Radiolabeled antibodies directed at CD45 for conditioning prior to allogeneic transplantation in acute myeloid leukemia and myelodysplastic syndrome

Abstract

While allogeneic hematopoietic cell transplantation (HCT) may offer the best chance of cure for patients suffering from aggressive hematological malignancies such as acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome, successful outcomes for the subgroup of patients with high-risk disease remain disappointing and lag behind those of lower-risk patients. Because relatively high rates of relapse are an important contributor to these poor outcomes, efforts have explored approaches to increase the cytotoxic effects of treatment. Relapse rates have been shown to improve with the addition of increased doses of total body irradiation (TBI) and/or the introduction of additional chemotherapy to a HCT conditioning regimen. However, the increase in TBI dose and/or additional chemotherapy has also been associated with a significant increase in life-threatening toxicities, resulting in no change in overall survival. Radioimmunotherapy (RIT) has been employed as an adjunct to HCT where targeted delivery of radiation may allow for further escalation of therapy to reduce relapse with minimal toxicity. In this review we describe these efforts, including the benefits of escalating the dose of radiation to sites of hematologic disease prior to HCT, the various cellular targets for antibody-mediated delivery of radiation, as well as the rationale for incorporation of various radionuclides such as alpha emitters and beta emitters into the preparative regimen prior to HCT. Lastly, newer novel approaches such as pretargeted RIT (PRIT) are described as a method to further increase delivery of targeted radiation to hematological tissues while sparing noninvolved organs.

Keywords

antibodies CD45 hematopoietic cell transplantation radioimmunotherapy

Introduction

Total body irradiation (TBI) has been an integral element of preparative regimens prior to hematopoietic cell transplantation (HCT) because of its therapeutic impact on malignant cells and its immunosuppressive effects that facilitate donor stem-cell engraftment. Despite the widespread application of myeloablative doses of TBI, many individuals treated with high-dose therapy will still relapse, suggesting a suboptimal degree of malignant cell killing. Consequently more intensive preparative regimens with higher doses of TBI and/or chemotherapy have been investigated as a means to decrease rates of relapse. However, these more aggressive treatment approaches are often met with increased regimen-related toxicities and associated mortality, yielding no significant improvement in overall survival (OS). Research efforts have thus focused on targeting escalated doses of therapy to sites of disease to reduce relapse and minimize nonspecific organ toxicity. Monoclonal antibodies (Abs) have been widely used as biologic vehicles to target a cytotoxic effector to cancer cells. A variety of Abs have been developed to target tumor-specific antigens on both solid and hematological malignancies. In the case of myeloid neoplasms, CD33, CD66, and CD45 have been the most extensively investigated antigenic targets for radioimmunotherapy (RIT) as an adjunct to standard transplant preparative regimens for HCT. These Abs have been linked to cytotoxic agents or therapeutic radionuclides that can be targeted toward the malignant cell.

Various radionuclides with differing physical characteristics have been used in RIT, including alpha and beta emitters, as well as reagents that induce cell killing via electron capture or internal conversion. Distinctive emission profiles are associated with each radionuclide that may make one radionuclide over another particularly suitable for a unique clinical scenario (e.g. minimal residual disease versus large tumor burdens). Each radionuclide conveys different energy levels, effective distances of energy dispersion, and biologic half-lives, with significant associated differences in therapeutic efficacy, tolerability, and toxicity (Table 1). In general, beta-emitting agents impart lower average energy levels compared with their alpha counterparts, yet beta emitters such as yttrium-90 (⁹⁰Y) have a much longer mean path length in tissue than alpha emitters such as bismuth-213 (²¹³Bi). The properties of the beta emitters, for example, may be ideal for the treatment of macroscopic disease because of the significant crossfire or bystander effect achieved primarily by the ionizing particles, whereas an alpha emitter may be most appropriate for nonbulky disease, such as leukemia. In each of these scenarios, the energy from the local delivery of ionizing radiation deposited in the nucleus of the cell leads to single- or double-strand DNA breaks, in addition to point mutations, induction of apoptosis, and to cell cycle arrest. The accumulation of DNA damage imparted by RIT thus results in the potential for cell death that is not dependent on properties of cell kill mediated by the antibody itself or by mechanisms of host cellular immunity.

Table 1.

Characteristics of selected radioisotopes.

