Molecular Pathways: Targeted a-Particle Radiation Therapy · 22.01.2013 · Molecular Pathways...

Molecular Pathways

Molecular Pathways: Targeted a-Particle Radiation Therapy

Kwamena E. Baidoo, Kwon Yong, and Martin W. Brechbiel

AbstractAn a-particle, a 4He nucleus, is exquisitely cytotoxic and indifferent to many limitations associated with

conventional chemo- and radiotherapy. The exquisite cytotoxicity ofa-radiation, the result of its highmeanenergy deposition [high linear energy transfer (LET)] and limited range in tissue, provides for a highly

controlled therapeutic modality that can be targeted to selected malignant cells [targeted a-therapy (TAT)]with minimal normal tissue effects. A burgeoning interest in the development of TAT is buoyed by the

increasing number of ongoing clinical trialsworldwide. The short path length rendersa-emitters suitable fortreatment and management of minimal disease such as micrometastases or residual tumor after surgical

debulking, hematologic cancers, infections, and compartmental cancers such as ovarian cancer or neoplastic

meningitis. Yet, despite decades of study of high LET radiation, the mechanistic pathways of the effects of

this modality remain not well defined. The modality is effectively presumed to follow a simple therapeutic

mechanism centered on catastrophic double-strand DNA breaks without full examination of the actual

molecular pathways and targets that are activated that directly affect cell survival or death. This Molecular

Pathways article provides an overview of themechanisms and pathways that are involved in the response to

and repair of TAT-induced DNA damage as currently understood. Finally, this article highlights the current

state of clinical translation of TAT as well as other high-LET radionuclide radiation therapy using a-emitterssuch as 225Ac, 211At, 213Bi, 212Pb, and 223Ra. Clin Cancer Res; 19(3); 1–8. �2012 AACR.

BackgroundAn a-particle is a naked 4He nucleus; therefore, it is

relatively heavier than other subatomic particles emittedfrom decaying radionuclides and nuclear reactions such aselectrons, neutrons, and protons. With a þ2 charge, a-par-ticles aremore effective ionization agents, have a high linearenergy transfer (LET), in the range of 100 KEV/mm, and arehighly efficient in depositing energy over a short range intissue (50–100 mm). An a-particle deposits �500 timesmore energy per unit path length than an electron orb�-particle. Unlike low-LET radiation (conventional x-,g-, and electron-like radiation), the cytocidal efficacy ofa-particle radiation is indifferent to dose fractionation, doserate, or hypoxia and also overcomes the resistance to che-motherapeutics encountered in conventional chemo- andradiotherapy. The a-emitting radionuclides that are medi-cally relevant and available for potential clinical useat this time are 211At, 212Bi, 213Bi, 225Ac, 223Ra, 212Pb, 227Th,and 149Tb.The use of a-particle radiation as a therapeutic modality

was recognized almost concurrently with the discovery of

particle radiation by Rutherford in 1898 from whichevolved the use of radium radionuclide brachytherapyapplications (1). Although there are several isotopes ofradium, 223Ra (Alpharadin) has recently moved to theforefront for clinical translation to treat bone metastases(vide infra; ref. 2), whereas 224Ra has had application in thetreatment of bone diseases such as ankylosing spondylitis(3). However, the targeting of radium radionuclide reliessolely upon the physicochemical nature of this element,which dictates the innate unaided biodistribution proper-ties of the radium ion and as such does not qualify as atargeted a-therapy (TAT). For TAT, a molecular target ischosen and the a-emission delivered to that chosen loca-tion and site. In fact, at this time, the necessary chemistry toconduct TAT with radium is not yet available (4).

