Targ Oncol DOI 10.1007/s11523-014-0324-y

REVIEW

The status of radioimmunotherapy in CD20+ non-Hodgkin’s lymphoma Evan D. Read & Peter Eu & Peter J. Little & Terrence J. Piva

Received: 27 January 2014 / Accepted: 19 May 2014 # Springer International Publishing Switzerland 2014

Abstract Rituximab, the CD20-directed antibody, has become a standard component of treatment regimens for patients with B cell non-Hodgkin’s lymphoma (NHL). The use of rituximab has resulted in greatly improved response and survival rates with less toxicity relative to standard chemotherapeutic regimes. However, relapse and recurrence is common, particularly in indolent varieties which remain incurable, requiring alternate therapeutic options. The subsequent coupling of β-emitting isotopes such as 131I and 90Y to anti-CD20 monoclonal antibodies (mAbs), including rituximab, has been steadily growing over the last decade and demonstrates even greater therapeutic efficacy with more durable responses. 177 Lutetium-labelled rituximab offers a number of convenient advantages over 131I and 90Y anti-CD20 mAbs for treatment of NHL, and a number of alpha-emitting isotopes lie at the frontier of consolidation therapy for residual, micrometastatic disease.

Keywords Radioimmunotherapy . Rituximab . NHL . Lymphoma . CD20 . Targeted alpha therapy E. D. Read (*) : P. J. Little Discipline of Medical Radiations, School of Medical Sciences, RMIT University, PO Box 71, Bundoora, VIC 3083, Australia e-mail: [email protected] P. Eu Department of Nuclear Medicine, Peter McCallum Cancer Centre, East Melbourne, VIC 3002, Australia P. J. Little Discipline of Pharmacy, School of Medical Sciences, RMIT University, Bundoora, VIC 3083, Australia T. J. Piva Discipline of Biomedical Science, School of Medical Sciences, RMIT University, Bundoora, VIC 3083, Australia

Introduction Non-Hodgkin’s lymphoma (NHL) is a collective term that describes a heterogeneous group of lymphoid neoplasms of B cell, T cell or natural killer cell origin. These diverse malignancies represent a considerable global health challenge, with NHL being the 8th most common cancer in men and the 11th most common cancer in women [1]. In Western countries, it is even more prevalent compared to less developed nations [1]. In Australia, NHL is the fifth and sixth most common cancer in men and women, respectively [2], with 4,690 new cases and an estimated 1,440 deaths in 2012 [2]. In the USA, NHL is the seventh most common cancer with 69,740 new cases and 19,020 deaths in 2013 [3]. As NHL has a complex range of presentations, clinical courses and responses to therapy, appropriate classification is critical to define the disease and to determine appropriate treatment. NHL is conventionally classified according to genetic markers, immunophenotype, morphology and clinical features, and in 2008, this was further refined by the World Health Organisation to better define ambiguous and heterogeneous varieties of the disease [4, 5]. Neoplasms of B cell origin are the most prevalent lymphoid malignancies with over 30 different entities and subtypes that represent over 85 % of all NHLs [6-8]. Diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) are the two most common NHL entities, accounting for 30–40 and 20 %, respectively [7, 8]. The aetiology of each B cell lymphoma reflects the development stage of the healthy lymphocyte when malignant clonal expansion occurs, so understanding NHL requires knowledge of the differentiation of these cells [5] (Fig. 1). Ly m p h o c y t e s b e g i n a s i m m a t u r e p l u r i p o t e n t haematopoietic stem cells in the bone marrow and differentiate into large and rapidly proliferating antigen-independent lymphoblasts [5]. The lymphoblasts themselves differentiate

Targ Oncol Fig. 1 The stages of B cell maturation. Stem cells (a) differentiate into lymphoblasts (b) which proliferate rapidly in the bone marrow. After differentiating again into immunologically naive lymphocytes (c), they migrate to the lymph nodes and lymphoid tissues. Upon presentation to an antigen, the cells again proliferate (d) and gain antigen specificity. Centrocytes (e) that express membrane immunoglobulin with an affinity for the antigen are positively selected to become either memory B cells (f) or antibody-producing plasma cells (g)