Isotope	Particle(s)emitted	Half life (t_½)	Path length	Medianenergy (MeV)	Advantages	Disadvantages	Development status
¹³¹I	β, γ	8 days	0.8 mm	0.66 (β)	+γ emission facilitates imaging +Easy conjugation to Abs	−Higher risk to healthcare providers −Easily dehalogenatedin vivo	Clinical trials targeting CD33 and CD45
⁹⁰Y	β	2.7 days	2.7 mm	2.3 (β)	+High-energy β emission	−Without γ emission not readily imaged	Clinical trials targeting CD33, CD66, CD45
¹⁷⁷Lu	β, γ	6.7 days	0.9 mm	0.5 (β)	+Small γ emission amenable to imaging +Low cost	−Lowest energy levels amongst β emitters	Preclinical studies targeting CD45
¹⁸⁸Re	β, γ	17 h	2.4 mm	2.1 (β)	+ β particles at therapy levels, and some γ particles for imaging	−Limited accessibility	Clinical trials targeting CD66
⁶⁷Cu	β	61.5 h	0.4–0.8 mm	0.6	+Small γ emission facilitates imaging, dosimetry	−Limited accessibility, costs	Clinical trials targeting HLA-DR
²¹³Bi	1α,2β	46 min	84 µm	5.84 (α)	+α particle with high potency, short path length	−Limited by extremely short in vivo half life	Preclinical studies targeting CD33, CD45
²¹¹At	1α	7.2 h	60 µm	5.87 (α)	+Short path lengths ideal for MRD	−Short path lengths may not be effective in bulky disease	Preclinical studies targeting CD30, CD45
²²⁷Ac	4α,2β	10 days	50–80 µm	5.75-8.38 (α)	+Short path lengths ideal for MRD +Longer t_½	−Short path lengths may not be effective in bulky disease	Preclinical studies targeting CD33

Abs, antibodies; MRD, minimal residual disease.

In this review we highlight efforts to improve outcomes of HCT for leukemias and myelodysplastic syndromes (MDSs) with adjunctive RIT, focusing on myeloid cell surface targets, the choice of radionuclide employed, and the current limitations of RIT prior to HCT. Newer approaches such as pretargeted (PRIT) to improve upon targeted radiation delivery to target sites are also described.

Radionuclides used in RIT

The radionuclide iodine-131 (¹³¹I) has been used extensively in clinical trials for targeted radiation therapy. One advantage to the use of ¹³¹I is its gamma component that provides gamma imaging that can be used to establish individualized dosimetric doses of radiation delivery. However, treating patients with ¹³¹I requires patients to remain in radiation isolation often for a number of days because of these gamma emissions. Thus, practical and logistical issues with the use of ¹³¹I have led to research and development of other radionuclides for more simplified therapeutic use (Table 1).

⁹⁰Y is a high-energy pure beta-emitting isotope (with a higher safety profile than ¹³¹I) that has more recently been coupled to monoclonal Abs for RIT in hematological malignancies [Jurcic, 2005; Mulford and Jurcic, 2004]. A major impetus to explore agents such as ⁹⁰Y has been that not every transplant center may be equipped with lead-lined rooms to manage the gamma emissions of ¹³¹I. Because ⁹⁰Y emits no gamma rays, however, it cannot be imaged directly for dosimetry estimates. Therefore, indium-111 (¹¹¹In) has been given as a surrogate radionuclide for biodistribution studies before a therapeutic dose of ⁹⁰Y-labeled Abs can be delivered. Given the promise of ⁹⁰Y use, biodistribution studies in macaques with ⁹⁰Y anti-CD45 Abs have been performed in preparation for human clinical trials. These nonhuman-primate studies with ⁹⁰Y anti-CD45 Abs have reliably predicted biodistribution in humans, with selective targeting of hematological tissues comparable to that achieved by ¹³¹I labeled Abs [Nemecek et al. 2005; Matthews et al. 1991].

Subsequently anti-CD45 Abs have been conjugated to ⁹⁰Y in phase I human clinical trials. One study used a ⁹⁰Y conjugated rat IgG2a monoclonal Ab (YAML568) that recognizes CD45. After receiving ⁹⁰Y YAML568 and a myeloablative conditioning regimen with TBI and cyclophosphamide (CY) or busulfan (BU), this study with eight patients showed that therapeutic infusion of the radiolabeled Abs was well tolerated and relatively easy to deliver, although the addition of supplemental unlabeled Abs was required to slow the clearance of radiolabeled Abs in order to achieve optimal biodistribution [Glatting et al. 2006]. In Seattle a phase II RIT clinical trial of ⁹⁰Y anti-CD45 Abs (⁹⁰Y DOTA–BC8) for the treatment of refractory acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and MDS is in the planning stages. It is the hope that the use of a ⁹⁰Y conjugate will offer expansion to other clinical centers that may lack the resources for handling large quantities of gamma-emitting radionuclides such as ¹³¹I.