A highly desirable goal in cancer therapy that has eludedclinicians is the ability to target malignant cells whilesparing normal cells. If significant differential targeting isachieved by the vector, then a toxic payload on the vectorwill deliver a lethal dose preferentially to those cells expres-sing higher concentrations of the target molecule, therebysparing nearby normal cells. TAT seeks to achieve this goalby using highly cytotoxic a-particle radiation carried tospecific sites of cancer by appropriate vectors. The shortpath length of the a-particle addresses the concern ofsparing normal tissue by limiting energy delivery, uponwhich cell killing depends within the cell where it is deliv-ered, and as indicated above, reverses resistance to chemo-therapy or conventional radiotherapy. The short pathlength also renders a-emitters suitable for treatment and

Authors' Affiliation: Radioimmune & Inorganic Chemistry Section, ROB,National Cancer Institute, NIH, Bethesda, Maryland

Corresponding Author: Martin W. Brechbiel, Radioimmune & InorganicChemistry Section, RadiationOncology Branch, NCI, NIH, 10 Center Drive,Building 10, Rm B3B69, Bethesda, MD 20892. Phone: 301-496-0591; Fax301-402-1923; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-0298

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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Research. on March 29, 2021. © 2012 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 10, 2012; DOI: 10.1158/1078-0432.CCR-12-0298

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management of patients with minimal disease such asmicrometastases or residual tumor after surgical debulking,hematologic cancers, infections, and cancers such as ovar-ian cancer or neoplastic meningitis that present as singlelayers or sheets of cells on compartment surfaces.

To make TAT possible, the development of monoclonalantibodies and other targeting vectors was required con-comitantly with the development of suitable conjugationchemistries that would securely sequester a-emitters suchas 225Ac, 211At, 213Bi, 212Pb (5–7). The physical limitationsabout what antibodies, peptides, or other targeting vectorsmight be labeled with an a-emitting radionuclide is onlylimited by their tolerance to the conjugation conditionsrequired for attaching chelating agents or other prostheticgroups (for sequestering the radionuclide) to the targetingvectors. It is necessary that the conjugation and labelingconditions lead to the retention of effective targeting prop-erties of the conjugate. As such, very few antibodies havebeen reported as difficult or impossible to radiolabel. It isimportant to choose chelating agents or prosthetic groupsthat bind the radionuclide strongly to limit dissociation ofthe radionuclide from the vector in vivo. However, the reallimitations of use reside in the actual applications of theradiolabeled product; for example, the optimal targetingtime profile versus radionuclide physical half-life can dic-tate choice of an a-emitting radionuclide or render it non-feasible. Thus, there is little point in treating a lesion thatrequires days to optimize antibody delivery with a radio-nuclide that has half-life measured in minutes. Matchinghalf-lives remains a significant criterion. Significant effortsabout the development of all of these have moved forwardto clinical investigation along with, in most cases, limitedinvestigations into the mechanisms of action.

Mechanisms of cell deathThe primary molecular target of ionizing radiation, and

specifically for high-LET a-particle radiation, has beenaccepted to be DNA (8). The seminal work of Soyland andHassfjell very clearly showed this wherein the physicalcellular path taken by an a-particle through cells definedcytotoxicity (9). Traversal through the cytoplasm failed tobe cytotoxic whereas traversal through the nucleus aswell asthe actual distance traveled through the nucleus was corre-lated to cytotoxicity. In addition, the high-LET effects werenot observed when cells were irradiated by b�-emissions orAuger electrons localized at the cell membrane or in thecytoplasm (10). An entire host of DNA damage can beexpected, including double-strand breaks (DSB), cross-link-ing, and complex chromosomal rearrangement (>3 breaksin >2 chromosomes) to which the high efficiency of celldeath may be attributed. The overall impact, however,exceedswhat canbe explained by ascribing the target simplyto DNA. Delayed toxicity attributable to increases in intra-cellular reactive oxygen species (ROS) as well asmitochron-drial involvement has been invoked to explain the extraeffects. Bystander effects in which DNA damage occurs incells adjacent to directly irradiated cells can also result fromextracellular ROS (11,12). Thus, complex multiple molec-

ular pathways are involved in the therapeutic application oftargeted a-particle radiation.