into small, immunologically naive lymphocytes in the bone marrow, and further maturation occurs in the lymph nodes and lymphoid tissue. When lymphocytes are exposed to an antigen, they increase in size and again proliferate rapidly (now known as centroblasts), undergoing somatic hypermutation which results in the cells having antigen specificity [5]. Mature non-dividing lymphocytes form in the germinal centre of the lymph nodes and are known as centrocytes. With the assistance of helper T cells, centrocytes that do not present antibodies with an affinity for the antigen will undergo apoptosis, while those that express affinity for the antigen differentiate into either memory B cells or antibody-producing plasma cells [5]. Aggressive B cell NHL neoplasms are the result of malignant transformation of the proliferative lymphoblasts (or centroblasts), while low-grade (indolent) lymphomas are the result of the malignant transformation of nonantigen-specific centrocytes which do not undergo apoptosis [5]. Aggressive NHLs are typically very sensitive and responsive to chemotherapeutic agents, and as such, a significant number of patients are curable [9-12]. However, this is not the case for indolent NHLs which are fatal and incurable, and relapse and recurrence are typical of the natural course of these cancers [12-14]. The sheer heterogeneity of B cell NHLs makes them a potentially overwhelming and complicated group of cancers to treat. However, over 95 % of these malignancies express the non-glycosylated phosphoprotein CD20 which is exclusively expressed on the surface of immature and mature B cell lymphocytes [15]. This transmembrane protein plays a role in cell cycle initiation and differentiation [15] and also serves as a calcium channel in the B cell plasma membrane [16]. The presence of CD20 on B cells is homogenous with >105

expressed per cell [17]. As such, CD20 is an attractive target for immunotherapy using anti-CD20 monoclonal antibodies to treat a wide range of NHL malignancies [18, 19].

Targeting the CD20 antigen with rituximab Rituximab is a recombinant chimeric IgG monoclonal antibody (mAb) that is directed against the CD20 protein [18]. There is evidence that rituximab engagement with the CD20 antigen induces B cell lysis through antigen-dependent cellular cytotoxic (ADCC) and complement-dependent cytotoxic (CDC) mechanisms [20, 21]. The mechanism of ADCC involves the recruitment of cytotoxic immune cells such as macrophages, neutrophils and natural killer cells, which leads to cell destruction of the rituximab-bound B cell. In terms of CDC, the rituximab-CD20 binding activates the complement protein cascade, which recruits macrophages and neutrophils through chemotaxis and enhances phagocytosis and also induces cell lysis by attacking and rupturing the cell membrane. In addition, the coupling enhances the influx of Ca2+ into the cell and induces apoptosis [16, 20, 22, 23], although the literature is conflicted regarding the nature of the mechanism(s) involved [23-26]. Lymphoid stem cells do not express the CD20 antigen, and as such, the depletion of healthy, mature B cells following treatment is only temporary, and so, haematopoietic stem cells are spared during treatment, and the B cell population is restored within 6 to 9 months [19, 27, 28]. The antibody administration is generally well tolerated, with the most common side effects of fever, chills, rashes and nausea related to administration [27, 28] possibly as a consequence of the rapid lysis of circulating B cells [20].

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Infusion reactions are managed by pre-medicating the patient with paracetamol and antihistamines or slowing the rate of infusion. The severity of reactions usually reduces with subsequent infusions, probably explained by a depleted B cell population following previous treatment [20]. Intravenous administration of exogenous proteins carries the risk of stimulating an undesired immunological response in the host [29]. Early monoclonal antibodies were rodent proteins, and the murine antibody may be recognised by the host immune system, resulting in the formation of human antimouse antibodies (HAMAs) [29]. The formation of HAMA limits the potential of repeat treatments as they result in the rapid clearance of the monoclonal antibodies and reduce therapeutic efficacy. In addition, there is an increased risk of infusion reactions ranging from benign symptoms such as rashes to serious adverse anaphylactic-type reactions and cardiopulmonary events [29]. Subsequent advances in recombinant genetic technology have resulted in the development of chimeric monoclonal antibodies, where the portion of the antibody recognised by the host immune system is humanised and, therefore, less likely to provoke an immune response from human anti-chimeric antibodies (HACAs) [30]. Davis et al. [31] found that HACAs were not detected in any of the 58 patients enrolled into their study of repeat rituximab treatments. The reported rate of HACA development for rituximab is 1 % [32].

