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PERSPECTIVE www.rsc.org/dalton | Dalton Transactions Targeted a-therapy: past, present, future?† Martin W. Brechbiel* Received 28th March 2007, Accepted 17th May 2007 First published as an Advance Article on the web 11th September 2007 DOI: 10.1039/b704726f Monoclonal antibodies have become a viable strategy for the delivery of therapeutic, particle emitting radionuclides specifically to tumor cells to either augment anti-tumor action of the native antibodies or to solely take advantage of their action as targeting vectors. Proper and rational selection of radionuclide and antibody combinations is critical to making radioimmunotherapy (RIT) a standard therapeutic modality due to the fundamental and significant differences in the emission of either a- and b-particles. The a-particle has a short path length (50–80 lm) that is characterized by high linear energy transfer (100 keV lm 1 ). Actively targeted a-therapy potentially offers a more specific tumor cell killing action with less collateral damage to the surrounding normal tissues than b-emitters. These properties make targeted a-therapy an appropriate therapy to eliminate minimal residual or micrometastatic disease. RIT using a-emitters such as 213 Bi, 211 At, 225 Ac, and others has demonstrated significant activity in both in vitro and in vivo model systems. Limited numbers of clinical trials have progressed to demonstrate safety, feasibility, and therapeutic activity of targeted a-therapy, despite having to traverse complex obstacles. Further advances may require more potent isotopes, additional sources and more efficient means of isotope production. Refinements in chelation and/or radiolabeling chemistry combined with rational improvements of isotope delivery, targeting vectors, molecular targets, and identification of appropriate clinical applications remain as active areas of research. Ultimately, randomized trials comparing targeted a-therapy combined with integration into existing standards of care treatment regimens will determine the clinical utility of this modality. Introduction Kohler and Milstein’s hybridoma/monoclonal antibody (mAb) technology resurrected the concept that antibodies might serve as magic bullets as proposed by Ehrlich. 1 Their seminal publication provided a clear opening towards the development of antibody targeted radiation. 2 In the 1980s, murine mAbs against tumor-associated antigens (TAA) generated multitudes of pre-clinical studies that provided proof-of-concept of the potential of cancer treatment using radiolabeled mAbs. These studies also demonstrated discordance in predictability of their therapeutic efficacy. Foremost was a seem- ingly inevitable patient production of human anti-murine imm- munoglobulin antibodies (HAMA) after one to three treatments. 3 Other factors limiting treatment included: (1) inadequate thera- peutic dose delivered to tumor lesions; (2) insufficient activation of effector function(s); (3) slow blood compartment clearance; (4) low mAb affinity and avidity; (5) trafficking to or targeting of normal organs; (6) heterogeneous antigen distribution on tumor cells; and (7) insufficient tumor penetration. 3 In part, these limitations were addressed by chemical modification of the mAb, but many of these challenges have been addressed with genetic engineering applied to eliminating HAMA by either production of chimeric mAbs, CDR grafting, or complete humanization of the protein. 4 Radioimmune & Inorganic Chemistry Section Radiation Oncology Branch, NCI, NIH Building 10, Room 1B40 10 Center Drive Bethesda, MD 20892- 1088, USA. E-mail: [email protected]; Fax: +1 (301) 402-1923 †Based on the presentation given at Dalton Discussion No. 10, 3rd–5th September 2007, University of Durham, Durham, UK. Investigators are now able to fully explore the real therapeutic potential of radiolabeled mAbs. With the elimination of many obstacles and a better understanding of the inherent limitations of mAbs, the active targeting and delivery vector of the radiation, many radiolabeled mAbs have been, or currently are being evaluated in Phase III trials. The FDA approved two radiolabeled mAbs for the treatment of non-Hodgkin’s lymphoma (NHL), Zevalin and Bexxar, making the approval of additional targeted radiation therapy products probable. 5 However, both agents are radiolabeled with b-emitters, 90 Y(t 1/2 = 2.67 d) and 131 I(t 1/2 = 8.07 d), respectively. Radionuclides that decay by emission of b-particles emit electrons with maximum kinetic energies of 0.3–2.3 MeV with corresponding ranges of 0.5–12 mm in tissue. This lengthy range reduces the need for cellular internalization and so targeting close to or at the cell membrane is sufficient. The range of b- particles, as compared to the diameter of cells, permits b-particles to traverse several cells (10–1000), an effect that has been termed “crossfire”. Crossfire is critical to b-particle emitter therapy to improve tumor dose homogeneity and to ensure a sufficient dose to each cell. 6 Single cell disease such as leukemia, micrometastases, post-surgical residual disease, and other disseminated types of cancer may not be curable with targeted b-particle therapy. Humm and Cobb reported that to attain a cell kill probability of 99.99% for single cells, several hundreds of thousands of b-decays at the cell membrane are required. 7 Concomitantly, a very large portion of the dose would also be deposited in the surrounding normal tissue by virtue of this same lengthy range. Therefore, the fundamental physics and radiobiology of b-particle radiation provides a poor 4918 | Dalton Trans., 2007, 4918–4928 This journal is © The Royal Society of Chemistry 2007 Published on 11 September 2007. Downloaded by California State University at Fresno on 05/08/2013 13:48:06. View Article Online / Journal Homepage / Table of Contents for this issue

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PERSPECTIVE www.rsc.org/dalton | Dalton Transactions

Targeted a-therapy: past, present, future?†

Martin W. Brechbiel*

Received 28th March 2007, Accepted 17th May 2007First published as an Advance Article on the web 11th September 2007DOI: 10.1039/b704726f

Monoclonal antibodies have become a viable strategy for the delivery of therapeutic, particle emittingradionuclides specifically to tumor cells to either augment anti-tumor action of the native antibodies orto solely take advantage of their action as targeting vectors. Proper and rational selection ofradionuclide and antibody combinations is critical to making radioimmunotherapy (RIT) a standardtherapeutic modality due to the fundamental and significant differences in the emission of either a- andb-particles. The a-particle has a short path length (50–80 lm) that is characterized by high linear energytransfer (100 keV lm−1). Actively targeted a-therapy potentially offers a more specific tumor cell killingaction with less collateral damage to the surrounding normal tissues than b-emitters. These propertiesmake targeted a-therapy an appropriate therapy to eliminate minimal residual or micrometastaticdisease. RIT using a-emitters such as 213Bi, 211At, 225Ac, and others has demonstrated significant activityin both in vitro and in vivo model systems. Limited numbers of clinical trials have progressed todemonstrate safety, feasibility, and therapeutic activity of targeted a-therapy, despite having to traversecomplex obstacles. Further advances may require more potent isotopes, additional sources and moreefficient means of isotope production. Refinements in chelation and/or radiolabeling chemistrycombined with rational improvements of isotope delivery, targeting vectors, molecular targets, andidentification of appropriate clinical applications remain as active areas of research. Ultimately,randomized trials comparing targeted a-therapy combined with integration into existing standards ofcare treatment regimens will determine the clinical utility of this modality.