The therapeutic benefit of a-radiation is cell death as aresult of the high dose and damage to DNA that is incurred.Cell death is brought about through a number of mechan-isms such as apoptosis, autophagy, necrosis, and mitoticcatastrophe. To ensure the maintenance of the integrity ofthe genome, the cell is endowedwith amyriad of redundantDNA repair mechanisms. Failure of these systems fromcatastrophic cellular injury results in cell death. A summaryof many of the cellular responses and pathways thatare involved in cell death and repair after DNA DSBs isdepicted in Fig. 1, which outlines some of the participatinggenes and complexes. DNA damage is possibly sensed bythe ATM/ATR system, which activates downstream com-plexes such as p53, PARP DNA-PK, and PI3K to controlcellular responses that regulate cell proliferation, DNAreplication, checkpoints, recombination, and the repair andregulation of DNA damage (10). Another group of kinasesare involved in cell death, and this group includes MAPK8.Cell-cycle checkpoints are generally observed and are asso-ciated with arrests to permit the performance of repairthrough various mechanisms, such as homologous andnonhomologous end joining followed by progression orinitiation of the apoptotic process. The pathway taken isdependent upon the degree of damage; higher percentagesof unrejoined DSBs remain after high-LET radiation dueto the more complex nature of these breaks being moredifficult to repair. A recent authoritative review provides anin-depth assembly of information about DNA DSBs due toionizing radiation and coordination between cell-cycleprogression and the relevant repair mechanisms; however,the aspects of the pathways that are applicable to TATremain unclear at this time (13).

Investigation of TAT mechanisms in vitroA limited number of reports of mechanistic investiga-

tions into TAT provide specific detail of the involvementof the various repair proteins. Many of the investigationshave been conducted using in vitro cell culture systems.Petrich and colleagues described a TAT study of a 211At-labeled anti-CD33 monoclonal antibody (mAb; �1:1,000molecules labeled) that directly compared the same mAbconjugated with calicheamicin (�1:1 molecules labeled).At effectively the same protein concentration, an equivalentdegree of DNA DSBs resulted (14). Dilution of the toxinconjugate to the 1:1,000 activity, use of unlabeled controlmAb, or control "free" 211At, resulted in no DNA DSBs inHL-60 cells. The degree ofDSBs from the 211At-labeledmAbwas shown to be dose dependent. Induction of radioactiv-ity-dependent apoptosis related to caspase activation wasalso observed. Taken as a whole, this study shows theexquisite potency of antibody-based TAT that also over-comes the resistance that has been seen with calicheamicinconjugates. One might speculate that this relative degree ofeffectiveness will carry though when it is compared withother toxin or drug conjugates.

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Human lymphocytes irradiated by Na211At have beenstudied to assess the relative expression of the radiationresponsive genes by Turtoi and Schneeweiss (15). Genesthat were investigated for their response to the high-LETradiation included BBC3 (B-cell lymphoma 2–bindingcomponent 3), CD69 (cluster of differentiation 69),CDKN1A (cyclin-dependent kinase inhibitor 1A), DUSP8(dual specificity phosphatase 8), EGR1 (early growthresponse 1), EGR4 (early growth response 4), GADD45A(growth arrest and DNA damage-inducible, alpha),GRAP (growth factor receptor–bound protein 2–relatedadaptor protein), LAP1B (TOR1AIP1; torsin A interactingprotein 1), IFNG (IFN- g), ISG20L1 (IFN-stimulated exo-nuclease gene 20 kDa–like 1), c-JUN (jun oncogene),MDM2 (mouse double minute 2), PCNA (proliferating cellnuclear antigen), PLK2 (polo-like kinase 2), RND1 (rhofamily GTPase 1), TNFSF9 (TNF superfamily member 9),and TRAF4 (TNF receptor–associated factor 4). The objec-tive of the study was to evaluate the potential of the genes asmeasures of a-particle biodosimetry. Although it is not aTAT study per se, the list of studied genes provides anindicator of the response and repair, proliferation, andgrowth factors, as well as pro-apoptotic factors that couldbe involved in the response to a-irradiation. With theexception of GRAP, all were dose dependently upregulatedover various ranges of exposure; GRAP, linked to transmis-sion of extracellular stimuli for induction of proliferation,

differentiation, or apoptosis, was significantly downregu-lated independent of dose.

