Technological Advances in Radioimmunotherapy

Clinical Oncology (2007) 19: 457e469 doi:10.1016/j.clon.2007.03.016

Overview

Technological Advances in Radioimmunotherapy J. L. J. Dearling, R. B. Pedley Cancer Research UK Targeting & Imaging Group, Department of Oncology, University College London (Hampstead Campus), London, UK

ABSTRACT: Radioimmunotherapy (RIT) is a method of selectively delivering radionuclides with toxic emissions to cancer cells, while reducing the dose to normal tissues. Although primary tumours can often be treated successfully with external beam radiotherapy or surgery, metastases often escape detection and treatment, leading to therapy failure, and these can be treated with systemic targeted therapies such as RIT. This review describes more recent developments in the field, including both technological developments from the laboratory and increasingly encouraging findings from clinical studies. Dearling, J. L. J., Pedley R. B. (2007). Clinical Oncology 19, 457e469 ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Key words: Antibody engineering, combination therapies, radioimmunotherapy, radionuclides, targeted radiotherapy

Introduction The use of antibodies for the clinical treatment of cancer is a rapidly growing field. Radioimmunotherapy (RIT) is a branch of molecular medicine in which an antibody is used to deliver a therapeutic radionuclide to a tumour in order to selectively kill cancer cells. An advantage of RIT is that it can target small metastatic lesions that are undetected by conventional scanning, and would otherwise remain untreated. In this overview we discuss the factors underpinning RIT, some recent technological developments that have improved its efficacy, and give examples of its clinical application. Access to the tumour is one of the chief challenges in RIT. Tumours produce a chaotic vascular system in which blood flow is slow, and can be interrupted or even reversed. The ratio of tissue cells to vascular support is lower than most normal tissues. These effects create areas of tumour hypoxia, which are relatively resistant to radiation therapy and therefore reduce the efficacy of RIT. They also pose challenges to the delivery of the systemically delivered radioimmunoconjugate (RIC) to its target antigen. The antigen is a molecule expressed on the cancer cell surface to which the antibody binds and is subsequently retained in the tumour. To achieve selectivity of uptake in the tumour, the antigen must be expressed uniquely, or at least predominantly, on cancer cells. Antigen expression throughout the tumour can be heterogeneous, again contributing to poor distribution of the radionuclide and absorbed dose throughout the tumour, and reducing therapeutic success. A revolution in molecular biology applied to antibody engineering has increased the forms of antibody-based constructs available, allowing investigation of the ways in 0936-6555/07/190457þ13 $35.00/0

which antibody characteristics can be modified to accommodate these problems of delivery and efficacy. We will now consider how research in the field has improved our understanding of RIT, and allowed us to identify desirable RIC characteristics for use in clinical trials.

Pre-clinical Investigations Antibody Engineering The main properties of antibodies that have been manipulated to optimise efficacy are size, affinity and avidity, and immunogenicity. Antibody size In the investigation of optimal features of the RIC, a range of molecules has been constructed, which are depicted in Fig. 1. Antibodies are relatively large molecules with long circulation times and require an extended period to localise to the tumour. Retention in the circulation restricts their usefulness in antibody-based imaging, as well as increasing toxicity of the RIC by extending exposure of the bone marrow to the radionuclide. The revolution in antibody engineering came when it was shown that expression of the VH and VL domains, linked by a chemically or genetically introduced amino acid chain or a disulphide bridge, produced a molecule, termed an scFv (single-chain Fv is conventionally abbreviated to scFv, though is more correctly sFv), which retained antigen binding [1]. scFvs and their derivatives are now the basis of much antibody engineering. Being smaller molecules, scFvs are more suited to imaging, with short circulation times, lower absolute localisation to the tumour

ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

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Fig. 1 e A schematic of antibody-derived molecules discussed in this review. The formats are as follows: (A) whole antibody (domains labelled); (B) scFv-Fc; (C) antibody deleted of its CH2 domain (DCH2); (D) minibody (scFv-CH3); (E) small immunoprotein (SIP); (F) scFv (VHeVL form, linker behind); (G) diabody represented in stylised (left) and associated (right) forms; (H) streptavidin-based tetrameric molecule d a scFv linked to a streptavidin molecule is shown on the left, the four associated molecules on the right. Antigen-binding regions are indicated using white arrows.

and rapid excretion by the kidneys. Reducing the size below the filtration threshold of the kidney (!60 kDa) increases renal excretion of the RIC and therefore toxicity to this organ. Adams et al. [2] showed that in surgically nephrectomised mice, scFvs achieved similar tumour uptakes to whole antibodies. In extended observations, damage to the kidneys after treatment with radiolabelled scFvs was shown [3]. The relationship between size and delivery to the tumour was illustrated by the work of Yokota et al. [4], who compared tumour targeting for a range of molecules. It was reported that the tumour uptakes (%ID/g at 24 h) of the IgG, F(ab0 )2, Fab0 and scFv were 27.2, 19.2, 3.7 and 1.7, respectively. Having developed from monomeric to dimeric formats, tetrameric molecules have been shown to have superior pharmacokinetics compared with whole antibodies. Goel et al. [5], Wittel et al. [6] and Chauhan et al. [7] all reported on the blood kinetics and tumour targeting of tetramers with positive conclusions. Generally, tumour uptake of the tetramer ([sc(Fv)2]2) is similar or improved compared with the whole antibody, but blood clearance is more rapid, resulting in more favourable tumour to normal tissue ratios. Tetramers can also be constructed from two scFvs with an Fc region, which then dimerises, or by attaching an scFv to the end of a molecule, such as streptavidin, which naturally forms tetramers. Domain-deleted molecules. In designing an optimal molecule, compromises have to be resolved. Smaller molecules have a shorter circulation time and better penetration into the tumour, but also achieve lower absolute tumour uptake. Reintroducing domains, complete with their own functionality, can result in improved pharmacokinetics and therapy. Antibody-derived molecules of particular interest are the minibody, the (scFv-CH3)2 (or DCH1) and DCH2 and the small immunoprotein (SIP). Minibody. The minibody is a dimeric molecule comprising the T84.66 (a carcinoembryonic antigen [aCEA]) scFv

