of 69% and 46%, respectively. The survival for nonmalignant patients was also impressive with similar 2-year OS and DFS of 94%.53 The use of 188Re-labeled anti-CD66 RIT before allo-SCT seems to provide higher NRM and higher risk of nephropathy after transplant. Indeed, in the study by Lauter and colleagues, using a reduced-intensity conditioning regimen, NRM was 23% at 2 years. The NRM was also superior to 20% (28.6%) in the study by Koenecke et al54 in which 21 patients with high-risk AML or MDS received either myeloablative or reduced-intensity conditioning regimen after the RIT. Moreover, Ulm’s team has already reported cases of renal toxicity with the use of 188 Re-anti-CD66 RIT.55-57 For instance, in the latest study published,56 including 114 consecutive patients, nephropathy developed only in the 188Re-anti-CD66 group and not in the 90Y-anti-CD66 group due to significantly lower absorbed renal doses (four vs seven Grays) for the 90 Y-labeled group. The superior efficacy of anti-CD66 RIT as part of the conditioning regimen before allo-SCT remains hypothetic, as no prospective comparative study has been published so far. It may be interesting to compare this strategy in older patients with leukemia, where the feasibility of such approaches has been well documented52,58,59 and higher risk of relapse is the rule. Also, in one of these “older” studies,58 the 2-year OS was

142

Table 2 Clinical Studies of RIT in Acute Leukemias Target Disease

Agent

SCT Treatment Activity Conditioning

CD 33

RR-AML

131

No

–

1.85-7.7 GBq/m2

RR-AML

131

Yes

Cyc/Bu

4.4-8.51 GBq/m2

RR-AML

131

No

–

1.85-2.59 GBq/m2

RR-AML RR-AML or UT-AML 4 60yrs RR-AML

213

– Cyt

10.36-37 MBq/Kg 18.5-46.25 MBq/Kg

CD45

CD 66

I-M195 I-M195

Bi-HuM195 No Bi-HuM195 No

213

No. of Patients

Outcome

References

Not reached Not reached Not reached – 37 MBq/ Kg 110 MBq/ Kg 10.5 Gy to liver

24

Schwartz et al27

19

20 BM blasts reductions 8 secondary SCT 18 CR; 6 relapses; 10 NRM

7

DFS 8 m; OS 28 m

Jurcic et al28

18 31

Jurcic et al35 Rosenblat et al32 10 BM blasts reductions 3 with Jurcic et al34 o 5% remaining 13 Relapses 7 NRM (OS: 65m) Matthewset al39

AcHuM195 131 I-BC8

No

–

18.5-150 KBq/Kg

Yes

AML

131

Yes

Cyc TBI 12 Gy Cy Bu

Target 3.5-12.25 Gy to liver Target 5.25 Gy to liver

AML or MD 4 50 years

131

Yes

Flu TBI 2 Gy Target 5.25 Gy to liver

24 Gy to liver

AML or MD o 50 years

131

Yes

Flu TBI 2 Gy o43 Gy to BM

Not 15 reached

AML or MD 55-65 years

90

Flu/Mel

–

AML or MD

Pediatric AML or MD ALL and NB

225

I-BC8 I-BC8

I-BC8

Y-BW250/ Yes 183 188 ReYes BW250/183 90 Y-BW250/ Yes 183

58

12

35 Gy to BM, Gy to kidney, and liver

–

14

20 Gy to BM, Gy to kidney, and liver

–

22

–

21

–

AML

188

–

ALL

46

–

188

ReYes BW250/183 90 Y-hLL2 No

25

8

AML 55-75 y

ReYes BW250/183

35 Gy to BM, Gy to kidney, and liver

18

–

185-740 MBq/m2

Not reached

Jurcic et al28

14 BM blasts reductions 24 BM blasts reductions

3-Year DFS 61% 3-Year NRM 21% 1-Year OS 41% 1-Year DFS 40% 1-Year NRM 22% 1-Year OS 73% 8 Relapses after SCT 2 NRM 4 1 year 1-Year OS 70% 55% Relapses 25% NRM 2-Year OS 69% 2-Year DFS 46% 6 Relapses 2-Year OS 41% 2-Year DFS 41% 2-Year NRM 23% 4-Year DFS 43% 4-Year NRM 28,6% 2CR; 1CRp

Pagel et al40 Pagel et al41

Mawad et al43

Ringhoffer et al52

Schulz et al53

Lauter et al58

Koenecke et al54 Chevallier et al63

BM, bone marrow; CR, complete response; Cyc, cyclophosphamide; Cyt, cytarabine (partially cytoreductive); Flu, fludarabine; MD, myelodysplasia; Mel, melphalan; NB, neuroblastoma; PR, partial response; RR, refractory or relapsed; UT, untreated.

C. Bodet-Milin et al.

CD22

I-M195

DMT

RIT for acute leukemia 40% for the 65-year median age population with advanced myeloid malignancies including AML. This was comparable to the survival of a younger historical cohort receiving the same conditioning without RIT, suggesting an advantage of using RIT as part of the conditioning regimen to reduce relapse after transplant in this older population. In conclusion, like anti-CD33 or anti-CD45 RIT, anti-CD66 RIT may achieve a pronounced antileukemic effect with tolerable toxicities. However, it may not be the optimal radioimmunoconjugate as its mechanism is indirect with no direct fixation on the leukemic blasts. These results may be improved, for example, by patient-specific-based pharmacokinetic models as suggested recently by the study of Kletting et al,60 or by combination with other antibodies targeting other surface leukemic antigens. This strategy (as for anti-CD33 and anti-CD45 RIT) should also be tested not only as part of the conditioning regimen before allo-SCT but also in other indications (Table 2).