Several studies about the molecular mechanisms of 213Bi(212Bi) TAT have been reported. Macklis and colleaguesreported on the observation of the classic patterns of apo-ptosis, membrane blebbing, chromosomal condensation,and characteristic DNA fragmentation displays frommurine EL-4 lymphoma cells undergoing TAT with 212Bi(16). Supiot and colleagues reported on treating multiplemyeloma cells (LP1, RMI 8226, and U266) with a 213Bi-labeled anti-CD138 antibody in combination treatmentwith paclitaxel or doxorubicin (17). Interestingly, althoughpretreatment with either drug resulted in G2–M arrest, onlyone cell line showed an increase in DNA fragmentation(comet assay). No increase in apoptosis was observed in allof the studied cell lines. Although radiosensitization fromcombination therapy was noted, involvement of apoptosiswas ruled out as a mechanism for cell death. Seidl andcolleagues have executed far more extensive studies target-ing d9-E-cadherin with 213Bi-labeled d9mAb using humangastric cancer cells (HSC45-M2; ref. 18). These studiesshowed that cell killing was dose dependent Visible effectsof a-irradiation of HSC45-M2 cells were evident in theformation of micronuclei and severe chromosomal aberra-tions. However, cell death was not inhibited by z-VAD-fmkand thus was independent of caspase-3 activation, and themode of cell death was therefore concluded to be different

Figure 1. Mechanisms of celldeath by a-radiation. Irradiationof cancer cells by a-radiationproduces DSBs that evoke amyriad of cellular responses andpathways that include apoptosis,mitotic catastrophe, autophagy,necrosis, cell-cycle arrest, andDNA repair. Many genes andproteins are involved in thesepathways, some of which aredepicted here. When cell deathoccurs by autophagy, Becklin,LC3, ATG1, ATG5, and ATG7 areinvolved. CDK1 and cyclin B areinvolved in mitotic catastrophe,whereas RIPK1, TRAF2, PARP,and calpains are involved innecrosis. Associated with theATM/ATR and Ku/DNA-PKcscomplexes are a host ofdownstream systems that resultin cell-cycle arrest, apoptosis, orDNA repair by nonhomologousend joining or homologous repair.

Becklin, LC3,ATG1, ATG5,

ATG7

CDK1,cyclin B

RIPK1, TRAF2,PARP, calpains

DSB

ATM/ATRKu/

DNA-Pkcs

NecrosisNHEJHRRCHK1/CHK2

GADD45

Mitoticcatastrophe

Autophagy

Apoptosis

G2 phase

Cell death

M phase

DNA repair

Cell-cycle arrest

TP73

α-Irradiation

© 2012 American Association for Cancer Research

Targeted a-Particle Radiation Therapy Mechanisms

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from apoptosis. Seidl and colleagues also conducted geneexpression profiling and a time course microarray for thewhole genome (19). Of the 682 to 1,125 genes that showedupregulation and 666 to 1,278 genes that showed down-regulation at one time point each, 8 genes appeared to beupregulated and 12 genes were downregulated throughoutthe course of study. Of those that were upregulated,COL4A2, NEDD9, and C3 had not been previously foundto be linked to high-LET radiation response; complemen-tarily, this observation held for the downregulated WWP2,RFX3, HIST4H4, and JADE1. This discovery process alsoyielded genes that were not previously associated with anybiologic process or molecular function; ITM2C, FLJ11000,and MSMB were consistently upregulated, whereas HCG9,GAS2L3, and FLJ21439 were complementarily downregu-lated. Such findings bring to light additional new targetsthat might be involved in the selective eradication of malig-nant cells and provide further insight into mechanisms andpathways of response to a-emitter–based therapies.