Treatment of CD20+ non-Hodgkin’s lymphoma with rituximab Rituximab was the first chimeric antibody approved for clinical use, and thus, there is a wealth of literature surrounding its use in treating NHL [10, 13, 18, 28, 31, 33-36]. Most of the therapies involving rituximab have concentrated on treating low-grade and indolent lymphoid neoplasms, such as FL, and on relapsed or refractory aggressive lymphoid neoplasms, such as DLBCL [11, 13, 28, 34]. Conventional treatment involves a chemotherapeutic regimen combining cyclophosphamide, vincristine, prednisone and doxorubicin, otherwise known as CHOP [28]. It has been shown that this chemotherapeutic approach achieves initial response rates of 50 % for patients with indolent lymphoid neoplasms [8]. However, in these neoplasms, conventional chemotherapeutic treatments have not increased overall survival rates [18, 34, 35], and with subsequent treatments, both the magnitude of the response and the duration of the response decrease [8]. For DLBCL, 50 % reduction in nodal masses [37], and overall responses are the sum of CR and PR. In a followup study, Czuczman et al. [13] showed that the median time to progression was less than 7 years, and 42 % of the patients were still in remission after 9 years. Even though a cure had not been found, the R-CHOP regimen provided a durable response for low-grade NHL. Although the addition of rituximab to conventional chemotherapy has improved patient outcomes, only a proportion of patients with either intermediate or high-grade NHL are cured [13]. Patients with low-grade and indolent NHL are rarely free of disease, and although their median survival is 7 to 8 years, they usually suffer multiple relapses and eventually become refractory to therapy [11, 13]. Similarly, the prognosis is also poor for patients with relapsed intermediate or high-grade NHL [11]. Approximately 50 % of patients are reported to have an innate resistance to rituximab, with another 60 % developing resistance after treatment [19, 21]. In view of these therapeutic shortcomings, new treatment regimens are required. One such strategy is the combination of radioisotopes with anti-CD20 antibodies.

Radioimmunotherapy of CD20+ non-Hodgkin’s lymphoma Malignant B lymphocytes are inherently radiosensitive, and radioimmunotherapy with anti-CD20 antibodies has shown a considerable promise in patients with relapsed and refractory NHL [38]. Yttrium-90 ibritumomab tiuxetan (Zevalin) and 131 I tositumomab (Bexxar) are two commercial murine antiCD20 monoclonal antibodies that have demonstrated superior therapeutic responses compared to NHL patients treated with rituximab alone [11, 39, 41-43]. Controversially, in 2013, Bexxar was discontinued by the manufacture GlaxoSmithKline because of limited and declining use, despite its clinical success in improving patient outcomes [44]. In radioimmunotherapy, anti-CD20 antibodies are coupled to particulate-emitting radioisotopes in order to exploit the targeting properties of the monoclonal antibodies and deliver a radiologic payload specifically to CD20+ NHL cells (Fig. 2). The β emissions from radioisotopes such as 90Y and 131I penetrate several hundreds of cell layers, creating a

Targ Oncol Fig. 2 Monoclonal antibodies can be used to deliver radiologic payloads to specific targets. This illustration is of the chimeric antibody rituximab that is directed against CD20+ lymphoid neoplasms, which has humanised domains to reduce immunogenicity

“crossfire” effect useful for inducing apoptosis in malignant cells not directly bound to the radioimmunoconjugate (Fig. 3). This makes radioimmunotherapy valuable for treating bulky or poorly vascularised tumour masses [45], although surrounding healthy tissue is also irradiated, which will be discussed later. Fig. 3 The “crossfire” effect is useful for the killing of malignant cells that are not directly bound by the antibody

Yttrium-90 is a radiometal that is chelated to the bifunctional ligand tiuxetan, which is covalently linked to the murine anti-CD20 antibody ibritumomab. Witzig et al. [39] undertook a study of 143 patients with relapsed or refractory follicular, transformed or low-grade NHL who were randomised to receive either four weekly infusions of rituximab or a single