linked to the human IgG1 CH3 domain. Hu et al. [8] reported the construction of two forms, one with an extended linker (the ‘flex’ minibody), and showed localisation to tumour of the radioiodinated molecules. Circulation half-lives were slightly different, which probably contributed to the improved tumour uptake of the flex minibody (33%ID/g) compared with the shorter linker (17%ID/g, both at 6 h). In a later study, the 111In-labelled form displayed similar tumour targeting to the radioiodinated form of the minibody, but high uptakes in kidney and liver of either the molecule or its catabolite(s) precluded further study for use as either an imaging agent or a vehicle for RIT [9]. DCH2. The DCH2 molecule has a complete antigenbinding region and dimerises to a molecular weight of around 120 kDa. Although reduced tumour localisation of the radiolabelled molecule is often reported, the greatly increased rate of blood clearance leads to improved tumour to normal tissue ratios. Mueller et al. [10] reported construction of a CH2deleted antibody (ch14.18-DCH2). Rapid clearance from the blood was reported for the radioiodinated form d the DCH2 had similar pharmacokinetics to the F(ab0 )2, but localised to melanoma xenografts more rapidly and with better tumour to normal tissue ratios than the intact antibody. Gillies and Wesolowski [11] reported on the antigen-binding characteristics of this molecule. In competition assays it was found that the DCH2 form competed for antigen better than the whole antibody itself. This was attributed to increased flexibility at the hinge region. Slavin-Chiorini and coworkers [12] found that the a and b blood T1/2 of CC49 monoclonal antibody were 2.4 and 48.9 h, reduced to 1.7 and 7.8 h for its DCH2 derivative, and with tumour uptakes of 13.3 and 1.4%ID/g at 24 h, respectively. Similarly, Chinn et al. [13] found that the [111In]-DTPA-labelled whole antibody (CC49) had biexponential clearance of T1/2 a of 1.5 h and b of 162 h, whereas the DCH2 clearance was reported as monoexponential with a half-life of 5.4 h. Other metal labellings (177Lu and 188Re)

TECHNOLOGICAL ADVANCES IN RADIOIMMUNOTHERAPY

of DCH2 molecules and parental antibodies have shown similar behaviour [14,15]. The importance of the shorter circulation times of the DCH2 was emphasised in a preclinical RIT experiment by Rogers et al. [16]. In an intraperitoneal model of cancer, and using intraperitoneal injection, the therapeutic effect of whole antibody (HuCC49) and its DCH2 derivative labelled with 177Lu using N,N0 ,N00 ,N000 -tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA) was investigated. Mice treated with the DCH2 survived 67.5  7.5 days compared with controls at 32  3.3 days. Mice given the same amount of radioactivity (3  300 mCi) using the whole antibody as the vehicle died within days from toxicity. scFv-Fc. Re-incorporation of the Fc region (by using, for example, the (scFv-Fc)2 molecule) takes advantage of the FcRn antibody recycling system in the kidney, maintaining circulating levels of the RIC. In vitro binding of the FcRn to the antibody Fc region can be decreased by changing one or two key amino acids (as described below) and can also be increased by a factor of 10 through as few as three point mutations [17]. Shu et al. [18] showed that the dimerised, 120 kDa DCH1(DCL) molecule was capable of binding to antigen, and confirmed retention of a functional Fc region capable of eliciting cytolysis. Improved tumour uptake of the DCH1 molecule was shown by Slavin-Chiorini et al. [19], although blood levels were also higher. With the equivalent DCH2, tumour uptake was lower, but blood clearance was much faster, resulting in improved tumour to blood ratios, whether the antibody was labelled with 125I or 177Lu. Greater tumour localisation of the (scFv-Fc)2 molecule compared with the minibody [20] has been reported. Tumour uptake of the 64Culabelled minibody at 18 h was 4.2  0.5%ID/g, whereas (scFvFc)2 achieved 11.8  1.0%ID/g at 21 h. In a study aimed at optimising FcRn binding for improved pharmacokinetics, Kenanova et al. [21] mutated the FcRn binding site of scFvFc molecules. All five mutants had shorter circulation times than the parent scFv-Fc, but a double mutant had the shortest (e.g. at 72 h the tumour to blood ratio for parent scFv-Fc was 0.6 : 1, and for the double mutant was 11.7 : 1). The biodistribution of radioiodinated and metal-labelled scFvFcs was found to be different (e.g. 111In versions had higher liver uptake), possibly because the radioiodinated molecules were dehalogenated [22]. Although the double mutant had lower tumour uptake (peak at 28%ID/g at 12 h post injection (p.i.)) than a single mutant (peak at 44.6%ID/g at 24 h p.i.), high normal tissue localisation of the single mutant RIC led to poor ratios, so the double mutant is currently under investigation as a vehicle for RIT. Small immunoprotein. L19-SIP (molecular weight w80 kDa) was constructed using the CH4 region of Ige-S2 attached to the VH and VL domains of the L19 scFv [23]. It binds to its target, the extracellular domain B of fibronectin, which is associated with angiogenesis, divalently. The biodistribution of the 125I- and 111In-labelled scFv, SIP and whole antibody led to the conclusion that the iodinated SIP was the best vehicle for therapy, followed by the whole antibody [24]. In the subsequent therapy study, the [131I]SIP exhibited superior therapy compared with the iodinated