Investigation of TAT mechanisms in vivoEven fewer investigations of themechanisms of cell death

implicated in TAT have been conducted in vivo. The studiesof the mechanisms that apply in vivo are extremely impor-tant because they are more relevant to the actual tumorenvironment. However, these studies are exceedingly chal-lenging and expensive to conduct. Recent studies by Yongand colleagues related to 212Pb, an in vivo generator of 212Bi,targeted to HER2 by conjugation to trastuzumab is, to thebest of our knowledge, the first study to actually investigatethe in vivo tumor response at the cellular level. (20). In thisstudy, mice bearing human colon cancer LS-174T intraper-itoneal xenografts were treated with trastuzumab radiola-beled with 212Pb and compared with several controls.Significant apoptosis induction and DNA DSBs wereobserved after 24 hours. In addition, Rad51 protein expres-sion was found to be downregulated, indicating delayedDNA double-strand damage repair as compared with con-trols. The cell cycle was also affected, resulting in G2–Marrest, depression of the S-phase fraction, and depressedDNA synthesis that persisted beyond 120 hours whereasDNA synthesis appeared to recover in the control tumors by120 hours. The 212Pb TAT also delayed open chromatinstructure and expression of p21 until 72 hours, suggesting acorrelation between modification of chromatin structureand induction of p21. A second study from Yong andcolleagues examined the impact of TAT combination ther-apy wherein gemcitabine, a standard-of-care therapeuticfor pancreatic cancer and a well-defined radiosensitizer,was administered before the 212Pb TAT in the same animaltumor model system (21). The 212Pb TAT treatment againincreased the rate of apoptosis in S-phase–arrested tumors.In this instance, 212Pb TAT administered after pretreatmentwith gemcitabine abrogated G2–M arrest, which was asso-ciated with inhibition of Chk1 phosphorylation and incre-ased apoptosis. This combination therapy also resultedin reduced DNA synthesis, enhanced DNA double-strandbreaks, accumulation of unrepaired DNA, and with down-

regulation of Rad51, all correlating with a blockage in DNAdamage repair. Again, modification in the chromatin struc-ture of p21was indicated. Changes in the H3K4/H3K9 ratioindicated transcriptionally repressed chromatin states anddelayed open DNA structure as a result of the failure ofadequate p21 induction. Thus, the impact of catastrophicdouble-strand DNA destruction as a result of high-LETa-particle traversal of the nucleus included significant inter-ference with the homologous repair mechanism throughthe downregulation of Rad51, inhibition of Chk1 phos-phorylation, chromatin modification, apoptosis, and per-turbation of the cell cycle.

Clinical–Translational AdvancesA strong case can bemade for the use of TAT in the clinic.

With exquisite and effective targeting of DNA, the principalmolecular target, TAT could deliver better outcomes thanthe ongoing ravening horde of "molecular targeted" drugs.In cancer therapy, the simple facts are that �50% of ther-apies incorporate radiation as one of the more efficaciousforms of therapy (22, 23) and combination therapies out-perform single modalities (24). The response rates that canbe achieved with proper application of RIT are difficult toachieve otherwise and strongly suggest that TAT, whenapplied properly, could prove to be a significant therapeuticmodality to incorporate in the clinic (25). Many of theoverarching obstacles to clinical translation of TAT, how-ever, include high costs of the radionuclide, unresolvedchemistry, limited availability of the radionuclides, tradi-tional opposition to and fear of radioisotopes, and real ormere imagined perceptions as opposed to the use of more"traditional" drugs.

Appropriate use of TAT is defined by a combination of theradionuclidic properties, including actual emissions andhalf-life, the choice of targeting vector, scale of disease, andaccessibility of disease by the targeting vector such that thea-emitting radionuclide might be delivered within a real-istic time frame and targeted volume or disease presenta-tion. Thus, use of a-emitting radionuclides is envisioned asbeing exceptionally potent and appropriate for the treat-ment of small lesions andmetastases; locoregional or com-partmentalized diseases of similar presentation; and readilyaccessible diseases such as leukemia and lymphoma. Fur-thermore, because of the limited range of the a-particle,normal tissue toxicity is expected to be quite low when aTAT strategy is used. Finally, although it is generally accept-ed that there is no effective resistance to a-particle lethalityand no oxygen or hypoxia limitations to efficacy, makingsuch therapies extremely potent in the therapeutic arenas,Haro and colleagues provide a study on induced resistantclones of HL-60 cells to high-LET radiation (26). Althoughthis study did not concern TAT per se with the a-emissionoriginating from an 241Am source, it showed that it is pos-sible to have a population of tumor cells that might berefractory to TAT (26). Regardless of these attributes, a verylimited number of clinical trials have been executed to dateevaluating TAT (Table 1). However, there has been anincrease in this activity recently, particularly spurred by the