Anti-CD22 RIT for Treatment of Acute Lymphoid Leukemia Although some cases of patients with ALL receiving antiCD45,39,42 anti-CD66,51,56 or anti-CD5261 RIT as part of the conditioning regimen before allo-SCT have been reported (among many patients with AML), there was no specific study dedicated to the use of RIT in ALL until recently. Indeed, after publishing preliminary results in the form of a case report,62

143 our group was the first to report, this year, the results of a phase I dose-escalation study of 90Y-radiolabeled-epratuzumab tetraxetan (90Y-DOTA-hLL2) RIT in 17 patients with relapsed or refractory ALL.63 CD22 is highly expressed in B-ALL.22,24 As such, the anti-CD22 humanized antibody, epratuzumab (Immunomedics, Inc., Morris Plains, NJ), is under active investigation in adult and pediatric ALL.64,65 Epratuzumab acts through antibody-dependent cellular cytotoxicity, CD22 phosphorylation, and inhibiting proliferation following cross-linking.66 Epratuzumab has little antileukemic activity in monotherapy but showed improved activity when radiolabeled with 90Y, as demonstrated by two previous studies in the setting of B-lymphoma. Morschhauser et al11 reported the results of anti-CD22 90Y-RIT in 64 patients with relapsed or refractory NHL showing an overall response rate of 62% and a median progression-free survival of 9.5 months. Results were better for low-grade lymphoma, and the dose of 20 mCi/m2 (740 MBq/m2) twice 1 week apart was recommended for future studies. In a phase 2 trial, the French Goelams-Lysa group also tested anti-CD22 90 Y-RIT as consolidation following R-CHOP chemoimmunotherapy in elderly patients with aggressive NHL, showing an estimated 2-year event-free survival of 78.7% and an estimated 2-year OS of 90.1%. RIT toxicity consisted of grade 3-4 hematologic toxicity in 83.6% of patients whereas severe nonhematologic toxicity consisted of grade 4 gastrointestinal tract infection in one patient (1.6 %) and grade 4 infection in three (4.9%).67

Figure An example of biodistribution of radiolabeled anti-CD22 epratuzumab. Whole-body images (illustrate the biodistribution of 111In-epratuzumab at 4 hours (D0), 1 day (D1), 4 days (D5), and 7 days (D7) after infusion. Early images at 4 hours and 1 day demonstrate blood-pool activity in the heart and large blood vessels. Between day 1 and day 4, blood-pool activity faded and liver and spleen uptake decreased, whereas BM activity increased and persisted up to day 7.

C. Bodet-Milin et al.

144 In our phase 1 study, 17 relapsed or refractory CD22þ BALL patients were treated with 4 increasing dose levels. RIT infusion was well tolerated overall. As in AML studies, postinjection imaging revealed significant uptake in areas of leukemic involvement: bone marrow, liver, and spleen (Fig.). One case of DLT (aplasia lasting 8 weeks) was documented at level 4, but the MTD was not reached. The most common grade 3-4 adverse events were pancytopenia (n ¼ 1 at level 2, n ¼ 1 at level 3, and n ¼ 6 at level 4) and infections (n ¼ 3 at level 1, n ¼ 1 at level 2, and n ¼ 5 at level 4). One BCR-ABL molecular CR was documented at level 2 whereas 1 CR and 1 CR with incomplete platelet recovery were observed at level 4 (1 Phþ ALL and 1 Ph- ALL). Overall, two of the three responders received a second RIT cycle with no toxicity. Responses lasted between 7 and 12 months. Because one DLT and two CR were documented at level 4 whereas almost all patients exhibited profound pancytopenia and severe infections, we currently recommend this level of two times 370 MBq/m2 1 week apart/cycle for further phase 2-3 studies. Such a strategy could allow more patients to receive allo-SCT after achieving CR. Thanks to two French grants, we would soon test anti-CD22 90Y-RIT as part of a reduced-intensity conditioning regimen for CR1-CR2 ALL patients, and a phase 2 study comparing RIT vs chemotherapy in relapsed or refractory B-ALL is ready to start. A third trial is in preparation to test RIT to reduce high MRD before transplantation. Finally, this strategy may be enhanced by combining RIT to anti-CD20 immunotherapy in dual CD22-CD20-expressing B-ALL patients, as suggested for patients with lymphoma,7 or by using α-RIT, as α-emitters have been found to be more toxic to isolated target cells, because cell toxicity is achieved with only few disintegrations at the cell surface.17

Conclusion Radiolabeled mAbs demonstrated encouraging results in the treatment of acute leukemias. Antileukemic effects with tolerable toxicities have been reported using anti-CD33, antiCD45, and anti-CD66 RIT for patients with AML and using anti-CD22 for patients with ALL. Promising results have been observed using α or β emitters with various clinical objectives: obtaining cytologic CR in relapsed or refractory disease, decreasing MRD before allo-SCT, or intensifying the conditioning regimen of allo-SCT. α-RIT is more adapted to target isolated tumor cells and MRD, and β-RIT allows treatment of reasonably larger tumor burden. Randomized clinical trials should be performed to identify the benefits and the role of both α and β RIT in the several clinical presentations of acute leukemias better.

Acknowledgments The authors would like to thank David Goldenberg, Immunomedics, Morris Plains, NJ, and William Wegener, Immunomedics, Morris Plains, NJ, for their collaboration over many years in the field of immunotargeting.

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RIT for acute leukemia

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

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