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whole antibody in terms of tumour growth control and extension of survival. A number of therapy studies in different model systems have since been reported, and the antibody has recently entered clinical trials. In addition to reintroducing the Fc region into the molecule, other methods of extending serum half-life have been reported. For example, albumin is also maintained in the circulation, and its use as a foundation for RICs has been shown [25]. Similarly, a bispecific antibody with one of the arms binding to albumin has been reported, thus creating an albumin-containing molecule in vivo. The bispecific molecule (AB.Fab4D5) also binds to erbB2, and had better tumour uptake, lower kidney localisation and more uniform distribution in the tumour than either Trastuzumab or Fab4D5, from which it was derived [26]. Affinity and avidity The affinity of an antibody is a measure of its binding to the target antigen. This is a function of non-covalent bonds formed between amino acids on the complementarity determining regions (CDR) loops of the VH and VL domains of the antibody and those on the surface of the antigen, in addition to other forces, including hydrophobic interactions. Manipulation of this interaction changes the affinity of the interaction. The avidity of the molecule is a measure of the binding affinity of the antibody, taking into account the valency of the interaction. The avidity is therefore always equal to or greater than the affinity. Although high affinity is a requirement for good tumour localisation [27,28], there seems to be a point at which further increases in affinity do not increase uptake at the target site [29]. For example, reduced tumour uptake and limitations on penetration of IgG into the tumour tissue can result from increasing the antibody affinity [30,31], particularly when large amounts of protein are given. Similar observations have been made in the development of smaller molecules, where the relationship between affinity and tumour localisation of scFvs was investigated [2]. It was found that non-specific scFvs achieved no localisation (as would be expected), scFvs with low affinity had low tumour uptake, and, surprisingly, that scFvs with very high affinity also achieved low tumour localisation. Occupation of antigen proximal to the tumour vasculature has been suggested as the basis of this effect for both whole antibodies and scFvs. If the antibody is retained on the antigen surrounding blood vessels then further antibody leaving the vascular lumen will not be able to bind, so it will return to the circulation, and total antibody uptake will be restricted. This is the binding site barrier effect, first reported by Weinstein’s group [32]. An optimum affinity of the antibody (around 109 M [33]) for its target antigen has been suggested, above which no further advantage is gained and specific localisation might be lost. Two scFvs can be joined by shortening the linking moiety, so that intramolecular association is not favoured and molecules form intermolecular association, creating a diabody. Affinity maturation has been shown to be relatively ineffective compared with using the diabody [34]. As

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affinity of the diabodies increased, tumour uptake decreased. So, for example, the scFv form of the C6G98A antibody had a KD of 361  109 M and tumour uptake at 24 h of 0.19%ID; its diabody form had a KD of 5.6  109 M and a tumour uptake of 7.07  0.89%ID/g. The diabody form of C6ML3-9 had a KD of 0.49  109 M, but tumour uptake at 24 h of 3.18  0.52%ID/g (all experiments used radioiodinated molecules). In a later study, Adams et al. [35] showed that improved tumour retention of diabodies is not due to their increased size, which gives them longer blood circulation times, but to the greater functional affinity of the molecule. Use of the diabody molecule as a vehicle for RIT has been reported [3]. Immunogenicity Foreign proteins, antibodies included, elicit an immunogenic response. In the case of RICs, this results in localisation of the radionuclide to the liver, causing radiotoxicity, and precluding further treatment. Immunogenicity has been addressed by removing portions of the molecule that could be recognised as foreign. This can be achieved using either a gradualist approach, changing solvent-exposed features of the molecule known to be immunogenic (‘veneering’) or by transplanting the CDR loops of the antigen-binding site to a human antibody.

Radionuclide There are three radioactive emissions relevant to RIT: a particles are essentially helium nuclei, with a short range but high toxicity; b particles are electrons with a greater range in the tumour but lower toxicity than a particles; Auger electrons have a very short range and are only toxic when produced in the cell, preferably close to the nucleus (see Table 1). To date, the most widely used nuclides in RIT are b emitters. They lend themselves to RIT partly through practical considerations, such as cost and availability. Their chemistry makes them more amenable to attachment to proteins under physiological conditions. Additionally their physical half-lives allow their distribution from a central production centre and, once injected, are commensurate with the pharmacokinetics of whole antibodies. a particles are of greatest current developmental interest (for further discussion see [36]). Although they are very toxic on a per decay basis compared with b particles, challenges to their routine clinical use remain [37,38]. Radionuclides emitting a particles tend to be from the more exotic end of the periodic table, require significant infrastructure for generation, have short physical half-lives, challenging chelating chemistry, and troublesome daughter nuclides. The a- and b-emitting radionuclides will form the focus of our review. Pre-clinical studies of a-emitting radionuclides have been reported. One of the challenges to the use of a emitters is to develop appropriate chelating strategies and bifunctional chelators to keep them attached to the antibody [39,40]. For example, Zhang et al. [137] reported

Table 1 e Radionuclides used for imaging and therapy in radioimmunotherapy. Radionuclides with imaging emissions are used for tumour identification and dosimetry (pre-scouting) before the use of a ‘matched’ radionuclide for therapy Radionuclides

Emission energy (MeV)