Introduction

Kohler and Milstein’s hybridoma/monoclonal antibody (mAb)technology resurrected the concept that antibodies might serve asmagic bullets as proposed by Ehrlich.1 Their seminal publicationprovided a clear opening towards the development of antibodytargeted radiation.2

In the 1980s, murine mAbs against tumor-associated antigens(TAA) generated multitudes of pre-clinical studies that providedproof-of-concept of the potential of cancer treatment usingradiolabeled mAbs. These studies also demonstrated discordancein predictability of their therapeutic efficacy. Foremost was a seem-ingly inevitable patient production of human anti-murine imm-munoglobulin antibodies (HAMA) after one to three treatments.3

Other factors limiting treatment included: (1) inadequate thera-peutic dose delivered to tumor lesions; (2) insufficient activation ofeffector function(s); (3) slow blood compartment clearance; (4) lowmAb affinity and avidity; (5) trafficking to or targeting of normalorgans; (6) heterogeneous antigen distribution on tumor cells; and(7) insufficient tumor penetration.3 In part, these limitations wereaddressed by chemical modification of the mAb, but many of thesechallenges have been addressed with genetic engineering appliedto eliminating HAMA by either production of chimeric mAbs,CDR grafting, or complete humanization of the protein.4

Radioimmune & Inorganic Chemistry Section Radiation Oncology Branch,NCI, NIH Building 10, Room 1B40 10 Center Drive Bethesda, MD 20892-1088, USA. E-mail: [email protected]; Fax: +1 (301) 402-1923† Based on the presentation given at Dalton Discussion No. 10, 3rd–5thSeptember 2007, University of Durham, Durham, UK.

Investigators are now able to fully explore the real therapeuticpotential of radiolabeled mAbs. With the elimination of manyobstacles and a better understanding of the inherent limitations ofmAbs, the active targeting and delivery vector of the radiation,many radiolabeled mAbs have been, or currently are beingevaluated in Phase III trials. The FDA approved two radiolabeledmAbs for the treatment of non-Hodgkin’s lymphoma (NHL),Zevalin and Bexxar, making the approval of additional targetedradiation therapy products probable.5 However, both agents areradiolabeled with b-emitters, 90Y (t1/2 = 2.67 d) and 131I (t1/2 = 8.07d), respectively.

Radionuclides that decay by emission of b-particles emitelectrons with maximum kinetic energies of 0.3–2.3 MeV withcorresponding ranges of ∼0.5–12 mm in tissue. This lengthy rangereduces the need for cellular internalization and so targetingclose to or at the cell membrane is sufficient. The range of b-particles, as compared to the diameter of cells, permits b-particlesto traverse several cells (10–1000), an effect that has been termed“crossfire”. Crossfire is critical to b-particle emitter therapy toimprove tumor dose homogeneity and to ensure a sufficient doseto each cell.6 Single cell disease such as leukemia, micrometastases,post-surgical residual disease, and other disseminated types ofcancer may not be curable with targeted b-particle therapy. Hummand Cobb reported that to attain a cell kill probability of 99.99%for single cells, several hundreds of thousands of b-decays at the cellmembrane are required.7 Concomitantly, a very large portion ofthe dose would also be deposited in the surrounding normal tissueby virtue of this same lengthy range. Therefore, the fundamentalphysics and radiobiology of b-particle radiation provides a poor

4918 | Dalton Trans., 2007, 4918–4928 This journal is © The Royal Society of Chemistry 2007

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Page 2: Targeted α-therapy: past, present, future?

tumor-to-normal-tissue dose ratio for treatment of single celldisease. Selection of an a-particle emitter over a b-particle emittercreates a scenario wherein such diseases may be treatable withtargeted radionuclide therapy.

Energy deposition per unit path length in tissue for a-particles isfar higher than for b-particles, due to the greater mass and chargeof the a-particle, which is a mono-energetic, high-energy heliumnucleus (4He). The average energy imparted per unit path length,termed linear energy transfer (LET), is 60–230 keV lm−1 for a-particles and is therefore classified as high LET radiation.8 Forcomparison purposes, the LET values for b-particles are typicallybetween 0.1 and 1 keV lm−1; low-LET radiation. A single a-particle traversal of the cell nucleus has a 20–40% probabilityof killing the cell.9–11 A typical a-particle kinetic energy of 5–9 MeV results in a 50–90 lm range in tissue, or to ∼2–10 celldiameters. Delivery of an a-emitting radionuclide close to, or atthe cell membrane, remains sufficient for therapy with a-particlesto kill targeted malignant cells. In a single cell disease scenario,taking into account omnidirectional decay and geometry, only afew hundred a-particle decays at the cell membrane are requiredfor a 99.99% level of cell kill with a correspondingly significantdecrease in collateral toxicity to normal tissue.7 Thus, given agood targeting vehicle suitable for a a-particle emitter, a highlylocalized and cytotoxic radiation dose can be delivered to cancercells with minimal damage to the surrounding normal tissue.

Additionally, high LET radiation is well established as beingfar more lethal to cells than low LET radiation.12–16 Differences

Martin W. Brechbiel received a B.A. in 1979 from GettysburgCollege and a M.S. in 1982 from the University of Delawareunder the guidance of Professor Harold Kwart. After working forFMC Corp., he joined the National Cancer Institute (NCI) in1983. Thereafter, he worked to develop novel bifunctional chelatingagents for sequestering radionuclides and their conjugation toimmunoproteins under the direction of Dr Otto A. Gansow whilesimultaneously obtaining a PhD from American U. in 1988 withProfessor Thomas Cantrell. He remained with the NCI and in 2001was appointed as the Section Chief of the Radioimmune & InorganicChemistry Section. His research group’s activities span the range ofcontinuing development of novel chelating agents for radionuclides,the development of contrast media for MRI, EPR, and CT imaging,SPECT and PET imaging agents, and the development of rationallydesigned targeted a-therapy regimens.

Martin W. Brechbiel

between high and low LET radiation is often described throughtheir relative biological effectiveness (RBE). RBE is defined as theratio between a given test radiation dose and a reference radiationdose (originally 250 kV X-rays) wherein the test and referenceradiation doses result in equal biological effect. RBE values forin vitro and in vivo cell survival of 3–8 have been reported fora-particles.18,23,24 The primary cause for higher cell toxicity hasbeen hypothesized as originating from the increased frequency ofclustered DNA double-strand breaks (DSBs) observed with highLET radiation.13,17–19 The cytotoxicity of a-particles has also beenshown to be independent of both dose rate and oxygenation statusof the irradiated cells.12 Low LET radiotherapy is less effective onhypoxic cells and at low dose rates.12

Radionuclides

There are >100 radionuclides that emit a-particles. However, theoverwhelming majority either decay too quickly or too long to beof meaningful therapeutic use, or no viable chemistry exists fortheir use, or there is no viable supply. Therefore, this discussionwill be limited to those a-emitters that fall within the boundariesof reasonable use and have been investigated in animal models orhumans.