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rituximab/90Y ibritumomab tiuxetan treatment (pre-treatment with rituximab prior to the radioimmunotherapy depletes the healthy B cell lymphocyte population to improve the tumour targeting of the radiolabelled anti-CD20 antibody and subsequently lowers the non-specific whole-body absorbed dose). The OR in the 90Y ibritumomab tiuxetan group was 86 % compared to 56 % in the rituximab group, while the CR in the former group was 30 % compared to 16 % in the latter group [39]. Similar results were observed in another study with OR at 83 % and CR at 65 % [46]. In 2000, Kaminski [11] conducted a study in 59 chemotherapy-relapsed/refractory patients and observed a 71 % OR in patients who had received 131 I-labelled tositumomab, compared to the response rate of 52 % to their last chemotherapy treatment. Forty-two patients in this study had low-grade or transformed low-grade NHL, and the results were even more striking. An overall response of 83 % was achieved with 131I tositumomab, compared to a 50 % OR to their last chemotherapy treatment. In these patients, their duration of response increased from 8 months following chemotherapy to 11.7 months following 131I tositumomab. However, HAMA was detected in 10 of the 59 patients (17 %). In a later study, 76 patients with untreated follicular lymphoma received 131I tositumomab as their initial treatment; however, HAMA was detected in 63 % of the patients [40]. Patients who were treated with 131I tositumomab were pre-treated with unlabelled antibody to improve tumour targeting, which could explain the high rates of HAMA and demonstrates the limited scope of using murine anti-CD20 antibodies in front-line therapy. The development of HAMA in patients receiving 90Y ibritumomab tiuxetan has been reported in 1 % of patients who had relapsed or were refractory to chemotherapy [39]. This could be explained by prior heavy chemotherapeutic treatment compromising their immune response [39] and the pre-treatment with rituximab instead of unlabelled ibritumomab, which differed to the approach used in 131I tositumomab trials. Efforts to optimise therapeutic efficacy must be balanced against the potential non-specific cytotoxic effects to bystander healthy tissue caused by the β emissions administered to the patient [47]. Several studies show that the principal toxicity of the 90Y and 131I radioimmunoconjugates is haematological, and thus, the radiosensitive red bone marrow is the doselimiting organ, with the irradiation to this tissue, leading to neutropenia and thrombocytopenia [42, 43, 48-52] and risking myelodysplasia [11]. Dosimetric imaging is required to measure uptake and retention in the tumour mass and healthy organs and allow absorbed dose estimation to predict both tumour response and toxicity. Dosimetry relates a prescribed absorbed dose (measured in gray) to the administered activity (measured in becquerels), which, theoretically, can be achieved by performing pre-therapy imaging using diagnostic activities of the radioimmunoconjugate. This was possible for 131I

tositumomab [11, 52] as 131I emits 364 keV γ emissions. However, the process is less direct for 90Y as it is a pure β emitter with no γ emissions and cannot be imaged. 111Indium has similar chemical properties and a similar half-life to 90Y but has γ emissions suitable for imaging. As such, 111In can be used as a diagnostic surrogate for 90Y and be compounded to ibritumomab using tiuxetan, allowing the biodistribution of the anti-CD20 antigen to be mapped and potentially permit dosimetric estimations. There have been several studies that have substituted 111In for 90Y, aiming to correlate dosimetric and pharmacokinetic factors to tumour response and haematological toxicity [42, 49, 51]. Whole body, liver, spleen, lung, kidney and bone marrow absorbed doses were used to estimate the uptake and residence times, determined at several time points via scintigraphic imaging. However, neither tumour response nor haematological toxicity was predicted by the dosimetric imaging, which may be due to differences in bone uptake of 111In ibritumomab tiuxetan and 90Y ibritumomab tiuxetan [49]. Hence, 111In ibritumomab tiuxetan is only used to confirm tumour avidity for the radioimmunoconjugate [43], and a fixed, weight-adjusted dosing schedule is used for determining administered activity, given the patient is haematologically stable with 98 %, although the technique required a 24-h labelling process using DOTA-NHS-ester, with 18 h required to prepare the DOTArituximab immunoconjugates and 3 h of incubation for radiolabelling. Forrer et al. [62] described a technique that required only 3 h to radiolabel rituximab to 177Lu using p-SCN-Bn-DOTA and achieved a labelling efficiency of 99 % after 15 min of incubation that was >97 % stable at 7 days. Both techniques were successful in creating a cold kit formulation that could be stored and radiolabelled as required. Audicio et al. [63] determined that their DOTA-NHS-rituximab immunoconjugate could be safely stored up to 6 months in kit formation for routine reconstitution. In a phase I/II trial, 31 patients with relapsing follicular, mantle cell or other indolent B cell lymphomas were enrolled with 90 % of patients in stage III or IV [59]. All patients had been heavily pre-treated with a combination chemotherapy, external beam radiation therapy and/or rituximab. The trial was a dose escalation study with the primary objective to determine the maximum tolerated dose of 177Lu-rituximab, which was achieved at 1,665 MBq/m2. The pattern of haematological toxicity was similar to the results observed using 131I and 90Y radioimmunoconjugates. Tumour responses were seen in all patients, and although only 11 patients had survived at the time of results analysis, four patients were in remission with one having been in remission for 8 years. This study established the feasibility of treating B cell lymphomas with 177Lu-rituximab.