Physical T1/2

Imaging (g) 99m Tc 111 In

0.142 0.173, 0.247

6.01 h 2.805 days

Imaging (bþ) 64 Cu 72 As 86 Y 89 Zr 124 I

1.675 1.17 1.479 0.9 1.53

12.701 h 1.1 days 14.7 h 3.27 days 4.18 days

Therapy (Auger) 111 In 123 I 125 I

0.86 1.234 0.179

2.805 days 13.2 h 60.1 days

Therapy (a) 211 At 212 Bi 213 Bi 225 Ac

5.980 6.051 8.537 5.3e5.8

7.21 h 1.009 h 0.8 h 10 days

Therapy (b) 67 Cu 77 As 90 Y 131 I 177 Lu 186 Re 188 Re

0.58 0.226 2.282 0.606 0.497 0.973 2.118

2.58 days 1.6 days 2.67 days 8.04 days 6.75 days 3.78 days 16.94 h

high stomach uptake of 211At, presumably following loss from the antibody. In this study, the specific antibody showed advantage over the non-specific in terms of tumour growth control. Chelating agents with improved in vivo stability have since been developed [41]. Characterisation of astatinated and iodinated antibodies against erbB2 have confirmed that the RICs retain antigen-binding ability and have similar biodistributions [42]. In other studies using a emitters, 213Bi-labelled scFv produced similar tumour growth control for both the specific and non-specific molecules [43]. Dose from the nuclide in the vascular lumen is relevant in this instance due to the short half-life of the nuclide. Kennel et al. [44] investigated the use of 225Ac coupled to monoclonal antibody 201B in mammary carcinoma colonies. Although high tumour uptake of radionuclide was found (e.g. at 1e4 h O 300%ID/g), actinium was released from the chelator 1,4,7,10,13,16-hex aazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA) and accumulated in the liver. In the therapy study, the administered dose was limited by toxicity. Death through wasting syndrome in mice given 1 mCi or more was reported with a dose-dependent relationship. In a subsequent study [45], the whole antibody (CC49) and domain-deleted (HuDCH2CC49) 125I and 225Ac biodistributions were similar.

TECHNOLOGICAL ADVANCES IN RADIOIMMUNOTHERAPY

Therapy studies were hampered by the chelation of the daughters of 225Ac. The lethal dose was found to be 0.5 mCi, and injected activities of 0.25 and 0.5 mCi of 225Ac had little effect on tumour growth. Daughter nuclides also proved problematic in the study of Su et al. [46] applying a 212Pb/212Bi generator to pre-targeted RIT (PRIT). Although good tumour uptake (O25%ID/g) was achieved, 212Bi was released from the chelate (DOTA) after the 212Pb decay.

Improving Radioimmunotherapy In addition to the refinement of the molecular vehicle and choice of radionuclide, a number of other measures have been explored with the intention of improving RIT, either by increasing residence of the nuclide at the target site or amplifying its cytotoxicity. Methods of RIC administration that maximise tumour toxicity but reduce normal tissue dose, such as dose fractionation or PRIT, have been adopted clinically. One of the drawbacks to rapid elimination of the low molecular weight radionuclide vehicle in PRIT is clearance via the kidney, so consideration of methods for reduction of the kidney dose is relevant, as are techniques of investigating the absorbed dose in normal tissues using pre-scouting. Finally, methods of altering tumour biology, such as increasing antigen expression or adapting the vasculature, and also the response to radiotoxicity by combination with radiosensitising agents, have been explored and are discussed.

Dose Fractionation In this technique, the dose is given in a number of fractions, reducing normal tissue toxicity and allowing a higher cumulative dose to be given. A variety of fractionation regimens have been developed, and studies have reported both against and in its favour [47e51]. Different findings probably relate to different patterns of administration and model systems. For further discussion see [52]. A recent report of a-emitting nuclide-based fractionated RIT (213Bi-MAb against d9-E-cadherin, [53]) showed that two administrations of half the single dose was more effective in a pre-clinical model of diffuse gastric cancer. Although RIC uptake has been reported to be reduced, possibly due to vascular damage decreasing antibody access [54], clinical experience has been largely positive.

Pre-targeted Radioimmunotherapy This technology has been used extensively in the clinic. It unites the high absolute localisation and prolonged blood circulation times of the large, whole antibody molecule with the rapid systemic elimination of the small protein radionuclide delivery system. There are a range of different ways of applying this technique and this example is illustrative (for a review see [55]). The antibody is given attached to a molecule such as avidin. This localises to the tumour and then a secondary antibody can be used to clear the first antibody from the vascular space, or alternatively

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extra-corporeal affinity adsorption could be used. Next, a radionuclide-conjugated molecule, which binds to that attached to the antibody, in this example biotin, is introduced. The radioactivity is retained wherever the first antibody was bound, but due to its small size is cleared relatively quickly from the normal tissues via renal excretion, thus reducing toxicity. Recent developments in this field include the production of new chelating and targeting molecules. Novel trifunctional chelators have been reported that bind to antibody, chelate a metal, and have a biotin moiety for use in extracorporeal affinity adsorption [56,57]. In a technique allied to PRIT, affinity enhancement system, bispecific molecules bind the target antigen with one arm of the antibody and the hapten carrier bearing the radionuclide with the other. A trispecific molecule, with one site for hapten binding and two for CEA binding, has also been reported [58].