Although not as heavily explored thus far, another radionuclide under investigation has been lutetium-177 (¹⁷⁷Lu). ¹⁷⁷Lu has energy levels (0.5 MeV) on a par with ¹³¹I (0.6 MeV) and both have a relatively similar physical half-life (t_½ = 6.7 days for ¹⁷⁷Lu compared with 8 days for ¹³¹I). Path lengths of each agent are also comparable, with the path length of ¹⁷⁷Lu being approximately 0.9 mm and the path length of ¹³¹I about 0.8 mm. The biggest difference however is that ¹⁷⁷Lu does not emit high-energy gamma rays that would warrant the strict isolation needed for ¹³¹I. In addition to its potentially therapeutic beta emissions, ¹⁷⁷Lu does emit a small amount of low-energy gamma emission that would make this radionuclide amenable to direct imaging. ¹⁷⁷Lu can also be produced with highly specific activities that are relatively inexpensive, making it an attractive therapeutic agent when costs are considered. Another beta-emitting isotope similar to ¹⁷⁷Lu is rhenium-188 (¹⁸⁸Re). Like ¹⁷⁷Lu, ¹⁸⁸Re emits both beta particles for therapeutic effects and a smaller gamma emission that makes the radioisotope amenable to imaging. However, ¹⁸⁸Re has limited availability limiting its therapeutic application. Consequently, studies to better characterize ⁹⁰Y and ¹⁷⁷Lu are ongoing in preclinical models.

Similarly, two copper isotopes share properties that may make them attractive in targeted RIT. Copper-67 (⁶⁷Cu) emits beta energy (0.6 MeV) comparable to ¹³¹I, which is within range for therapeutic benefit. Copper-64 (⁶⁴Cu) emits positrons in addition to its beta emissions, where the positrons could be used for positron emission tomography to estimate absorbed doses. Unfortunately, quantities of these two radioisotopes are currently limited and furthermore remain costly, which may hinder any significant use of these radionuclides in preclinical and clinical trials.

Because beta emitters have relatively longer path lengths of dispersed energy that can affect more distant nonspecific organs, alpha emitters such as ²¹³Bi, actinium-227 (²²⁷Ac), and astatine-211 (²¹¹At) with shorter path lengths have also been investigated. Experts have postulated that the longer path length of beta radionuclides may be more appropriate for bulky diseases such as lymphoma, while shorter path length alpha agents may be more appropriate when disease is limited to isolated tumor cells and cases of minimal residual disease. Leukemia is a scenario where malignant blasts could be targeted by alpha emitters with minimized cytotoxic effects on distant uninvolved tissues. Alpha radionuclides can deliver high energy over a very short distance (∼50–80 µm) and when combined with their high linear transfer energy characteristics, they have the potential for highly effective tumor cell kill and decreased risk of relapse for patients with leukemia.

Previously, ²¹³Bi labeled anti-CD45 Abs were shown to effectively replace TBI as conditioning for HCT in a canine model that showed sustained marrow engraftment [Bethge et al. 2004; Sandmaier et al. 2002]. Subsequently, to better characterize myelosuppression and toxicities, both ²¹³Bi and ²¹¹At were conjugated to anti-CD45 Abs as TBI replacement in a conditioning regimen using a mouse model [Nakamae et al. 2009]. This study suggests that smaller doses of radiation from ²¹¹At anti-CD45 are capable of myeloablation with less nonspecific toxicity as compared with ²¹³Bi conjugated Abs. Although renal toxicity remains a concern in RIT employing alpha emitters because of the potential radiation effects from renal excretion of high-energy daughter molecules, radiation-induced renal toxicity was not observed with either radionuclide in this study. Nonetheless, further development for clinical applications with these radionuclides remains limited by the short physical half-lives of these radionuclides, ranging from 0.7 hours for ²¹³Bi up to 7.2 hours for ²¹¹At.