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progress associated with Alpharadin (vide infra). Theremainder of this discussion will focus on clinical achieve-ments and progress associated with each a-emittingradionuclide.The number of TAT clinical trials conducted with 211At

has been quite limited. In part, this is a direct consequenceof the limited number of production sites for this radionu-clide (6). Nonetheless, Zalutsky and colleagues investigatedthe feasibility and safety of this therapy in patients withrecurrent malignant brain tumors using a chimeric anti-body, ch81C6, that targets tenascin, a glycoprotein over-expressed in gliomas, as the vector for the first 211At TAT trial(27, 28). A total of 18 patients were treated with 211At-TATadministered into a surgically created resection cavity(SCRC). This compartmentalized therapy strategy resultedin no reported cases of dose-limiting toxicity, no toxicity ofgrade III or higher with 96.7% of the 211At decays being

retained within the SCRC. Results of the study were quiteencouraging. Eight of 14 patients with recurrent glioblas-toma multiforme survived for 12 months, 2 survived for 3years, and no patient required repeat surgery for radio-necrosis. These results show that this application with211At-TAT was attainable, safe, and associated with thera-peutic benefit for patients with recurrent central nervesystem tumors. There have been no follow-up studies as yet.

A second 211At-TAT clinical trial is ongoing at the Uni-versity of Gothenburg in Sweden. Andersson and col-leagues investigated the pharmacokinetics and dosimetryof 211At-TAT in a phase I study in patients with recurrentovarian carcinoma. In this trial, the delivery vehicle was a F(ab’)2 fragment of antibody MX35 which targets the sodi-um-dependent phosphate transport protein 2b (NaPi2b) inhuman cancer cells (29). To date, 9 patients have beeninfusedwith 211At-TAT via a peritoneal catheter to assess the

Table 1. Clinical trials using a-particle emitters

Trial Cancer type Radioimmunoconjugate Outcome Reference

Zalutsky and colleagues Glioblastoma 211At-ch81C6 18 patients treated; 14patients survived 12 mo

27, 28

Andersson and colleagues Ovarian cancer 211At-MX35-F(ab')2 9 patients treated; nosignificant toxicity

29

The Scheinberg group AML 225Ac-HuM195(225Ac-lintuzumab)

18 patients treated; trialexpanded to multicenterphase I/II

31, 32

Heeger and colleagues B-cell non-Hodgkinlymphoma

213Bi-labeled anti-CD19 andanti-CD20-CHX-A"-DTPA

9 patients treated;2 patients showedresponse; limited toxicityin 2 patients

33

The Allen group Melanoma 213Bi-mAb 9.2.27 22 patients treated; 6%CR;14% PR; 50% stabledisease

34

The Scheinberg group AML 213Bi-HuM195(213Bi-lintuzumab)

18 patients treated; 14patients had reductionsin marrow blasts

35

Jurcic and colleagues AML 213Bi-HuM195(213Bi-lintuzumab)

31 patients treated; marrowblast reductionsobserved at all doselevels

36

The Merlo group Glioblastoma 213Bi-substance P 5 patients treated; Barthelindex improved for 2patients

37, 38

Areva Med LLC Ovarian 212Pb-TCMC-trastuzumab

3 patients treated; studyongoing; no furtherinformation available

39

Parker and colleagues Castration-resistantprostate cancer andbone metastases

Alpharadin(223Ra chloride)

292 patients treated;median overall survivalincreased by 4.5 mocompared with placebogroup

40

NOTE: This is strictly speaking not a TAT trial per se but uses 223Ra2þ. Alpharadin is not an immunoconjugate but is included herebecause 223Ra is an a-emitter.Abbreviations: AML, advanced myeloid leukemia; CR, complete response; PR, partial response.






strategy of intracavitary administration. Results have shownthat 211At-TAT by intraperitoneal administration is feasibleand that therapeutic doses in microscopic tumor clusterscan be achieved without significant toxicity to the patient.