Multivalency Multivalency has been reported as an advantage in RIT, retaining the radionuclide for extended periods in the radiosensitive outer regions of the tumour [59]. One way of achieving this is by attaching the scFv to molecules that spontaneously form multimers, such as streptavidin. Sato et al. [60] reported the use of a streptavidin tetramer. Although much of the cell-bound tetramer was internalised (about 60% within 6 h), therapy response was achieved at levels of radioactivity below the maximum tolerated dose (MTD). Cell internalisation can be related to the cell line rather than the targeting molecule [61], so these results need not hinder further development of this technique. The use of a tetrameric targeting moiety (scFv-streptavidin) and the site of injection (intraperitoneal injection into an intraperitoneal model) resulted in good localisation and very low levels of nuclide in the kidney (e.g. 6%ID/g between 24 and 72 h) [62]. As the kidney absorbed dose can be a concern in PRIT, this is a significant observation. Peptides can replace the biotin or hapten radionuclide vehicle [63], and can be labelled with a number of a- or b-emitting nuclides, allowing the combination of nuclide and targeting system most suited to the size or site of disease.

Reducing Kidney Uptake Because of the negative charge of the basement membrane of the glomeruli, positively charged catabolites of the RIC may be retained in the kidney, increasing the toxic absorbed dose to this radiosensitive organ. The administration of basic amino acids such as lysine reduces the accumulation of radionuclides in the kidney [64e66]. However, this does not apply in all cases. Rutherford et al. [67] reported that the use of lysine to decrease kidney uptake of two 67Cu-labelled F(ab0 )2 fragments worked for one but not the other. Given that there was no difference in their isoelectric focusing (IEF), it could be suggested that it is not the overall formal charge of the RIC

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that dictates kidney accumulation. Regions of net positive charge could influence the association of the molecule to the glomerular membrane, leading to tubular resorption and lysosomal breakdown. When high kidney uptake is encountered and metabolites are studied, it is often the positively charged, lysine-conjugated form of the radiolabelled bifunctional chelator (a chemical designed to form a covalent bond with the antibody and chelate a radiometal) that is identified. One method of addressing this is to place a triglycine linker between the bifunctional chelator and the antibody, so that the terminal catabolite will instead be negatively charged and kidney uptake reduced. This has been shown to be effective, and in some cases slowed blood clearance and increased tumour uptake [68]. One of the areas of PRIT that would gain from improvement is the clearance of non-tumour localised radionuclideeDOTAebiotin through the kidney. This is not decreased by lysine administration, although succinylation of the scFv-CC49-SA fusion protein has been shown because the molecule was rendered anionic (pI about 4.4) [69].

Pre-scouting In this technique, the targeting system of choice is labelled with a nuclide with emissions suited to imaging, or a small amount of nuclide is given to the patient to confirm localisation and provide dosimetry data for patient-specific treatment. Nuclides with similar chemistry, allowing more direct comparison, are referred to as ‘matched pairs’, examples being 99mTc and isotopes of rhenium. Some elements have different nuclides with both types of emission, such as the positron emitter 124I and therapeutic 131 I, or 62Cu or 64Cu and 67Cu. Some radiometals, such as 111 In and 90Y, have also formed partnerships. The imageable emission of 111In can be compared with the X-rays produced by Compton scatter of 90Y b emissions. However, a note of caution has been sounded by the work of Lovqvist et al. [70]. In a direct comparison of 86Y (positron emitter) and 111 In it was found that the initial biodistribution (by imaging) was similar, there was significant variance at later time points, relevant to antibody pharmacokinetics, and that 90Y uptake was higher in the tumour and also bone tissue, with obvious implications for dosimetry. The production of 89Zr and chelates for use in immunodetection has also been developed [71], and clinical application in the detection of metastases has recently been shown [72].

Combination with Other Agents Increasing antigen expression levels increases tumour uptake and the retention of RICs. The administration of interferon resulting in a two-fold increase in RIC uptake has been shown [73,74], as has the increased homogeneity of tumour expression of the antigen TAG-72, again leading to raised localisation of radionuclide, after interferon treatment [75]. This is now approaching routine in the use of CC49 and derived molecules, as we shall see in the clinical section. Penetratin, a peptide, improves the retention of radionuclide in the tumour, although the mechanism is yet

to be conclusively established [76]. TAT also increased tumour nuclide retention, but a combination of both peptides increased lung uptake. Autoradiographic studies indicated that radionuclide was more homogeneously distributed in penetratin-treated tumours. Increasing vascular permeability by combining RIT with external beam radiotherapy (EBRT) has also been shown to improve localisation [77]. DMXAA and combretastatin improve the therapy response by disrupting the vasculature, starving some cells in the centre of the tumour and increasing radionuclide uptake [78e80], in addition to their inherent cytotoxicity. Tolerance to a wide range of chemotherapeutics in combination with RIT has been reported [81]. Gemcitabine in combination with PRIT led to a radiopotentiating effect and a significant advantage in pre-clinical studies [82]. Mechanistic investigations into the combination of paclitaxel with RIT concluded that much of the effects were due to cell cycle arrest at the radiosensitive G2-M point [83,84], in addition to some anti-angiogenic effects detected in vivo [84]. Clinical studies were limited to low dosage by toxicity (60 mg/kg compared with 120e140 mg/kg for use as a single agent) [85]. RIT ([177Lu]-CC49 given with interferon) given with paclitaxel (up to 100 mg/m2) produced 4/17 measurable responses (partial response [PR]), and 4/27 with nonmeasurable disease had progression-free survival of 18e37 months [86].