The impact of RIT on disease relapse risk after HCT

For the last century, the focus of the therapeutic use of ionizing radiation has been to improve localization of the radiation dose. Reducing radiation-induced injury and increasing efficacy have led to encouraging results. However, in the treatment of hematologic malignancies, curative radiotherapy has had limited applications until the advent of HCT and its concomitant use of radiation as part of cytoreductive preparation. The impact of total radiation dose on reducing relapse rates after HCT was most appreciated in the early 1990 s [Clift et al. 1991, 1990]. These early studies found that in both chronic myeloid leukemia patients transplanted in chronic phase, and AML patients transplanted in first remission, a higher TBI dose of 15.75 Gy resulted in lower post-HCT relapse rates when compared with a lower dose of 12 Gy. Despite these advances, toxicities associated with the dose of radiotherapy continue to be substantial, as these studies show an increased nonrelapse mortality, resulting in no difference in OS appreciated among those treated with either higher or lower doses of TBI. An update of these studies evaluating a minimum of 7.5 years beyond the posttreatment period confirmed substantially higher relapse rates when lower TBI doses were used during HCT conditioning [Clift et al. 1998].

Over the past two decades, researches have been focusing on developing more precise ways to deliver ionizing radiation to uncontrolled proliferative neoplastic blood cells. Monoclonal Abs have been primarily used for this purpose by serving as a biological vehicle that can be conjugated to therapeutic radionuclides and be delivered directly to target sites of disease. In particular, when attached to radioisotopes, Abs can deliver impressive cytotoxic payloads to malignant cells. In Seattle, the radionuclide ¹³¹I conjugated to Abs has been deployed in multiple clinical trials in the treatment of both lymphoma and leukemia for nearly 20 years. Early trials at the Fred Hutchinson Cancer Research Center (FHCRC) reported favorable results with high-dose RIT utilizing radiolabeled anti-CD20 Abs for the treatment of lymphoma patients when used in combination with autologous stem cell rescue. In a pioneering study, anti-CD20 Abs labeled with therapeutic ¹³¹I was infused in 19 patients with relapsed B-cell lymphoma who had achieved favorable biodistribution of the Abs, followed by autologous HCT [Press et al. 1993]. This approach resulted in complete remission (CR) in 16 patients and partial remission (PR) in three patients. Many of these high-risk patients have remained in CR without the need for additional therapy for several years.

Targeting radiation to hematological disease targets

These early and exciting results opened the doors to increased study of RIT treatment for other hematologic malignancies including acute leukemias and MDS. Numerous trials have since attempted to achieve delivery of higher doses of radiation by targeting radiation to leukemic tissues to cytoreduce disease while minimizing radiation effects on nonaffected organs. These approaches have explored delivering radiotherapy to myeloid leukemia sites of disease by targeting CD33, CD66, and CD45 antigens.

Most myeloid leukemic cells but not hematopoietic stem cells express CD33, making it a logical target for early exploration of RIT. Initial studies at the FHCRC using an anti-CD33 Ab (p67) conjugated to ¹³¹I as part of a HCT preparative regimen in AML patients, however, highlighted the limitation of this RIT approach [Appelbaum et al. 1992]. In a small pilot study, less than half of AML patients achieved a favorable biodistribution of radioactivity as gauged by serial quantitative gamma camera imaging. Those patients for whom the biodistribution study estimated a radiation-absorbed dose delivered to the marrow and spleen to be greater than to any normal organ were considered to have favorable biodistribution and were eligible to receive a therapy dose of radiolabeled Abs. In this study, those few patients that achieved favorable biodistribution of radiolabeled Abs went on to receive a therapeutic dose of ¹³¹I anti-CD33 Abs in combination with a standard preparative regimen of high-dose CY and myeloablative doses of TBI. While the small number of patients in this study tolerated the treatment well, the data indicated a short residence time of radiation in the marrow due to internalization of the CD33 antigen and subsequent release of free ¹³¹I from the cell, resulting in a suboptimal supplemental dose of radiation delivered to the marrow. Another possible contribution to modest supplemental radiation dose is that saturation of CD33 binding with radiolabeled Abs happens at a low Ab concentration, likely limiting the total radiation dose that can be delivered to the target cell [Scheinberg et al. 1991].