Table 1 Summary of β-nuclides used in radioimmunotherapy of CD20+ NHL Nuclide

Half-life (days)

γ (keV)

Abundance (%)

β Energyav (keV)

β Energymax (keV)

β Rangeav (mm)

β Rangemax

177

6.7 8.04 2.7

113, 208 364 –

6.4, 11 82 –

113 187 934

497 610 2,290

0.22 0.36 3.8

2.5 mm 3 mm 12 mm

Lu I 90 Y 131

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Treating residual and micrometastatic disease with targeted alpha therapy

Table 2 Physical properties of candidate α-emitting nuclides for targeted alpha therapy of NHL Nuclide

Despite the improved patient outcomes from radioimmunotherapy using β-emitting nuclides, a cure for indolent NHL remains elusive [65]. Beta emissions are useful for larger tumour volumes due to their range in tissue achieving a crossfire effect into surrounding malignant cells not bound by the antibody [66, 67]. However, the absorbed dose in tumour volumes decreases as the tumour size decreases, and β particles are less effective in isolated and small clusters of malignant cells, particularly in circulating tumours such as leukaemia and NHL, which allows the disease to linger [68, 69]. Alpha particles have a short range of only a few cell diameters and have a high density of ionisations (linear energy transfer (LET)) which is 100 to 1,000 times that for β emissions [70], with a subsequently higher relative biological effectiveness (RBE). For instance, the LET of 90 Y is 0.2 keV/μm, compared to 97 keV/μm for astatine-211 (211At). Nearly all released energies are deposited within the tumour [71], and a single α emission is capable of killing the cell through direct irreparable double-strand DNA breaks rather than free radical-mediated damage [72-74]. These αemitting nuclides have the potential to induce cytotoxicity more selectively and efficiently than can be achieved with β particles [47, 66]. Thus, α emitters are ideal for the consolidation treatment of residual disease following β-emitting radioimmunotherapy, even in tumours that have been resistant to chemotherapeutic agents and radiation [75]. The short range also limits the toxic effects of radiation to bystander non-malignant cells [76]. Several α-emitting nuclides have been investigated as potentially suitable candidates for NHL radioimmunotherapy, including 211 At, bismuth-213 (213Bi) and thorium-227 (227Th) [68, 75, 77-80]. Actinium-225 (225Ac) has not yet been applied to targeted alpha therapy (TAT) of NHL but has shown a promise in the treatment of acute myeloid leukaemia [67]. The physical properties of each α-emitting nuclide are shown in Table 2. A significant concern for each of the α emitters is that each nuclide has a cascade of radioactive daughters, all involving α emissions in decays toward stability, particularly 225Ac [81] and 227Th [82] which each has several unstable progenies. Daughter radionuclides do not generally exhibit the same chemical characteristics as the parent, and coupled with the recoil from energetic α emissions from the nucleus, they dissociate from the carrier ligand and localise elsewhere in vivo, potentially causing toxicity in nontargeted tissues [66, 79, 83, 84]. For some tumour types, the antigen-antibody complex is internalised by the malignant cell, which may effectively trap the daughter products in the cytoplasm which can continue to exert a radiologic effect [67, 85]. However, the CD20

Progeny

211

At 211

Po 207 Bi 225

Ac 221

Fr At 213 Bi 217

213

Bi 213

Po Tl 209 Pb 209

227

Th 223

Ra Rn 215 Po 211 Pb 211 Bi 207 Tl 219

Half-life

α Energy (MeV)

β Energy (keV)

7.2 h 516 ms 32 years 10 days

6 7.5 – 6

– – – –

4.9 min 32.3 ms 45.6 min 45.6 min 4.2 μs 2.2 min 3.25 h 18.7 days 11.43 days 3.96 s 1.78 ms 36.1 min 2.17 min 4.77 min