Pre-clinical Summary In this section, the basic components of RIT and some recent developments have been discussed. Its successful clinical application depends on the implementation of measures learned from pre-clinical studies. Success also depends on the appropriate use of RIT in responsive diseases and with a suitable end point. Given that an inverse relationship exists between tumour size and RIC uptake, best practice is to use RIT against micrometastatic deposits [87], which are undetectable using current medical imaging technology. This makes the assessment of successful RIT difficult, but it has been observed that RIT-treated patients tend to have longer progression-free survival, suggesting that RIT is achieving a degree of success [88]. Clinical application is developing from single treatment, intravenous injection with standardised radionuclides, to multi-treatment and locally delivered RIT with improved combinations of vehicle and radionuclide, resulting in improved, encouraging responses.

Clinical Application Having described the basis of RIT, and the advances that have been made at the pre-clinical level, we will now report on a selection of the clinical experience to date. Although an effort has been made to make this overview as topical as possible, we also intend for it to serve as an illustration of the range of techniques and approaches that have been used, and of the increasingly encouraging results that have been obtained.

TECHNOLOGICAL ADVANCES IN RADIOIMMUNOTHERAPY

Blood-borne cancers are an ideal target for RIT, being inherently radiosensitive, disseminated and highly accessible. Solid tumours are proving more of a challenge, but positive experiences are starting to be reported. As has been previously stated, a lot of ‘negative territory’ [89] has been defined. Reasons for poor results in the past include: phase I trials involving patients with advanced cancers that have proved refractory to other treatments; variations and difficulty in response assessment; variations in the patient population, nuclide, chelate and protein used, and doses of protein and radionuclide given. The therapy regimen also varies and includes a further barrier to success d although EBRT and chemotherapy are given in cycles, RIT was often given as a single administration. This might have had some basis in the immunogenicity of murine-derived antibodies, precluding multiple administrations of antibody. Now that a number of strategies to reduce immunogenicity are available, dose fractionation is becoming the norm.

Haematological Cancers In February 2002, RIT therapies targeting the CD20 molecule on B cells, for the targeting of non-Hodgkins lymphoma, were approved by the US Food and Drug Administration for clinical application. These have been reviewed elsewhere (e.g. [90]) and we summarise experience with those molecules for consideration with clinical use of others. Non-Hodgkins lymphoma B cell-based diseases, such as non-Hodgkins lymphoma, can be targeted using antibodies raised against CD20, which these cells often over-express. CD20 was chosen as a target because binding does not alter its expression, and apoptosis can be elicited through antibody interaction. The anti-CD20 therapies, Bexxar and Zevalin, are the current success stories of RIT. Bexxar ([131I]-tositumomab) and Zevalin ([90Y]-ibritumomab tiuxetan) recognise different epitopes on the CD20 molecule. Use is indicated in patients with disease that has proved refractory to treatment and patients who have relapsed after chemotherapy. For Zevalin, dosimetry is based on pre-scouting using [111In]labelled antibody, whereas for Bexxar the dose is based on body mass. As both were derived from murine antibodies, there are reports of immunogenic responses (human antimouse antibodies; HAMA) for both Bexxar (w5e10%) and Zevalin (w2%). Due to the expression of CD20 on normal B cells, as well as on those involved in disease, ‘cold’ antibody is given before the RIC in order to improve biodistribution. Although encouraging results have been reported at low doses [91,92] it is at higher doses, requiring bone marrow support, that therapy has been achieved [93]. It has been shown that Zevalin can be used safely in patients with prior myeloablative therapy, although response rates were modest in this feasibility study [94]. As has been seen in other cases, patients with bulky tumour (O5 cm) are less likely to respond to [90Y] anti-CD20 therapy than those with non-bulky (!5 cm) disease [95]. A pragmatic report of therapy using these molecules has been published [96].

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The antibody Lym-1 was raised against CD22 (human leukocyte antigen HLA-DR), which is expressed in O95% of B cell tumours. It does not require the pre-infusion step with cold antibody as CD22 is expressed at lower levels on normal B cells. The development of Lym-1 use has included the use of low-dose therapy, high-dose therapy and fractionation, and comparison of the iodinated and copper-labelled antibody. Gaze [97] reported that the use of [131I]-labelled Lym-1 led to responses in 30 out of 45 patients. A high-dose trial with autologous support involving 19 patients with Hodgkin’s lymphoma led to 16 complete responses (CRs) and two PRs. Treatment at a lower level of radioactivity (i.e. below the MTD of 100 mCi/m2) led to seven CRs and four PRs out of 21 patients [98]. Using a low-dose, fractionated regimen in patients with non-Hodgkins lymphoma (n ¼ 25) or chronic lymphocytic leukaemia (CLL) (n ¼ 5) led to favourable responses. The goal was to achieve O300 mCi administered dose of [131I]-labelled antibody. There was transient toxicity in 28% of 176 doses. HAMA was encountered, but only interrupted therapy in 10% of patients, whereas thrombocytopenia was the dose-limiting toxicity. Tumour regression was reported in 83% of patients. Most patients (94%) receiving a dose O180 mCi responded to therapy [99]. A fractionated regimen using copper-labelled antibody ([67Cu]-2IT-BAT-Lym-1) in patients pre-loaded with antibody used 60 mCi/m2 for each of the first two doses. Toxicity was thrombocytopenia and neutropenia, and although this was a pilot study there was a CR (1/3) lasting 12 months [100]. Liver received the highest radiation dose from copperlabelled Lym-1 (a single dose of [67Cu]-labelled antibody at 50e60 mCi/m2) although no hepatotoxicity was seen. Use of this radionuclide resulted in a higher peak concentration in tumours compared with radioiodine [101]. Splenomegaly has also been reported to have been treated with radiolabelled Lym-1 antibody [102], possibly due to the radiotoxic effect on malignant lymphocytes. Leukaemia Treatment options can be common to both lymphoma and leukaemia due to shared characteristics. Rituximab has been investigated in CLL, and Epratuzumab and Lumiliximab are also under development. The treatment of leukaemia has included therapy with b and a emitters, combination with more conventional therapies such as whole body irradiation and chemotherapy, as well as application to treat residual disease. The expression of CD33 on early myeloid progenitor cells and myeloid leukemic cells led to the development of antibody M195. This, and the humanised version HuM195, have elicited responses when labelled with either 131I or 90 Y [103]. This can be used to reduce large leukaemic tumour burdens, but myelosuppression has been encountered at O135 mCi/m2 necessitating bone marrow support. When this therapy was combined with chemotherapy outcome improved further. [131I]-M195 has also been shown to be effective in the treatment of residual disease. Patients in remission after retinoic acid therapy remained