Investigators at Memorial Sloan Kettering Cancer Center have more recently reported on phase I/II studies using a different anti-CD33 Ab conjugated with a different radioisotope. HuM195 is a humanized anti-CD33 Ab that has been conjugated to an alpha-emitting radionuclide, ²¹³Bi [Rosenblat et al. 2010]. A total of 31 patients with untreated or relapsed AML were treated with cytarabine followed by infusion of ²¹³Bi-HuM195 to determine the maximum tolerated dose (MTD) and therapeutic effects of ²¹³Bi-HuM195. Of the 25 patients who received the MTD or higher, there were two patients with CR, two patients with incomplete platelet recovery, and two patients with PR, for an overall response rate of 24%.

Another target antigen, CD66, has also been investigated as this antigen has been found to be highly expressed on maturing hematopoietic cells. However, CD66 is not expressed on AML blasts; therefore, its therapeutic effect would be largely mediated by the crossfire of radiation given off from neighboring cells targeted by the radiolabeled anti-CD66 Abs (the bystander effect). In phase I/II trials, an anti-CD66 monoclonal Ab was conjugated to ¹⁸⁸Re and used as part of a HCT conditioning [Bunjes et al. 2001]. Bunjes and colleagues investigated ¹⁸⁸Re-labeled anti-CD66 Abs as part of conditioning regimens prior to HCT in 36 patients with high-risk AML. Relapse rates were 20% for those individuals undergoing HCT in remission, compared with 30% for those patients not in remission at the time of HCT. Similarly better disease-free survival (DFS) was seen in patients in remission at time of HCT. DFS at 18 months was 45% overall, with patients undergoing transplantation in remission having a significantly higher DFS compared with those patients who were not in remission at the time of transplant (67% versus 31%).

Anti-CD66 Abs have since been conjugated to ¹⁸⁸Re as well as ⁹⁰Y and used as part of a reduced-intensity HCT regimen for older patients with AML or MDS [Ringhoffer et al. 2005]. Although this approach was well tolerated in this older AML/MDS population, the cumulative incidence of relapse was high (55% at 30 months post-HCT). For patients not in remission at the time of their HCT the risk of relapse was 60%, compared with 42% for those patients in CR1 or CR2. Although the difference was not statistically significant due to small numbers and relatively short follow-up periods, 6 of the 8 patients treated with ¹⁸⁸Re whereas only 3 of the 12 patients treated with ⁹⁰Y ultimately relapsed.

CD45 targeted RIT clinical trials

Our group in Seattle has primarily focused on targeting CD45 for RIT of leukemias and MDS. CD45 is a cell surface antigen expressed on most hematologic tissues, including 85–90% of ALL and AML cells, but CD45 is not found on nonhematopoietic tissues [Andres and Kadin, 1983; Omary et al. 1980]. CD45 is expressed at a relatively high copy number (∼200,000 binding sites per cell) and is not internalized nor significantly shed after binding BC8 Abs on human lymphoma cells [Press et al. 1994], nor when binding a different anti-CD45 Ab (J.33) in B-cell lines [Sieber et al. 2003]. Early studies demonstrated that an ¹³¹I-labeled anti-CD45 Ab could selectively deliver higher radiation doses to lymphohematopoietic tissues in the macaque model [Matthews et al. 1991]. Table 2 highlights the results of key clinical studies targeting CD45.

Table 2.

¹³¹I anti-CD45 radioimmunotherapy as part of conditioning prior to allogeneic HCT in leukemia and MDS.

Disease	Number of patients	Key findings
AML, MDS, ALL [Matthews et al. 1999]	34	• Favorable biodistribution in majority of patients
AML 1st remission [Pagel et al. 2006]	43	• 3-year nonrelapse mortality 21%, DFS 61% • Mortality hazard ratio of 0.65 when compared with similar registry patients transplanted with BU/CY alone
High-risk AML, advanced MDS [Pagel et al. 2009a] • ≥50 years of age	68 in dose escalation study 31 treated at MTD	• CR in all patients • Well tolerated, toxicities mostly from fludarabine, TBI or calcineurin inhibitors • 100% donor derived CD3, CD33 cells by 1-month post-HCT in all patients • 1-year survival 45% for patients treated at MTD
High-risk AML, advanced MDS (ongoing study) • ≥18–50 years of age	14 in dose escalation	• All patients achieved full donor chimerism by day 28 post-HCT
High-risk AML, advanced MDS (ongoing study) • haploidentical donors	8 in dose escalation	• 6/8 treated patients achieved CR by day 28 post-HCT • All patients 100% donor chimerism by day 28 post-HCT

AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia; MTD, maximum tolerated dose; DFS, disease-free survival; BU, busulfan; CY, cyclophosphamide; CR, complete remission; TBI, total-body irradiation; HCT, hematopoietic cell transplantation.