6 7 6 6 8 – – 6 5.8 6.9 7.5 – 6.7 –

– – 444 444 – 659 198 – – – – 449 – 491

antigen-rituximab complex in NHL does not internalise, and so, the fate of the daughter radionuclides needs to be considered in dosimetric calculations. Astatine-211 There are no stable or long-lived isotopes of astatine, and so, any understanding of its compounding characteristics using conventional analytic techniques is limited [47, 69, 86]. Despite this, 211At has been successfully conjugated to a variety of compounds including monoclonal antibodies, such as rituximab [68, 69, 78, 87, 88]. Astatine is attractive because it has an optimal LET value of 97 keV/μm, which is close to the value where the RBE for ionising radiations is highest. Astatine is a halogen, and synthesis of 211At-labelled molecules exploits its halogen properties [68, 88], but radiolabelling techniques that exploit its metallic characteristics have also been described [89, 90]. Adoption of 211At radiopharmaceuticals is fairly confined due to the fact that few centres have the capability of αbombardment production in a 22–28.5-MeV cyclotron [47, 66, 91], and the short half-life of 7.2 h does not make it amenable to long-distance shipping. In addition, the short half-life renders it unsuitable for radioimmunotherapy using systemically administered antibodies, given the duration of time antibodies need to concentrate at their targets to optimise therapeutic efficiency [92]. Experiences with 211At-rituximab are limited to preclinical studies, and while initial studies demonstrate efficient and stable labelling characteristics and

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excellent cell-killing capabilities [68, 88, 93], it was noted that 211 At radiopharmaceuticals are reportedly more unstable in vivo relative to their radioiodinated counterparts [69]. Given the extreme cytotoxic effects of α emissions, 211At dissociated from the antibody is sequestered in the thyroid and stomach which makes systemic administration of this nuclide problematic [69]. Bismuth-213 Bismuth-213 is conveniently acquired as a daughter product from a 225Ac generator and reaches a secular equilibrium with 225 Ac within 300 min, but 90 % is available at 150 min [72]. This means that therapeutic quantities of 213Bi can be eluted regularly and on demand [66, 72, 94]. Another advantage of the generator system and the long half-life of 225Ac (10 days) means that it can be imported internationally to clinical centres [95]. However, the decay scheme and short half-life (46 mins) of 213Bi presents considerable technical challenges for the application of radiolabelled products. Bismuth-213 decays to 209 Pb via two branches: (a) by β emission to 213Po and subsequently to 209Pb or (b) 209Tl via α emission and subsequently to 209Pb via β emission [66]. The resultant 209Pb then decays to the stable 209Bi via β emission. The in vivo fate of the daughter radionuclides and their impact on toxicity remains to be investigated. Jurcic et al. [67] and Cherel et al. [96] established the feasibility of using 213Bi-labelled antibodies for consolidation therapy of isolated malignant cells and small clusters of residual disease in acute myeloid leukaemia and myeloid myeloma, respectively. Investigations into the treatment of NHL using 213Bi-rituximab are mostly limited to in vitro evaluations of its cytotoxicity on CD20+ NHL cells [75, 77, 80, 97, 98]. Vandenbulcke et al. [77] compared 213Bi-rituximab targeting α exposure to external γ irradiation in CD20+ NHL cell populations and determined that at absorbed doses between 2 and 7 Gy, TAT was more effective at inducing apoptosis. Roscher et al. [75] observed that 213Bi-rituximab reactivated apoptotic pathways in NHL cells that have become resistant to both chemotherapy and indirect radiological effects of β and γ radiation. The short half-life of 213Bi requires rapid labelling techniques to allow a minimal loss of radionuclide during the preparation process. In a method described by McDevitt et al. [99], Ma et al. [100] reduced the preparation time from 25 to 10 min to compound 213Bi with rituximab using CHX-A DTPA. However, the relatively long duration required for radioimmunoconjugates to localise is also compromised by the short half-life of 213Bi [97, 98]. Park et al. [98] proposed a pre-targeting radioimmunotherapy (PRIT) strategy to hasten the localisation time of 213Bi in the treatment of CD20+ NHL. PRIT has previously been described for 90Y radioimmunotherapy of NHL [101] as a strategy to decrease

the amount of time the radioactivity circulates in the blood stream to minimise non-specific irradiation to normal organs. Park et al. [98] adopted this strategy to achieve rapid and specific localisation of 213Bi at the site of the tumour. The multistep technique involved rituximab-streptavidin conjugates to pre-target NHL tumour cells followed by administration of 213Bi-labelled DOTA-biotin. Compared to the control group using 213Bi directly labelled with anti-CD20 antibodies, it was observed that after 90 min, the level of radioactivity in the NHL cells was eightfold higher in the PRIT group relative to the control group.