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disease free for 8 months compared with 3 months without RIT. Use of these b-emitting labelled antibodies can lead to prolonged myelosuppression. In order to avoid this toxicity, the antibody was labelled with the a emitter 213Bi [104]. Patients with relapsed or refractory acute myeloid leukaemia or CLL were given between 10.36 and 37 MBq/kg. Transient myelosuppression was encountered with a median recovery of 22 days. The vast majority of the radionuclide localised to and was retained in sites of leukemic involvement, including bone marrow, liver and spleen. Biodistribution was more favourable with this nuclide d the absorbed dose ratios between these sites and the rest of the body were around 1000 better than with b-emitting nuclides. Treatment results reported included a reduction in circulating blasts in 14/15 patients and a reduction in bone marrow blasts in 14/18 patients. RIT for CLL is often limited by the extent of bone marrow involvement. The use of Bexxar and Zevalin is restricted to those patients who have less than 25% bone marrow involvement. However, favourable responses to Lym-1based RIT have been reported [105]. In addition, the use of RIT (aCD45 antibody BC8 labelled with 131I) was given as an adjuvant to total body irradiation and chemotherapy [106]. A similar combination of chemotherapy, total body irradiation and RIT for acute lymphoblastic leukaemia and myelodysplastic syndrome has shown potential [107]. Treated acute lymphoblastic leukaemia can recur in the central nervous system without disease elsewhere. This type of compartmentalised disease recurrence is ideal for RIT therapy using site of injection, and responses to such intrathecal administration have been reported (5/6 patients [108]). Epratuzumab is a humanised version of the aCD22 antibody mLL2 with around 90% of the sequence replaced. A trial using this antibody in conjunction with 90Y (185 MBq/ m2 over two to four infusions) reported high and durable objective response rates [109], with only minor toxicity.

Non-haematological Cancers The clinical application of RIT in the context of nonhaematological cancers has been less successful than in haematological cancers. However, the following are examples of the increasingly encouraging results that are being achieved. Prostate Early stage clinical trials in the treatment of metastatic prostate cancer have reported a reduction in diseaseassociated markers and relief of bone pain, such as a phase I trial using 90Y-2IT-BAD-m170 (aMUC-1 antibody targeting aberrant sugars on cancer cells) [110]. In a second study, m17 was used with a combination of 111In/90Y, for dosimetry and therapy, respectively. Patients were given 10.5 GBq, leading to relief of pain in 7/13. The combination of RIT and paclitaxel led to objective responses in 4/6. Neutropenia was identified as the dose-limiting toxicity [111]. Further

refinements have been reported in the use of high-dose RIT using 111In/90Y-labelled m170. The cathepsin linker was used and the therapy was given in combination with paclitaxel (75 mg/m2). Bone marrow suppression was encountered at 12 mCi/m2 (for prostate cancer patients) and 22 mCi/m2 (for breast cancer patients). Cyclosporin did reduce, but failed to eliminate, the immunogenic response to the antibody [112]. Reductions in disease-associated markers can also indicate that therapy has been achieved. For example, a phase I trial using [177Lu]-J591 reported a decrease in prostate-specific antigen (PSA) levels lasting 3e8 months in 11% of patients (4/35), whereas in 46% of patients (16/35) the PSA level was stabilised for a median of 60 days [113]. Similarly, in a trial using [90Y]-J591, 7% of patients (2/29) had a reduction in PSA levels lasting up to 8.5 months, whereas 21% (6/29) experienced a stabilisation in PSA levels [114,115]. Finally, a phase II trial treating metastatic prostate cancer with the anti-TAG72 antibody CC49 labelled with 131I at 75 mCi/m2 in combination with interferon led to a reduction in tumour size in addition to pain relief [116]. Ovarian Again the importance of the appropriate use of RIT has been shown. Stewart et al. [117] used the site of injection (i.e. intraperitoneal introduction) to treat metastatic ovarian carcinoma. A total of 31 patients were grouped according to tumour size. Those with microscopic disease had a good probability of achieving a CR (3/6), whereas only 2/15 with macroscopic tumours !2 cm and none of the group with larger tumours (O2 cm) achieved a CR. Combinations of chemotherapy with radiolabelled versions of the CC49 antibody have recently shown promise. Interferon, paclitaxel and [177Lu]-CC49 monoclonal antibody achieved PRs in 4/17 patients [86], whereas a feasibility study combining paclitaxel, interferon alpha 2 beta and [90Y]CC49 gave only minor toxicities [118]. Breast There are a number of reports of breast cancer RIT studies using a range of antibodies. Targeting of HER2, a molecule expressed on the surface of around 30% of breast cancer cells and correlating with poor prognosis, has yet to be applied successfully [119], partially due to heterogeneous distribution throughout the tumour. Other target molecules include CEA, using for example T84.66 ([90Y]-DTPA-cT84.66) given in a range from 15 to 22.5 mCi/m2 to six patients, which led to temporary responses [120]. Transient decreases in measurable disease were reported when [90Y]-MX-DTPA BrE-3, targeting the MUC-1 molecule, was used, although at higher doses HAMA responses were elicited [121]. Colorectal Although the primary tumour is often identifiable, and usually surgically removed, metastases, primarily to the