149Tb

149Tb (t1/2 = 4 h) is a lanthanide that decays via a complicated set ofmechanisms: a-decay (17%), b+-decay (4%), and electron capture(79%) (Fig. 1). Production has been at the CERN spallationfacility and small amounts have also been produced using a 10-mV tandem accelerator using the 141Pr(12C, 4n)149Tb reaction at 70MeV. Separation and purification from the target materials hasproved challenging when faced with the levels of purity requiredfor RIT applications.20,21 Parts per million contamination cancompletely compromise radiolabeling protocols.

Fig. 1 Decay scheme for 149Tb.

211At

211At is a cyclotron produced radionuclide by virtue of bombard-ment of a bismuth target with a-particles in a cyclotron via the 207Bi(a, 2n)211At nuclear reaction.22 Isolation from the cyclotron targetis routinely performed by means of dry distillation procedures.23,24

Few institutions, however, possess a cyclotron of adequate energyrange that is capable of producing 211At.

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211At (t1/2 = 7.2 h) decays through a branched pathway with eachbranch resulting in the production of an a-particle in its decay tostable 207Pb (Fig. 2). The a-particles from 211At have a mean energyof 6.8 MeV with a mean LET of 97–99 keV lm−1. Because ofits relatively long half-life, 211At-labeled constructs can be usedeven when the targeting molecule does not gain immediate accessto tumor cells. Additionally, its daughter, 211Po, emits K X-raysthat allow photon counting of samples and external imaging forbiodistribution studies. This radionuclide, by virtue of behavinganalogously with iodine halogen chemistry, is also not retainedas well as other a-emitting radiometals post-internalization intocells, which is a factor to be considered.25

Fig. 2 Decay scheme for 211At.

212Bi

212Bi (t1/2 = 60.6 min) emits an a-particle with a mean energyof 7.8 MeV from the decay of 228Th to stable 208Pb (Fig. 3). Agenerator that uses 224Ra as the parent radionuclide provides foron-site production of 212Bi for radiolabeling targeting vectors suchas mAbs since the half-life is too short for realistic transportationbetween sites.26 The 224Ra actually originates from weaponsdevelopment and is extracted from 229Th, currently at PacificNorthwest Laboratories with the 228Th originally being purifiedfrom 232U.26 One daughter from the decay of 212Bi, 208Tl, emits a2.6-MeV c-ray that requires heavy shielding to minimize radiationexposure to personnel, thereby limiting the clinical utility of thisradioisotope. However, it is unclear what level of shielding is really

Fig. 3 Decay scheme for 212Pb and 212Bi.

necessary in a clinical setting due to the combination of both actualdosing schedules and short half-life. After 212Bi has been selectivelyeluted from the ion-exchange resin of the 224Ra generator either inthe form of chloride or the tetraiodide complex, the isotope can beused after pH adjustment to radiolabel mAbs, peptides, or othervectors conjugated with a suitable bifunctional chelating agentsuch as the C-functionalized trans-cyclohexyldiethylenetriaminepentaacetic acid derivative, CHX-A′′ DTPA (Fig. 4).27–30

Fig. 4 Structures of DTPA, cyclic dianhydride of DTPA, CyDTPA,1B4M-DTPA, and CHX-A′′ DTPA.

212Pb

212Pb (t1/2 = 10.2 h) is actually a b−-emitter and is the immediateparental radionuclide of 212Bi. Its inclusion here is justified since212Pb has been evaluated as an in vivo generator for the productionof 212Bi thereby effectively extending the half-life of 212Bi to ∼11 h(Fig. 3). However, during the decay processes, ∼ 30% of the formed212Bi is released from the chelation environment.31 Nonetheless, thecombination of greater efficacy as compared to 212Bi on the basisof lCi vs. mCi lowered administered dose, and issues of availabilityvs. cost, all combined with appropriate usage continue to promotethe use of this radionuclide as a viable therapeutic within specificlimitations. 212Pb is available from the same 224Ra generator thatfacilitates the production of 212Bi, and may be selectively eluted bycontrolling the pH of the HCl eluant from that same ion-exchangebased generator system vs. 212Bi for labeling mAbs.26 Concerns

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regarding the 2.6-MeV c-ray from the 208Tl daughter are greatlydiminished due to decreased dose levels combined with half-life.

213Bi

213Bi is also available from a very similar generator based tech-nology from its parent radionuclide 225Ac dispersed onto a cation-exchange resin to prevent charring and decomposition of resin dueto the confined radiation flux.32,33 The source of 225Ac in the UnitedStates is currently limited to Oak Ridge National Laboratorieswhere the source materials extend back to 225Ra extracted from229Th which again has its origin in weapons development from233U.8,34 213Bi decays to stable 209Bi by emitting an a-particle and2 b−-particles (Fig. 5). Additionally, a 440-keV photon emissionallows biodistribution, pharmacokinetic, and dosimetry studiesto be performed. Similarly to 212Bi, after elution from the 225Acgenerator, 213Bi is readily conjugated to mAbs, peptides, or othervectors that have been modified with a suitable bifunctionalchelating agent, such as CHX-A′′ DTPA.27–30

223Ra

223Ra (t1/2 = 11.4 d) can be provided in a generator form fromthe 227Ac (t1/2 = 21.8 y) parent and is also available from uraniummill tailings in large quantities. Similar to 225Ac (vide infra), 223Raultimately provides for the emission of 4 a-particles through itsdecay scheme and daughters (Fig. 6).35 Because of inherent bone-seeking properties, cationic 223Ra may be a promising candidatefor the delivery of high-LET radiation to cancer cells on bonesurfaces. A Phase I clinical study demonstrated pain relief andreduction in tumor marker levels in the treatment of skeletalmetastases in patients with prostate and breast cancer.36 Devel-opment of chelation chemistry actively targeted 223Ra continues tobe pursued, however, the retention and biological trafficking of thedecay process daughters remains a problematic challenge. The firstdaughter in the 223Ra decay pathway is 219Rn, a gaseous productthat would pose a serious challenge to control in vivo. Thus, thebiodistribution and targeting as well as those issues pertainingto control and trafficking of the decay daughters remain underinvestigation.

Fig. 5 Decay scheme for 225Ac and 213Bi.

Fig. 6 Decay scheme for 223Ra.