Thorium-227 Thorium-227 is a therapeutically attractive nuclide because it can be produced in clinically relevant quantities in a generator from β decay of 227Ac, which in turn is produced from thermal neutron irradiation of the abundantly available stable 226 radium [74, 82]. The 227Ac generator system has already been successfully implemented clinically to harvest large-scale quantities of 223Ra [82] for the alpha therapy of metastatic bone disease. Radium-223 is a daughter product of 227Th. Despite the relatively long (18.7 day) half-life of 227Th, with a biological half-life of 8 days relative to the 3.2 day biological half-life of rituximab, the immunoreactivity of 227 Th-rituximab is maintained for a prolonged period of time [79]. The dose rate of 227Th was initially considered possibly too low, allowing time for DNA repair and repopulation of malignant cells [74]. However, 227Th-rituximab is three times more effective at killing CD20+ NHL cells than external Xray radiation per unit dose [74]. Therefore, 227Th may be more suitable for TAT than the shorter lived isotopes 211At and 213Bi which depart most of their energy before the radioimmunoconjugates have a chance to localise on the plasma membranes of these cells. Thorium-227 is conjugated to rituximab in a two-step process using the bifunctional chelate p-SCN-benzylDOTA [79, 82]. Initial efforts to radiolabel 227Th with rituximab achieved low yields ranging between 6 and 17 % [82]. This low yield was mitigated by the abundant quantities of 227Th that can be produced for clinical purposes. The daughter 223Ra dissociates from the immunoconjugate as a result of its differing chemical characteristics to 227Th, and the recoil experienced during α emission, and primarily localises in the bone when administered in vivo [82]. However, while 223Ra mainly irradiates bone surfaces, there is little exposure to the bone, and even with a half-life of 11.4 days, its clinical safety has been established with acceptable myelotoxicity [102]. However, the toxicity of 227Th-rituximab, free 227Th (which also localises in the bone),

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combined with 223Ra and its progeny radionuclides requires further evaluation.

clusters of micrometastatic malignant cells, particularly in residual disease of haematological cancers where conventional therapeutic options are not succeeding.

Actinium-225 Actinium-225 is the parent nuclide of 213Bi, and because of its 10-day physical half-life, it has been proposed as a potential candidate for radiolabelling antibodies for TAT [67, 83, 85, 103]. Actinium-225 has four α emissions during its decay cascade to stable 209 Bi. This, coupled with its long half-life, makes it 1,000-fold more lethal to malignant cells than its 213Bi analogues [103]. However, the possibility of finding a suitable chelating agent to remain bound to the daughter nuclides is low due to the immense recoil energies of α emissions and the differing parent-daughter chemical properties [66]. The α particle cascade is feasible for malignant cells which internalise the antibody-antigen such as acute myeloid leukaemia, because the daughter nuclides have short half-lives and are unlikely to escape the cells to cause non-specific toxicity [103]. Applications in NHL are more problematic as anti-CD20 antibodies are not internalised, and the daughter radionuclides could accumulate in other organs [84, 104, 105]. Bismuth is known to accumulate in the renal proximal tubule cells [104, 105], and francium has also been observed to rapidly accumulate in the kidneys of mice [84]. Jaggi et al. [84] reported morphological and functional changes in mice administered 225Ac-labelled antibodies, although they did suggest several techniques that could attenuate radiation-induced renal damage. These techniques include competitive metal blockade, diuresis and chelation of daughter nuclides to enhance renal excretion. Pharmacological intervention using angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists has also been demonstrated to reduce radiation nephrotoxicity in external beam radiation and might have a beneficial role in TAT with 225 Ac-antibodies [106].

Conclusion Radioimmunotherapy using 90Y- and 131I-radiolabeled antiCD20 mAbs has improved the outlook for patients with NHL, particularly for forms that have become refractory to conventional treatment. Lutetium-177 is a promising therapeutic radionuclide for treating small- and medium-sized tumour masses. The β emission profile is similar to 131I; however, it has a lower abundance of γ emissions to allow dosimetric imaging without the need to isolate the patient during treatment. Despite the challenges with α-emitting isotopes, they exhibit excellent potential in treating isolated and small

Conflict of interest The authors declare that they do have any conflict of interest that may inappropriately influence this work.

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