To date, only one clinical trial has used 225Ac. The target-ing vector for this trial is a humanized antibody, lintuzu-mab, that targets CD33 on acute myeloid leukemia (AML)cellswhichhadpreviously been investigated in clinical trialsfor RITwithb�-emitters and 213Bi TAT (30, 31) This trialwasbased on results of pharmacokinetics, dosimetry, and tox-icity obtained in cynomologus monkeys that indicated that225Ac TAT was feasible (30). The ongoing phase I clinicaltrial was initiated by the Scheinberg group at MemorialSloan-Kettering Cancer Center (New York, NY) with aprimary goal to define both safety and the maximumtolerated dose of 225Ac TAT in patients with advanced AMLthrough a dose-escalation series (31). The initial dose of 0.5mCi/kg, which is several orders ofmagnitude less than dosesroutinely used in RIT with b�-emitters, shows the extremepotency that this radionuclide delivers as a therapeutic.Eighteen patients with relapsed or refractory AML weretreated. The trial has been so successful in showing that225Ac TAT targeted by lintuzumab had antileukemic activityacross all dose levels that it is now being investigated in amulticenter phase I/II trial in combination with low-dosecytarabine for older patients with AML at Memorial Sloan-Kettering and the Fred Hutchinson Cancer Research Center(Seattle, WA). Additional centers are expected to open thestudy in the near future (32).

A somewhat larger number of TAT clinical trials havebeen initiated and executed with 213Bi, in part facilitated bythe availability of this radionuclide from an on-site gener-ator based on 225Ac. Heeger and colleagues at the GermanCancer Research Center in Heidelberg initiated a phase Idose-escalation trial to determine toxicity and feasibility aswell as dosimetry andpharmacokinetics. Nine patients withB-cell malignancies were treated with a 213Bi-labeled anti-CD20 antibody (33). Toxicity was limited to mild leuko-penia in 2 patients with 2 patients responding to thetherapy. This trial has been continued at the UniversityHospital D€usseldorf, in D€usseldorf, Germany.

The Allen group initiated a phase I dose-escalation 213BiTAT study for metastatic melanoma using mAb 9.2.27 totarget the core protein of chondroitin sulfate proteoglycanof cancer cells. A total of 22 patients with stage IV/in-transitmetastasis were treated (34). Patients showed disease reduc-tion at 8 weeks based on the tumor marker melanoma-inhibitory protein activity; 6% showed complete response,14% showed partial response, 50% stable disease, and 30%progressive disease, with no toxicity being registered duringthe study.

The Scheinberg group at Memorial Sloan-Kettering iscredited with the first proof-of-concept 213Bi TAT clinicaltrial again targeting CD33 with antibody HuM195 (lintu-zumab) to treat 18 patients with advanced myeloid leuke-mia in a phase I trial. Fourteen patients had reductions inthe percentage of bonemarrowblasts and had reductions incirculating blasts after therapy, all without detection of

significant toxicity (35). Rosenblat and colleagues con-ducted a follow-up study with 213Bi TAT wherein 13 newlydiagnosed patients and 18 patients with relapsed/refractoryAML were first treated with continuous cytarabine infusionfor 5 days (36). Myelosuppression was the primary toxicity,and 2 of 21 patients treated with the maximum tolerateddose died. At all dose levels, marrow blast reductions wereobserved and CD33 sites were found to be saturated by213Bi TAT lintuzumab.