225Ac

225Ac (t1/2 = 10.0 d) decays sequentially by a-emission throughthree daughter radionuclides, 221Fr (t1/2 = 4.8 min), 217At (t1/2 =32.3 ms), and Bi (t1/2 = 45.6 min), each of which then also emitsan a-particle (Fig. 5). 225Ac can be produced by the natural decayof 233U or by accelerator-based methods.34,35,37 Targeted 225Ac asa therapeutic, in theory may be as much as ∼1000 times morepotent than 213Bi-containing analogs by virtue of this a-particlecascade to a cancer cell.38 While this increased potency mightrender 225Ac more effective than other a-emitters, the biologicalfate of the free daughter radioisotopes in circulation after decay of225Ac is unresolved; the qualities of the chelation chemistry usedto sequester this element in vivo are equally unresolved.38–40

Radiolabeling-chemistry

One of the fundamental key aspects of targeted radiation therapyis the stable sequestration of the radionuclide in vivo.6 in vivostability of a radioconjugate is paramount to maximizing thedelivery of radiation to tumor while minimizing toxicity. All ofthe above radionuclides have specific biological sites of depositionwhich will then pose issues of unacceptable toxicity to normaltissue; 211At → thyroid, gut and lungs, 212Pb → red blood cellsand bone, 212Bi/213Bi → kidney, 223Ra → bone, 225Ac → bone andliver.8,22,38 Radioconjugates can also be susceptible to catabolismpost-internalization into target cells or to the direct effects ofradioactive decay. A variety of methods are used to conjugateradioisotopes to antibodies, dependent on the chemical nature ofthe radionuclide.

211At is generally treated as a halogen and like 131I can be usedto directly radiolabel mAbs by incorporation of an aryl carbon–astatine bond into the antibody using tyrosine residues.22 However,such chemistry results in labile products resulting in the lossof 211At. Methods have been developed in multiple laboratoriesbased on small “linker” molecules that create an aryl carbon–astatine bond involving an astatodemetallation reaction using atin, silicon, or mercury precursor.22,23,41 These small molecules havea reactive site for astatination whilst also possessing an active ester

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for protein or peptide modification and radiolabeling. Regardlessof these developments, most of these agents still suffer from somesmall measure of instability leading to an unacceptable loss of211At. Recent results indicate that this deficiency may be resolvedbut additional evaluation is necessary to confirm these results.42,43

The other radionuclides discussed above are metallic in natureand thus require chelation chemistry or bifunctional chelators forlinkage to antibodies. A sampling of bifunctional chelating agentsderived from DTPA include the cyclic dianhydride derivative,44

1B4M-DTPA (MX-DTPA, tiuxetan),45 and a family of trans-cyclohexyl derivatives that include the specific stereoisomer, CHX-A′′ DTPA (Fig. 4).28–30 The CHX-A′′ DTPA is an effectivechelator for 111In, 90Y, 177Lu, and to date is the only reportedDTPA derivative to form suitably stable complexes with eitherof the above bismuth radionuclides conjugated to mAbs orpeptides in vivo,28–30 resulting in radioimmunoconjugates thathave been used effectively in clinical trials.27 While studies of thephysical characteristics and coordination chemistry have not beenperformed on this specific enantiomeric form, CHX-A′′, studieson the parental ligand CyDTPA and DTPA and their Bi(III)complexes have been reported.46 Eight coordinate structures, bothsquare anti-prisms, for each complex were reported. However, theimpact of the trans-cyclohexyl ring was clearly noted by shorterBi–ligand bond distances, but more importantly by all eightelements of the coordination sphere originating from CyDTPAwhile one carboxylate donor in the DTPA complex originates froma neighboring molecule. This characteristic very easily translatesinto the significant differential in the in vivo complex stabilityfound for CHX-A′′ DTPA.

Numerous bifunctional analogs of the macrocyclic ligand1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) (Fig. 7)have been used effectively for labeling antibodies with 111In, 90Y,177Lu, 212Pb, and 212Bi.6,8,47–50 Structurally, DOTA complexes forlanthanides and other metal ions tend to be eight-coordinatesquare anti-prisms that exist in an equilibrium between isomericarrangements of the carboxylate arms and ring twists forms

Fig. 7 Structures of DOTA, and the bifunctional analogs C-DOTA,PA-DOTA, CHX-DOTA, lys-DOTA, and TCMC.

that saturate the coordination spheres about Bi(III) and Pb(II).However, the complex formation mechanism as reported forlanthanides can doubtlessly be extended to Bi(III) which correlateswith compromising the use of DOTA with 212Bi or 213Bi.51,52 Onesimply does not have the luxury of time when using short half-liferadionuclides except in selected cases where the formation ratecan be accelerated by heating; this option is not available whenusing proteins as the targeting vector and may be limited to smallmolecules and peptides.53

Unfortunately, despite also forming highly stable complexeswith 212Pb,54 the Pb(II) DOTA complex has been shown to bemore susceptible to acid catalyzed dissociation of Pb(II) than thecorresponding tetra-primary amide derivative, TCMC (Fig. 7).55

Loss of 212Pb post-internalization of mAb delivery to cells has beenreported as a source of marrow toxicity. Hence, TCMC continuesto be used for sequestration of 212Pb as opposed to DOTA. DOTAderivatives have also been evaluated for sequestration of 225Ac,however, and despite reported stability,38 this result conflicts withthe reported use of a bifunctional derivative of the 12-coordinationsite chelating ligand, 1,4,7,10,13,16-hexaazacyclohexadecane-N,N ′,N ′′,N ′′′,N ′′′′,N ′′ ′′ ′-hexaacetic acid (HEHA) (Fig. 8).39,40 Thedevelopment of Ac(III) chelation chemistry is considerably ham-pered by the simple fact that there are no stable isotopes ofthis element. As such, very little is actually known about thecoordination chemistry of Ac(III). One can infer or extrapolatefrom La(III), however the ionic radius is known to be ∼10% greaterfor Ac(III) (112 pm),56 and while a coordination number of eightmight be arrived at by this process, saturating the coordinationsphere may in fact require a greater number of donors moreeffectively distributed about the metal ion. Unfortunately, it iscompletely unclear that any research group is currently engagedin those fundamental studies required to define the coordinationchemistry of Ac(III).

Fig. 8 Structures of the bifunctional chelating agent BF-HEHA.

Chelation chemistry for 223Ra as noted above remains unre-solved for adequate in vivo sequestration of this element withcontrolled targeting via a biological vector, e.g. a mAb.57 Again,development of Ra(III) chelation chemistry for these applicationshas been considerably hampered by the simple fact that there areno stable isotopes of this element. Various chelating agents havebeen evaluated, but as yet none meet the minimal criteria for suchapplications.

A limited number of reports exist that evaluate chelationchemistry for 149Tb in vivo.58–60 Use of CHX-A DTPA has beenreported, yet no definitive in vivo stability and biodistributionstudies are available at this time using this chemistry. Evaluationsof in vivo pre-clinical efficacy of this radionuclide have been equallylimited.60 Contrary to several of the above listed radionuclides,

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Tb(III) is available in a stable form and as such a wealth ofknowledge is available in the literature on lanthanide complexationchemistry.61 Despite the abundant literature relating to lanthanidecoordination chemistry, both derivatives of DOTA and CHX-A′′

DTPA seem to be bifunctional chelating agents of choice whichmay also indicate that a little more development is required in thisregard.