Early phase I studies have utilized an anti-CD45 monoclonal Ab (BC8) coupled to ¹³¹I in combination with a high-dose CY and TBI conditioning regimen for HCT patients with AML, ALL, and MDS [Matthews et al. 1999]. Initial studies were performed in 41 individuals, with 37 (90%) patients showing favorable biodistribution results. In addition to high-dose CY and TBI, 34 patients received therapeutic doses of ¹³¹I anti-CD45 Abs at doses of 76–612 mCi. Of the 25 patients with AML/MDS who were treated, 7 were disease free at least 15–89 months (median 65 months) after HCT. Of the nine patients with ALL, three remained disease free at 19, 54, and 66 months after HCT. This study estimated a median DFS of 58 months in this extremely high-risk patient population. Overall, investigators were able to demonstrate a safe and tolerable method to increase supplemental radiation doses to bone marrow (estimated average of 24 Gy) and spleen (estimated average of 50 Gy) when combined with a conditioning regimen of CY and TBI.

Because higher radiation doses could safely be delivered to marrow and spleen with this approach, ¹³¹I anti-CD45 Ab was combined with BU and CY followed by matched related allogeneic HCT for patients with AML in first remission [Pagel et al. 2006]. Of 59 patients studied initially, 52 (88%) patients achieved a favorable biodistribution of trace ¹³¹I-labeled BC8 Abs, and 46 of these patients received a therapeutic dose of ¹³¹I-BC8 in addition to BU and CY. For the 46 individuals treated in this study the estimated 3-year survival was 63%, and 3-year DFS was 61%. The ¹³¹I-labeled Abs were estimated to deliver an average of 11 Gy to bone marrow and nearly 30 Gy to spleen. This patient group had a relatively low estimated relapse probability of 19%, with outcomes that compared favorably to similar patients who underwent HCT conditioning with BU and CY alone. The comparative group was taken from the Center for International Bone Marrow Transplant Registry (CIBMTR), and were 509 patients with AML in first remission who were conditioned with BU and CY alone. Even though the IBMTR patients were on average younger (mean age in CIBMTR group 28 years versus mean age 39 years in those treated with radiolabeled antibody), the hazard of mortality for those also treated with RIT was 0.65 times that of the hazard in the CIBMRT group treated with BU and CY (95% confidence interval [CI] 0.39–1.08; p = 0.09).

Myeloablative transplant conditioning regimens are difficult to tolerate due to the toxicity and ensuing immunosuppression, which has traditionally limited its applicability to younger individuals with limited or no comorbidities. However, older patients usually have significant comorbidities and may carry a significant burden of disease that has led to disappointing rates of survival using myeloablative HCT regimens [Lowenberg et al. 1998; Bennett et al. 1997; Ruutu et al. 1997]. Because engraftment has been shown to be feasible without complete myeloablation, reduced-intensity regimens have been developed to make HCT an option for older individuals. Reduced-intensity or nonmyeloablative regimens typically use lower doses of chemotherapeutics and TBI, which can more safely be used in older patients. In one study, 274 primarily older AML patients with a median age of 60, 94% of whom were in morphologic remission, received reduced-intensity allogeneic HCT using 2 Gy TBI with or without fludarabine followed by post-HCT immunosuppression [Gyurkocza et al. 2010]. In this study, the estimated 5-year relapse rate was 42% with a good proportion of individuals with some form of graft versus host disease. Despite the tolerability of the reduced-intensity approach, the majority of patients with high-risk disease relapsed, presumably due to suboptimal graft-versus-tumor effects in those with highly aggressive disease.