The Merlo group conducted a pilot 213Bi TAT trial usingsubstance P, a tachykinin peptide neurotransmitter whichtargets the neurokinin type-1 receptor (NK-1) which isconsistently overexpressed in grade 2, 3, and 4 gliomas(37, 38). In this pilot study, 5 patients were enrolled, andtreatments were administered with an implanted cathetersystem (intratumoral injection) Four patients received 1therapeutic cycle, and 1 patient received 4 therapeuticcycles. Again, the 213Bi TAT agent was retained at the targetsite without local or systemic toxicity being observed. Pre-therapeutic functional scores (Barthel index) for the 2patients with glioblastoma multiforme were 75 and 80,which after TAT improved to 90 of 100. Radiation-inducednecrosis and demarcation of the tumors were detected byMRI (38).

The first phase I clinical trial using 212Pb TAT, sponsoredby Areva Med LLC, opened at the University of Alabama inBirmingham in 2011. As one might expect, this trial isdesigned to determine dose-limiting toxicities and antitu-mor efficacy for treating intraperitoneal cancers, specifically,primarily ovarian cancer. Adverse events and immune re-sponse monitoring, as well as assessment of efficacythrough physical examination, radiographic imaging, andassay of tumor markers, are being followed. Pharmacoki-netics and excretion mechanism(s) from the peritonealcavity are being determined by g-camera imaging. Although3 patients have completed treatment in the first cohort, nofurther information is available at this time (39).

As noted earlier, Alpharadin (223Ra chloride) has beenevaluated in 2 phase I trials and 3 double-blind phase IItrials for castration-resistant prostate cancer and bonemetastases and is moving onward into phase III trials. Of292 patients treated with 233Ra, less than 1% experiencedgrade 4 hematologic toxicity, 4% had grade 3 anemia, lessthan 3% presented with grade 3 toxicity for platelets, neu-trophils, or white blood cells, and mild reversible neutro-penia was observed with repeated 223Ra treatments (40).There was no indication of renal or hepatic toxicity. In onetrial, median overall survival increased by 4.5 months ascompared with the placebo group.

ConclusionsThe limited clinical experience of targeted a-particle

radiation therapy has shown the potential of themodality for the treatment of smaller tumor burdens,micrometastatic disease, and disseminated disease wherea-emitters may be efficiently delivered. The rational scien-tific matching of disease presentation with realistic acces-

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sibility and delivery based upon physical considerations is akey criterion to their success; however, development andgrowth of clinical TAT have been principally compromisedby economics and limited supply issues.The targeting of DNA inherent in this modality is highly

efficacious, but events beyond the traversal of a cell byan a-particle require more study. The actual mechanismsby which cells die are not totally well defined. Additionalin vivo studies of the molecular mechanisms of response,repair, and cell death resulting from TAT treatment areclearly needed. A host of additional genetic pathwaysappear to be activated in vivo that simply have not beenrecognized or studied until relatively recently (41). Thestudies need to be conducted in relevant in vivo tumormodels inwhich assays of treated tumor tissue for pathwaysof response are investigated rather than the less relevant cellculture that predominate current experimentation. Combi-nation therapy studies need tobe conducted under the sameconditions. These data are of critical importance to grasp areal understanding of TAT that, like any other therapeuticmodality, will no doubt enhance its use and integration

with other therapies. A full mechanistic understanding ofthe therapy will accelerate its development and clinicaltranslation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.E. Baidoo, M.W. BrechbielWriting, review, and/or revisionof themanuscript:K.E. Baidoo, K. Yong,M.W. BrechbielAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Yong

AcknowledgmentsThe authors thank Diane Milenic for assistance in assembling the

manuscript.

Grant SupportThis research was supported by the Intramural Research Program of the

NIH, National Cancer Institute, Center for Cancer Research.

ReceivedOctober 15, 2012; revisedNovember 18, 2012; acceptedNovem-ber 27, 2012; published OnlineFirst December 10, 2012.

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Baidoo et al.





Published OnlineFirst December 10, 2012.Clin Cancer Res Kwamena E. Baidoo, Kwon Yong and Martin W. Brechbiel

-Particle Radiation TherapyαMolecular Pathways: Targeted

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