The vast majority of these bifunctional chelating agents havebeen functionalized with an aryl isothiocyanate group for conju-gation to amine moieties resident on proteins, peptides, or othervectors.6 Other reactive functional groups such as reactive halo-gens, maleimides, and active esters have been studied far less andseem limited to specific arenas of use. Almost all when used to formconjugates with proteins form random distributions reacting withavailable functionalities. Additionally, in the case of conjugatingchelating agents to proteins, e.g. mAbs, the number of chelatespossible per protein molecule has to be determined empirically toretain the biological activity of the protein. In the case of peptides,the chelating agent is generally at the N-terminus in a 1 : 1 formarranged synthetically. In general, bifunctional chelating agentsare reacted with the targeting vector first and after optimizationof the conditions to achieve acceptable conjugation conditions,radiolabeling of the conjugate is executed. Pre-radiolabeling of thebifunctional chelating agent prior to conjugation, which has beeninvestigated with 225Ac,38 tends not to be performed due to issuesof low conjugation yields versus radiolysis issues. Additionally, for225Ac both low complexation yields despite heating and then shortconjugation reaction times severely impact the specific activityof the 225Ac labeled protein product. Clearly, opportunities existin select areas of both coordination chemistry and conjugationchemistry. Both areas seems to be settled in regards to Bi(III)radionuclides as well as for 212Pb. The loss of formed 212Bi fromthe b−-decay of 212Pb seems an insurmountable problem,31 but onethat simply limits the viable applications of this radionuclide withchemistry that is equally adequate. One must seriously questionthe need for further derivatives of DOTA and DTPA for thatmatter since no significant gains arising from either basic structureseem forthcoming. No greater stability was needed nor has beenachieved by the continued production of DOTA derivatives inthis field and it is unlikely that any further improvements willbe achieved through the creation of additional DTPA derivatives.However, refinements in conjugation chemistry strategies continueto be needed to optimize yields and efficiency.

Development of novel ligands suitable for in vivo sequestrationof the both Ac(III) and Ra(II) remains an area of opportunitysince those ligands in use now were simply adopted from otherapplications or for chelating other radionuclides. Clearly, only withabsolute unequivocal complex stability of both 225Ac and 223Ra willresearchers be able to deconvolute and address issues surroundingthe fate and trafficking of the decay product daughters and thenbe able to determine if these radionuclides have a real place in thecancer therapy armament. Other than just stability, the inabilityto produce final radiolabeled products in high specific activitycontinues to challenge the use of metallic a-emitters.

Considerable progress has been made in the development oflinkers for forming 211At conjugates although it is clear thatthis effort is still asymptotic towards achieving complete suitablestability for intravenous administration. Clinical trials still treatingcavitary disease scenarios and pre-clinical studies continue to

demonstrate small, but meaningful levels of instability in vivo ofthe At–C bond.

Pre-clinical studies149Tb

There are limited in vivo evaluation studies reported using 149Tb nodoubt due to limitations associated with its production (see above).One study demonstrates high-efficiency elimination of leukemia ina SCID mouse model using the FDA approved anti-CD20 mAb,rituximab, labeled with 149Tb.60 RIT using 150 lCi (5.5 MBq) of149Tb-rituximab 2 days after intravenous administration of 5 × 106

Daudi cells resulted in >120 days of tumor free survival in 89%of treated animals. In contrast, all control mice (no treatmentor treated with 5 or 300 lg unlabeled rituximab) developedlymphoma disease dying within 43 and 118 days, respectively.Interestingly, one can see that high doses of rituximab had asignificant response, but no cure. This result very clearly definesthe potential impact and opportunities of combination therapy, inthis case integrating the effector functions of the targeting vectorwith targeted a-therapy.

When the study was terminated, 28.4% ± 4% of the long-liveddaughter activity from 149Tb decay remained in vivo; 91.1% waslocated in bone tissue with 6.3% in the liver. Significant daughterradionuclide activity was also determined to be in the spleen andinterpreted to be indicative of elimination of killed cancer cellsthrough the spleen. Alternatively, the spleen may well have been afocal point of the disease leaving those daughters retained in theorgan. While encouraging, one has to remain concerned about theimpact of retained daughter radionuclide and whether any latertoxicity effect might be generated by these radionuclides. Thisaspect is a recurring area of concern for many a-emitters that havemultiple daughter and/or branching decay pathways.

211At

Studies using 211At date back greater than 50 years, however,recent history defines the leading center for targeted therapy usingthis radionuclide to be Duke University. Investigators there haveextensively studied both 211At production and radiolabeling chem-istry whilst performing an array of pre-clinical studies developingnumerous small molecule linkers for astatination of proteins andpeptides.22,41 Studies with 131I-81C6, a chimeric antibody targetingtenascin, a glycoprotein over-expressed on gliomas, were extendedto 211At-81C6. Treatment of a neoplastic meningitis model with211At-81C6 prolonged survival compared with controls.62 Thesestudies were subsequently extended and extrapolated into one ofthe landmark clinical trials using targeted a-therapy (vide infra).

212Bi

Early reports on 212Bi-containing radioimmunoconjugates includestudies by Macklis et al.,44 and Simonson et al.63 Tumor-specificantibodies radiolabeled with 212Bi using either the cyclic DTPA di-anhydride or glycyltyrosyl-lysyl-N-e-DTPA, respectively, reportedprolonged survival of colon carcinoma-bearing mice as comparedwith controls. However, both of these studies noted significantrenal deposition of 212Bi.

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To overcome this deficiency, the value of the bifunctionalchelating agent, CHX-A DTPA,28 was defined by evaluationof 212Bi labeled anti-gp70 mAb 103A.64 Therapy of murineRauscher leukemia virus (RVL) resulted in decreased splenictumor growth and prolonged median survival. Additionally, 212Bi-anti-Tac (anti-CD25) was used to treat CD25+ tumor bearingmice which led to an enhanced tumor-free survival and preventeddevelopment of tumors in some animals. However, in mice withestablished tumors, this same therapy failed to produce anyresponse.65

212Pb

Ruble et al. studied the efficacy of 212Pb radiolabeled mAb 103Atreating RVL resulting in an histological cure in all animals with adose of 0.74 MBq (20 lCi). However, marrow toxicity originatingfrom the loss of the decay daughter, 212Bi, could not be obviatedby administration of a heavy metal chelator.66

Horak et al. investigated 212Pb-AE1-mAb targeting HER2 onovarian tumors in nude mice.67 Transient bone marrow toxicityand lengthy renal toxicity were observed after i.v. injection of 0.93MBq (25 lCi); doses of 1.48 MBq (40 lCi) resulted in acellularbone marrow and subsequent death of all animals. However, 0.37–0.74 MBq (10–20 lCi) of 212Pb-AE1 resulted in 100% tumorfree survival for 180 days when treating animals bearing a 3-day s.c. tumor; all controls developed tumors by day 20. Treatmentof larger tumors (15 and 146 mm3) resulted in no completeremissions.