Consequently, a trial of CD45-targeted radiation delivery in older patients with high-risk AML or advanced MDS was pursued to increase radiation doses delivered to disease sites [Pagel et al. 2009a]. In this study, 58 older patients (median age 63) with advanced AML (beyond first remission) or high-risk MDS (>5% marrow blasts) were enrolled in a phase I clinical trial investigating the well-tolerated reduced-intensity approach of fludarabine and 2 Gy TBI augmented by escalating doses of ¹³¹I anti-CD45 Abs to provide additional antileukemia therapy. These patients were largely ineligible for standard trials using reduced-intensity transplant regimens as 86% had refractory disease or were in florid relapse at the time of treatment. This approach led to a complete response in 100% of these high-risk patients, and all showed evidence of engraftment by 1 month after HCT. Seven of the 58 (12%) patients died before day 100 from nonrelapse etiologies. Dose-limiting toxicities observed (such as grade 3 renal insufficiency, grade 3 cardiac arrhythmias associated with infection, grade ≥3 pulmonary symptoms) at the various investigated doses established the MTD. The MTD was determined to be 24 Gy of radiation delivered to the liver, with an average of 36 Gy and 102 Gy estimated absorbed dose in the marrow and spleen, respectively. At the MTD, median OS and DFS were estimated to be 206 and 189 days, respectively. The estimated probability of relapse at 1-year was 40% with an estimated 1-year survival of 41%, which has been considered encouraging when compared with similar results among lower-risk AML patients transplanted using a regimen of fludarabine and TBI alone.

PRIT to further increase absorbed radiation doses while sparing normal organs

Although the radiation dose delivered to targeted hematological tissues via RIT has been shown to provide additional antitumor therapy to target tissues, many high-risk patients still relapse. Thus, many groups continue to explore a PRIT approach as a mechanism to further boost targeted radiation doses while minimizing nonspecific radiation exposure to normal organs. In one PRIT strategy, the targeting Abs such as anti-CD45 BC8 can be conjugated to streptavidin (SA) in an attempt to uncouple the delivery of the Abs from the delivery of the radiation [Pagel et al. 2009b; Weiden et al. 2000]. The Ab–SA conjugate, delivered as an unlabeled agent, can be allowed to localize to hematological targets over 24–48 hours. Unbound Ab–SA may be then cleared from the circulation by way of Ashwell receptors in the liver using a biotinylated clearing agent prior to a therapeutic dose of radiobiotin. In this model, the biotin moiety can be conjugated to a radiometal via a DOTA chelate. Radiobiotin can subsequently localize rapidly to the anti-CD45 Ab–SA conjugate pretargeted to malignant cells. Unbound radiobiotin can be rapidly eliminated through the kidneys into the urine, further reducing prolonged exposure of free radionuclide in normal organ tissues (Figure 1). It is likely that this strategy of PRIT will be limited to antigenic targets on lymphohematopoietic tissues that are negligibly internalized after binding of an Ab conjugate such as BC8–SA. Variations of this approach have also been explored to further optimize PRIT, including the use of fusion proteins engineered to form four single-chain Ab fragments linked to SA that can then bind radiolabeled DOTA–biotin [Lin et al. 2006]. The development of bispecific antibody fragment fusion proteins are refinements to the PRIT strategy that may improve upon the current PRIT approaches, where one arm of the single-chain is engineered for its specific target (such as CD45) and the other arm to target a therapeutic hapten such as radiobiotin [Sharkey et al. 2010].

Figure 1.

Schema of the pretargeted radioimmunotherapy (PRIT) approach. Antibody–streptavidin conjugate followed by clearing agent before infusion of therapeutic radiolabeled biotin. (This research was originally published in Blood. Pagel, J.M., et al. (2008) Eradication of disseminated leukemia in a syngeneic murine leukemia model using pretargeted anti-CD45 radioimmunotherapy. Blood 111: 2261–2268. © the American Society of Hematology).

Early preclinical efforts using this Ab–SA PRIT system have primarily utilized beta-emitting radionuclides. The selective targeting of radiation to disease tissue has repeatedly been shown to be superior with PRIT as compared with conventional RIT, including in a model of murine acute leukemia xenografts (Figure 2) [Pagel et al. 2009b]. Athymic mice with HEL leukemia xenografts were given conventional RIT (anti-CD45 DOTA–Ab labeled with fluorophore) or PRIT (anti-CD45 Ab–SA followed by a labeled biotinylated fluorophore). In vivo imaging studies showed that conventional RIT had most of the fluorescence in the blood pool at 12 hours while PRIT resulted in the majority of fluorescent activity remaining localized to the tumor. In a disseminated murine leukemia model where PRIT has been investigated, the Ab–SA PRIT approach has been shown to deliver over three times more radiation to murine hematological tissues than a conventional RIT approach with a directly labeled anti-CD45 Ab^.[Pagel et al. 2008]. Moreover, in a murine AML model our group has shown superior target-to-nontarget organ ratios of radioactivity using this PRIT approach as compared with conventional RIT. Using directly labeled ¹³¹I anti-CD45 Abs, marrow-to-normal organ ratios ranged as high as 65:1 for blood. When a PRIT approach was used with anti-CD45 Ab–SA followed by ⁹⁰Y DOTA–biotin, the marrow-to-blood ratio was over 220:1 when a clearing agent was used, and still 90:1 without the use of a clearing agent. In therapeutic studies syngeneic leukemic mice that were treated with pretargeted anti-CD45 Ab–SA conjugate and ⁹⁰Y DOTA–biotin had significantly prolonged survival compared with conventional anti-CD45 RIT. Given these encouraging results seen with PRIT, Good Manufacturing Practice grade reagents have been produced in an attempt to translate these strategies to clinical trials. A clinical trial is underway at our center investigating the safety and feasibility of using ⁹⁰Y DOTA–biotin pretargeted by BC8 Ab–SA conjugate in conjunction with HCT for patients with high-risk AML, ALL, or MDS. While it is too early to have meaningful results, this trial is accruing rapidly, with results of the first application of PRIT in humans greatly anticipated.