TECHNOLOGICAL ADVANCES IN RADIOIMMUNOTHERAPY

liver, lead to poor prognosis and form a significant challenge to successful therapy. RIT has therefore been used against micrometastases, in combination with surgical resection of major liver deposits, and as an adjuvant with other therapies, including EBRT and chemotherapy. Behr et al. [122] targeted small volume disease (%3 cm) using a single dose of [131I]-LMN-14 IgG (60 mCi/m2). Of 30 patients, five achieved objective responses, in addition to which three PRs and eight minor responses were recorded. Splitting the dose into fractions has been reported to produce lower bone marrow toxicity than for the same amount of radioactivity given as a single dose of 28 or 36 mCi/m2 [123]. Various methods of using the site of injection to increase uptake at the disease site have also been explored. The administration of RIC into the liver via the intrahepatic artery to target colorectal metastases resulted in uptake similar to that obtained after introduction into a peripheral vein [124]. However, intraperitoneal administration resulted in improved uptake in metastatic carcinoma, whereas introduction into the peripheral vein produced higher uptakes in lymph nodes and local recurrences [125]. Combining EBRT and RIT was shown in pre-clinical studies to not only improve therapeutic effect, but also to increase the uptake of radionuclide, possibly through vascular damage at the target site. A feasibility study, combining 131 I-F(ab0 )2 fragments with local EBRT to the liver, produced myelotoxicity and liver toxicity, but also produced positive tumour control, including a response in one patient and stable disease in 3/6 [77]. Addition of another systemic therapy to RIT has also been reported [126]. Patients whose disease had proven refractory to chemotherapy were given up to three cycles of 90Y-labelled aCEA antibody (16.6 mCi/ m2) in combination with 5-fluorouracil (700e1000 mg/m2/ day for 5 days). Tumour growth control was observed, and immunogenicity (human anti-chimeric antibodies (HACA) response) was reduced, possibly by the 5-fluorouracil, allowing repeat dosing. Pre-treatment of the patient population might affect results achieved with RIT. In a study giving escalating doses of 131I-aA33 to a patient group refractory to previous therapies [127], no objective results were recorded, and the MTD was reached at 75 mCi/m2. Brain Treatment of a brain tumour is very much dictated by the disease. The bloodebrain barrier greatly restricts access by large molecules such as antibodies, and most studies therefore use local introduction and PRIT. It is generally regarded that disease does not spread from the initial site [128,129], so success is often reported. Glioma patients were given 60e80 mCi/m2 a-tenascin antibody, after which radiographic responses were recorded in 25% (12/48) [130]. A further group of glioma patients were given 40e140 mCi 131I-labelled antibody, resulting in no major toxicities and 6/10 patients showed clinical improvement [131]. Although patients received antibody either intravenously or via the carotid artery, there was no significant difference in tumour localisation.

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In a separate study, the introduction of [131I]-labelled a-tenascin antibody into the surgical resection site resulted in a response rate of 51.6%, including 15% PR, 5% CR and 32% no evidence of disease (NED) [132,133]. The use of PRIT has achieved positive results [134]. Antitenascin antibody labelled with avidin was given (2 mg), then 90Y-biotin (MTD 30 mCi). Tumour growth control (stable disease) was achieved in 50% of cases (12/24 total, 7/16 glioblastoma multiforme and 5/8 anaplastic astrocytoma) with an objective response in 25% at a dose of 19e24.3 mCi. In a subsequent study group [135], 73 patients with glioblastoma multiforme were treated with PRIT given in this manner (38/73) or with chemotherapy (35/73), administered activity ranging from 370 to 925 MBq. RIT alone or in combination with temozolomide resulted in a median overall survival of 17.5 and 25 months and a progression-free survival of 5 and 10 months, respectively. In the absence of an increase in toxicity upon the addition of temozolomide, this study suggests an advantage in the combination of chemotherapy with RIT in this context.

Renal cell Brouwers et al. [136] reported the use of RIT ([131I]-cG250) in the treatment of renal cell carcinoma. A two-stage treatment regimen, including pre-scouting (222 MBq/m2), found an inverse correlation between tumour size and the absorbed dose to the tumour. Although most tumours received less than 10 Gy, smaller lesions (!5 g) absorbed more than 50 Gy. Of those patients (3/27) who received a lower administered dose (2220 followed after 3 months by 1110 MBq/m2), one had stable disease and two had progressive disease. Of the patients (15/27) who received a higher administered dose (2220 followed after 3 months by 1665 MBq/m2), four altered from progressive disease to stable disease and 11 had stable disease.

Summary and Conclusions The specialty of RIT has recently been through something of a rebirth. Experiences in the treatment of haematological cancers heralded the fact that RIC-based treatments could be used with clinical success. Improved application in more realistic settings, utilising the strengths of RIT in a wide range of cancers, has led to the heartening results described here, and points to its adoption as a valuable tool in the routine treatment of cancer.

Author for correspondence: J. L. J. Dearling, Cancer Research UK Targeting & Imaging Group, Department of Oncology, University College London (Hampstead Campus), London, UK. Tel: þ44-207794-0500; Fax: þ44-207-794-3341; E-mail: [email protected] Received 8 March 2007; received in revised form 13 March 2007; accepted 28 March 2007

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