Studies demonstrating feasibility of targeted a-therapy using212Pb-labeled trastuzumab to treat disseminated peritoneal diseasehave been reported.68 A pilot RIT experiment treating mice bearing5 d LS-174T (i.p.) xenografts determined a maximum tolerateddose (MTD) of 0.74–1.48 MBq (20–40 lCi). The median survivalof animals receiving 0.37 MBq (10 lCi) increased from 19 to56 days (p = 0.008). A multi-dosing regimen of 212Pb-trastuzumabincreased median survival of mice bearing 3 d LS-174T i.p.xenografts to 110 days using up to 3 monthly doses of 212Pb-trastuzumab.

Combination of gemcitabine as a radiosensitizing agent with212Pb-trastuzumab has been recently evaluated for treating dis-seminated peritoneal disease.69 A pilot study treated mice bearingi.p. LS-174T xenografts with gemcitabine (GEM) followed 24–30 hlater by either 0.19 or 0.37 MBq (5 or 10 lCi) of 212Pb-trastuzumab.Improved survival resulted with both doses of 212Pb-trastuzumab;31 to 51 d in the absence or presence of GEM with 0.19 MBq(5 lCi) of 212Pb-trastuzumab, and 45 to 70 d at the 0.37 MBq(10 lCi) dose vs. 16 d for untreated animals. A second studyexamined multiple doses of GEM combined with a single 212Pb-trastuzumab dose. One group of animals received a total of threedoses of GEM, one before and two weekly doses following 212Pb-trastuzumab. Median survival was 63 d whereas those receiving thesingle GEM dose before RIT experienced a median survival of 54d. This finding was further extended to one vs. two treatment cycleswherein a cycle consisted of sequential treatment with GEM, 212Pb-RIT, followed by one or two additional weekly doses of GEM. Thegreatest benefit, a median survival of 196.5 d, was achieved bytreating with two cycles of 212Pb-trastuzumab with two doses ofGEM.

213Bi

Kennel et al. investigated targeting blood vessels in lung tumors asa therapeutic approach with 213Bi labeled mAb’s 201B and 34A ina murine model with lung tumors of EMT-6 mammary carcinomaand IC-12 tracheal carcinoma.70 Animals with tumor cross-sections of 5–10 cell diameters (50–400 cells tumor−1) achieveda 100% cure level at a dose of 5.6–7.4 MBq (150–200 lCi), but thecure rate dropped markedly with larger tumors. Cured animalssacrificed after 73–75 days due to respiratory distress revealedsignificant lung damage, including fibrosis and edema.

Engineered mAb Hu-CC49DCH2 radiolabeled with 213Bi wasevaluated for efficacy treating mice with s.c. LS174T tumors(83.8 ± 31.5 cm3).71 Doses as high as 37 MBq (1.0 mCi) per animalwere administered i.p. with all of the animals exhibiting a tumorgrowth arrest and ∼50% of the animals being cured. Lower dosesof 18.5–27.8 MBq (500–750 lCi) provided positive responses with∼33% being cured, 33% responding with delayed tumor growthand ∼33% not responding. An MTD was not reached, however theresults for treating a solid tumor were very encouraging and mayalso indicate that contrary to accepted opinion that solid tumorsmay in fact be successfully treated with a-emitters, and that thelimitations to using 213Bi may in part lie within the properties ofdelivery vector.

The Allen group in Australia has performed several pre-clinicalstudies.72–75 Single and multiple dose toxicity and efficacy of 213Bilabeled plasminogen activator inhibitor type 2 (PAI2) in regressingprostate cancer model has been reported. Tumor growth wasinhibited with single doses of 947 or 1421 MBq kg−1 (25.6–38.4mCi kg−1), complete tumor growth inhibition was achieved ata total dose of 947 MBq kg−1 (25.6 mCi kg−1) given on fivesuccessive days.72 In a related study a single local or i. p. injectionof 213Bi-PAI2 completely regressed tumor growth and lymph nodemetastases. Control animals and one of five mice treated with 111MBq kg−1 (3 mCi kg−1) 213Bi-PAI2 developed metastases in thelymph nodes while no lymphatic spread of cancer was found inthe 222 MBq kg−1 (6 mCi kg−1) treated groups at 2 days and 2weeks post-cell inoculation.74

Anti-colorectal cancer mAb c30.6 radiolabeled with 213Bi wasfound to exhibit high tumor uptake and retention of the radiolabel,potentially offering a new approach for control of colorectalcancer.74

The 213Bi-PAI2 has also been evaluated for control of mi-crometastatic breast cancer treating a 2-day post-inoculation ofMDA-MB-231 breast cancer cells. A single local injection of 213Bi-PAI2 completely inhibited tumor growth while a single systemic(i.p.) administration resulted in dose-dependent tumor growthinhibition with up to 3.7 MBq (100 lCi) of 213Bi-PAI2 being welltolerated.75

Locoregional RIT of dissemination gastric cancer using 213Bigave good therapeutic results with a single administration thatwas dependent on the time interval between tumor inoculationand therapy.76 Single versus double i.p. injections of mAb d9MAblabeled with 213Bi were evaluated for therapeutic efficacy andtoxicity. Two applications of 0.37 MBq (10 lCi) of 213Bi-d9MAbat days 1 and 8 after tumor inoculation significantly prolongedmedian survival vs. a single injection.

The mAb, J591 radiolabeled with 213Bi was evaluated forefficacy in a murine model using LNCaP tumors.77 Cytotoxicity

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experiments achieved significantly improved median tumor-freesurvival (54 days) relative to the control (33 days), or no treatment(31 days). Reduction of PSA levels also correlated to tumorresponse.

Milenic et al. evaluated 213Bi-trastuzumab for treatment of dis-seminated peritoneal disease.78 A dose of 500 lCi was determinedto be the effective operating dose for future experiments based onchanges in animal weight treating 3 day LS-174T i.p. xenografts.Median survival increased from 19 days to 43 days and 59 dayswith 18.5 and 27.8 MBq (500 and 750 lCi), respectively. RITtargeting HER2 was proposed to be potentially beneficial even forthose patients currently ineligible for immunotherapy due to lowantigen expression.