Figure 2.

Fluorescent images of HEL xenograft-bearing athymic mice treated with either (A) conventional radioimmunotherapy (RIT) or (B) pretargeted radioimmunotherapy (PRIT). Mice were injected with either anti-CD45 antibodies (Abs) directly labeled with fluorophore (RIT, A) or were pretargeted with anti-CD45 Ab–streptavidin (SA) conjugate followed by clearing agent and then phycoerythrin–biotin (PRIT, B). Images of mice are shown at time 12 hours after injection of fluorophore. Arrows depict fluorophore in tumor (T) and blood pool (B). (C) Tumor-to-total body fluorescence ratios 8 hours after injection in conventional RIT and PRIT mice. (This research was originally published in Cancer Res. Pagel, J.M., et al. (2009b). Pretargeted radioimmunotherapy using anti-CD45 monoclonal antibodies to deliver radiation to murine hematolymphoid tissues and human myeloid leukemia. Cancer Res 69: 185–192. ^©American Association for Cancer Research).

With the success of PRIT, this approach has more recently been investigated using alpha emitters in a murine model of AML [Pagel et al. 2011]. In these studies, BALB/c mice bearing human leukemia xenografts were injected with anti-CD45 Ab–SA, followed by clearing agent and ²¹³Bi DOTA–biotin. Biodistribution studies demonstrated excellent localization of ²¹³Bi DOTA–biotin to tumor with minimal exposure to normal organs. In therapy studies, 80% of mice treated with 800 µCi of ²¹³Bi DOTA–biotin given 24 hours after infusion of anti-CD45 Ab–SA survived leukemia-free for >100 days with minimal toxicity.

Conclusion

Allogeneic HCT is an intensive and potentially curative therapeutic option for individuals with hematological malignancies. Despite gains made with HCT, disease relapse continues to negatively impact outcomes in individuals with aggressive or high-risk disease such as AML and MDS. While higher doses of chemotherapy or radiation have been associated with lower relapse rates, the toxicity of such augmented efforts may blunt the beneficial effects with ultimately no net positive impact on OS. In theory TBI may be conceivably replaced by some form of RIT in HCT conditioning regimens, leading to positive outcomes on transplant OS. Anti-CD45 RIT has shown promise and innovation as a means to increase the dose of cytotoxic radiation targeted to malignant cells, while sparing normal organs, with the goal of improving transplant survival outcomes. Opportunities for further optimization of CD45-targeted RIT exist, including exploiting alternate radioisotopes based on their physical properties and energy characteristics to tailor treatment to the desired goal. Newer strategies such as PRIT hold further promise in that radiation dose targeted hematological tissues may significantly be increased over doses delivered by conventional RIT, while minimizing radiation delivered to nonspecific organs. The continued combinations of innovative preclinical studies and supportive clinical trials suggest CD45-targeted RIT will continue to develop as a critical option for improving HCT outcomes for hematological malignancies.

Footnotes

This work was supported by the NIH (grant numbers K08 CA095448, RO1 CA138720, and PO1 CA44991), a SCOR grant from the Leukemia and Lymphoma Society and the Frederick Kullman Memorial Fund. JMP is a recipient of a Career Development Award from the Lymphoma Research Foundation and a Clinical Scholar of the Damon Runyon Cancer Foundation.

The authors declare no conflicts of interest in preparing this article.

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