Allogeneic marrow transplantation using myeloablative radia-tion preparative regimens causes significant toxicity despite beingpotentially curative for several malignancies. To reduce toxicityassociated with non-myeloablative regimens, 213Bi-labeled mAbstargeting CD45 and TCRaß were evaluated for immunosup-pression prior to marrow transplantation in a canine model.79,80

Administration of either radiolabeled mAb prior to transplan-tation combined with immunosuppressive agents resulted inengraftment of transplanted marrow and stable mixed chimerism.Toxicities included transient myelosuppression and liver enzymeabnormalities. Use of 213Bi radiolabeled mAbs for this applicationin humans however may be limited by estimated requirements of� 74 MBq kg−1 [2 mCi kg−1] doses.

225Ac

225Ac has been investigated for targeted a-therapy in attempts toharness the cascade of 4 a-emissions in the decay chain (Fig. 5)to provide a much greater radiation dose while also administeringfar less radionuclide. Increased potency, and hence impetus forthis investigation, lies within the long half-life and also that 225Acconjugates may act as in vivo generators. Both direct and indirectradiolabeling methods have been investigated and neither hasactually proven to be optimal for efficiently forming the requisitecomplex.38,40 Issues surrounding stability of the 225Ac complexremain debatable and, of far greater concern, is the control andfate of the decay product daughter radionuclides as the chelationchemistry cannot be expected to survive the decay event or evenremain suitable for the daughter elements.

The bifunctional derivative HEHA was evaluated for seques-tering 225Ac for targeted a-therapy (Fig. 8).40 HEHA conjugatedto mAb 201B determined that 225Ac was delivered to the lungefficiently, slowly released with an initial t1/2 = 49 h, andaccumulated in the liver. The decay daughters of 225Ac werealso released from the lung; levels of 213Bi, the third alpha-decaydaughter were deficient in the lungs and in excess in the kidney.Injected doses of 225Ac-201B of 0.037 MBq (1.0 lCi), deliveringa minimum calculated absorbed dose of ∼6 Gy to the lungs, waseffective in killing lung tumors, but also proved acutely radiotoxic.Animals treated with � 0.037 MBq (1.0 lCi) of 225Ac-201B dieddose dependently of a wasting syndrome within days. These studiesconcluded that 225Ac RIT was compromised not only by unstablechelation chemistry, but critically so by radiotoxicity associatedwith trafficking of the decay daughter radioisotopes.

The Sloan-Kettering group found that DOTA derivatives couldbe used by forming the 225Ac complex first followed by conjugation

to mAbs.38 The in vivo efficacy of 225Ac-J591 was evaluated in anintramuscular LNCaP tumor model. A single administration of225Ac-J591 12 days post-inoculation with tumor caused tumorregression and significantly improved (P < 0.0001) mediansurvival to 158 days compared to the mice treated on day 15(63 days) compared to controls (33 days). The mice survived atleast 10 months without measurable PSA levels or evidence oftumor at the time of death (293 days). Mice bearing a disseminatedhuman Daudi lymphoma were treated using 225Ac-B4 1 day post-tumor dissemination showed a dose dependent response in mediansurvival times: 165 days (6.3 kBq), 137 days (4.3 kBq), and 99 days(2.1 kBq) with the latter dose being significant versus controls (p =0.05). Approximately, 40% of mice treated with the highest dosewere tumor-free at 300 days.

An interesting target for targeted a-therapy is neovascularendothelium. E4G10, a mAb that targets vascular endothelialcadherin, was radiolabeled with 225Ac and evaluated in a modelof prostatic carcinoma.81 Treatment with 225Ac-E4G10 resulted intumor growth inhibition, decreased serum prostate specific antigenlevel, and markedly prolonged survival, which was enhancedby administration of paclitaxel. Immunohistochemistry revealeddecreased vessel density and enhanced apoptosis. Additionally,residual tumor vasculature appeared normalized without toxicitybeing observed in vascularized normal organs.

A major impediment to clinical use of 225Ac has been concernregarding radiotoxicity of systemically released daughter radionu-clides, particularly 213Bi which accumulates in the kidneys.82 Oralmetal chelation with 2,3-dimercapto-1-propanesulfonic acid ormeso-2,3-dimercaptosuccinic acid significantly reduced renal 213Biuptake; however, DMPS was more effective than DMSA. Theseresults were also confirmed in a non-human primate model.Renal 213Bi and 221Fr activities were significantly reduced bydiuretics, furosemide and chlorothiazide. Impact on renal 213Biactivity was further enhanced by combining DMPS with eitherchlorothiazide or furosemide (P < 0.0001). Lastly, liposomeshave been investigated for retention of both 225Ac along with thedaughter radionuclides formed.83 While 225Ac could be sequesteredin liposomes, retention of the daughter radionuclides was asize dependent phenomenon and was the basis for assemblyof liposomes of suitable size and complexity to perform tar-geted a-therapy via immunoliposomes. Based on this set ofpre-clinical data, a Phase I trial investigating the use of 225Ac-HuM195 for advanced myeloid leukemias has been initiated atSloan-Kettering.

Targeted a-therapy versus targeted b-therapy

Few studies directly compare the efficacy of a-emitters and b-emitters in vivo. Greater efficacy in inhibiting tumor growth andimproved survival rates have been demonstrated using a-emitters.A 213Bi-labeled Fab′ fragment of mAb CO17–1A has preventedgrowth of a human colon cancer xenograft while increasingsurvival compared to the 90Y-labeled Fab′.84

Pre-targeted RIT was developed to reduce the radiation dose tonormal organs while improving tumor-to-normal organ ratios.One pre-targeting strategy involves administering a mAb ortargeting vector conjugated to streptavidin, followed by ad-ministration of a biotinylated N-acetylgalactosamine-containing“clearing agent” to remove excess circulating antibody. Thereafter,

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radiolabeled biotin is infused to bind to the “pre-targeted” strepta-vidin at the tumor, leaving the unbound radiolabeled biotin to berapidly excreted.85 This strategy was used to treat disseminatedCD25+ adult T-cell leukemia DOTA-biotin radiolabeled witheither 213Bi or 90Y.86 Treatment with 213Bi reduced the levels of thesurrogate tumor markers b2lG and soluble CD25, and improvedsurvival while treatment with 90Y failed to improve survival andresulted in significant toxicity. This same strategy was extendedto a study of the anti-CD25 single-chain Fv-streptavidin fusionprotein followed by radiolabeled biotin achieving similar resultswith 7 out of 10 mice cured.87

Clinical studies211At

Based on pre-clinical results at Duke University, a Phase Idose-escalation trial of 211At-81C6 was initiated in patients withmalignant gliomas post-surgical resection of their tumor.88 Twelvepatients have been treated to date. Imaging (c-camera) showed99% of the 211At decays occurred within the tumor cavity,indicating high in vivo stability of the radioimmunoconjugate inthis intracavitary administration approach. Early results suggestthat 211At-81C6 as an adjuvant therapy prolongs survival in thesepatients.

213Bi

The 213Bi-labeled humanized anti-CD33 monoclonal antibody,HuM195, was translated to a landmark clinical trial at MemorialSloan-Kettering Cancer Center.89 Eighteen patients with advancedmyeloid leukemia were treated in a Phase I dose-escalation trialand with myelosuppression in all patients along with transientminor liver function abnormalities. Doses of up to 37 MBq kg−1

(1 mCi kg−1) were safely administered. Uptake of 213Bi was demon-strated by c-camera imaging to be in the bone marrow, liver, andspleen, without significant uptake in other organs, and most im-portantly, absent from the kidney. Absorbed dose ratios betweenmarrow, liver, and spleen and the whole body were 1000 timesgreater with 213Bi-HuM195 than with previously evaluatedHuM195 radiolabeled with b−-emitters. Fourteen out of 18 pa-tients had a reduction in the percentage of bone marrow blasts aftertherapy. There were no complete remissions thereby demonstrat-ing the difficulty of targeting an adequate number of 213Bi atomsto each leukemic blast at the specific activities used in this trial.

A Phase I/II study followed wherein patients were first treatedwith chemotherapy to achieve partial cytoreduction of theleukemic burden followed by 213Bi-HuM195.90 Greater than 20patients with acute myeloid leukemia were treated with cytarabine(200 mg −2 d−1 for 5 d) followed by 213Bi-HuM195 at 4 doselevels (18.5–46.25 MBq kg−1 [0.5–1.25 mCi kg−1]). Prolongedmyelosuppression was dose limiting at the highest dose level.Complete responses, complete responses with incomplete plateletrecovery, and partial responses were achieved at the two highestdose levels. These preliminary results indicate that sequentialadministration of cytarabine and 213Bi-HuM195 can lead tocomplete remissions in patients with acute myeloid leukemia.These studies have recently been extended to a Phase I study using225Ac.

Alternative delivery methods and uses

Peptides, as opposed to mAb targeted a-therapy, have also beenrecently investigated to take advantage of both rapid targetingwith cellular internalization combined with rapid clearance phar-macokinetics.

A melanoma-targeting peptide, (DOTA)-Re(Arg11)CCMSH,was radiolabeled with 212Pb for biodistribution and therapy studiescarried out in a B16/F1 melanoma-bearing murine tumor model.Treatment with 1.85, 3.7 and 7.4 MBq (50, 100, and 200 lCi)of 212Pb[DOTA]-Re(Arg11)CCMSH extended mean survival to 22,28, and 49.8 days, respectively, as compared with a 14.6-day meansurvival of the controls; 45% that received 7.4 MBq (200 lCi)surviving disease-free.91

The somatostatin analogue [DOTA0, Tyr3]octreotide (DOTA-TOC) was labeled with 213Bi. Significant decreases in tumor growthrate were observed in rats treated with >11 MBq (300 lCi) of 213Bi-DOTATOC 10 days post-inoculation with tumor compared withcontrols (P < 0.025). Treatment with >20 MBq (540 lCi) resultedin greater tumor reduction.53

While RIT applications have overwhelmingly been oncologic innature, Dadachova and co-workers opened up a novel arena tostudy the use of microbe-specific mAb 18B7 which binds to capsu-lar polysaccharides of the human pathogenic fungus Cryptococcusneoformans.92,93 When radiolabeled with 213Bi, biofilm metabolicactivity was reduced to 50% while unlabeled 18B7, 213Bi-labelednon-specific MAbs, and c- and b-radiation failed to have an effect.Their results indicate targeted a-therapy to be a novel option forthe prevention or treatment of microbial biofilms on indwellingmedical devices. These researchers have also investigated targeteda-therapy for infectious diseases for several fungal and bacterialinfections.

Prospects and conclusions

The role of radiolabeled mAbs in the treatment of cancer remainsa burgeoning enterprise. The majority of RIT trials have beenperformed with b−-emitting isotopes and this currently remainsthe case. Contrasting to b−-emitters, the shorter range and higherLET a-particles allow for more efficient and selective killingof individual tumor cells. And, while some experimental pre-clinical models have demonstrated that targeted a-therapy mayhave a significant impact on large tumor burdens, the majorityof pre-clinical and clinical trials thereafter clearly pursue theaccepted paradigm that RIT with a-emitters are best suited forthe treatment of small-volume disease. While both pre-clinicaland early clinical studies appear promising, several obstaclesobstruct the pathway to widespread acceptance and use oftargeted a-therapy. To traverse these obstacles, issues pertainingto supply and economics need to be resolved by the creationof new sources and methods for production of these medicallyvaluable radionuclides. Current supplies of those radionuclidesassociated with the 225Ac and 224Ra decay pathways remain limitedto those sources of naturally isolated by-products from weaponsdevelopment within the United States. Additional sources of bothreside within Russia and other locations and within recovery ofnuclear fuel materials during their reprocessing. Both reactorand accelerator routes to 225Ac have been proposed although ameaningful product remains to be delivered. Besides production

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of these radionuclides, assembly and transport of generators ofsufficient activity amounts to support clinical trials have yet tobe produced; current clinical use requires multiple generatorslinked together. Lastly, the costs of these radionuclides has morethan tripled in the past decade for a variety of reasons seriouslychallenging the ability of many researchers from both remainingin or entering the field. Production of 211At is also hampered byproduction limits both in amounts and feasible locations due tocyclotron energy constraints.

Chelation and linking chemistry, while partially resolved forsome radionuclides as noted earlier, remains a challenge for themultiple decay pathway radionuclides. The practical chemicalchallenges of studying the coordination chemistry of Ac(III) andRa(II) directed towards the requirements for these applicationsplace such studies well beyond all but a few facilities where itis equally unclear that such studies might ever be performed.Opportunities remain within the area of conjugation chemistry.Despite having traversed many chelation challenges, more efficientconjugation and radiolabeling protocols remain to be developedto produce more consistent products with higher specific activitiesto optimize therapeutic potentials. Better pharmacokinetic anddosimetry modeling techniques are also required that actuallyaddress the cellular micro-dosimetry aspect of targeted a-emitterssince their targets exist at the individual cell.

Novel delivery methods must be developed and carefully studiedand in part this approach has been initiated (vide infra). Carefuland comparable pre-clinical (and clinical investigations) will berequired to define optimal radioisotopes, dosing regimens, andtherapeutic strategies. This latter aspect is of particular importanceas it is obvious that while single dose administration protocolsdominate clinical investigation, the clinical reality remains thatRIT and targeted a-therapy will have to become integrated intoclinical standards of care, combined with both external beamradiation therapy and chemotherapies to become a real, acceptedtherapeutic modality.

Acknowledgements

This research was supported by the Intramural Research Programof the NIH, National Cancer Institute, Center for Cancer Re-search. I would also thank Diane Milenic and Barbara Keller fortheir critical reading of the manuscript and Kwamean Baidoo forhis assistance.

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4928 | Dalton Trans., 2007, 4918–4928 This journal is © The Royal Society of Chemistry 2007

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