Radioimmunotherapy of Cancer Using Monoclonal Antibodies to Target Radiotherapy

Current Pharmaceutical Design, 2000, 6, 1399-1418 1399

Radioimmunotherapy of Cancer: Using Monoclonal Antibodies to TargetRadiotherapy

Timothy M. Illidge* and Susannah Brock

From the School of Medicine, Cancer Sciences Division, Southampton University, Southampton SO16 6YD, UK

Abstract: After years of pre-clinical and clinical testing monoclonal antibodies (mAbs) finally offer newtherapeutic choices for patients with haematological and solid malignancies both as unconjugated antibodyand as vectors to target radionuclides in radioimmunotherapy (RIT). In recent years some of the mostexciting clinical data have come from the use of RIT in the treatment of lymphoma and haematologicalmalignancies and it would now appear highly likely that RIT will play a major role in the treatmentstrategies for these diseases. For the solid tumours there has also been considerable progress with RIT andmAbs have become a component of treatment protocols for breast cancer. This review highlights theimportant recent clinical progress that has been made with clinical RIT and provides some new insightsinto the important mechanisms of action of RIT in haematological malignancies.

INTRODUCTION effector mechanisms and their use as vectors for thedelivery of toxins, radionuclides and prodrugs.mAbs also have the potential advantage ofspecificity against both unique tumour-specific andtumour-associated antigens. Exquisite specificitieshave been identified in the idiotypic determinants onB lymphoma cells. For the tumour-associatedantigens these target antigens may not be tumourspecific, but rather normal cellular macromoleculesexpressed at increased density or in an atypicalcontext on the cancer cells. Specificity andmolecular architecture make mAbs attractive carriersfor radionuclides. In theory, antibodies with high invitro affinity should have a greater tumour uptakeand retention than lower affinity antibodies. Somepreclinical studies have indeed supported thishypothesis [2-5], however, in vitro binding affinitydoes not always correlate with in vivo activity [6].Unmodified mAbs facilitate the eradication ofantibody coated tumour cells by complementmediated lysis, phagocytosis or antibody-dependentcell mediated cytotoxicity (ADCC). ADCC andcomplement fixation depend primarily on the IgG1and IgG3 isotypes and antigen clustering alsoappears to be important for complement fixation.Genetically engineered chimeric monoclonalantibodies containing human Fc constant regionsand mouse variable regions have shown enhancedability to fix human complement and to induceADCC with less immunogenicity than murineantibodies. Non immune effector mechanism of

Monoclonal antibodies (mAbs) offer newtherapeutic choices for patients with haematologicaland solid malignancies both as unconjugatedantibody and as vectors to target radionuclides inradioimmunotherapy (RIT) [1]. After years of pre-clinical testing in recent years some of the mostexciting clinical data have come from the use of RITin the treatment of lymphoma and haematologicalmalignancies and it would now appear highly likelythat RIT will play a major role in treatmentstrategies for these diseases. Results in solidtumours are improving, however much progressremains to be made before RIT becomes acomponent of standard practice for commonmalignancies in the clinic. This article discusses thepotential and limitations of monoclonal antibody-based therapy and reviews the current clinical use ofRIT.

THE POTENTIAL OF ANTIBODIES

mAbs have the potential to trigger selectivetumour cell death via both their inherent immune

TMI is supported by grants from the Cancer Research Campaign, UK.

*Address correspondence to this author at the CRC Department ofMedical Oncology (Level F), Southampton General Hospital, SouthamptonSO16 6YD, United Kingdom (Tel 44 (023) 8079 6184; FAX 44 (023) 80783839); Email: [email protected]

1381-6128/00 $19.00+.00 © 2000 Bentham Science Publishers Ltd.

1400 Current Pharmaceutical Design, 2000, Vol. 6, No. 14 Illidge and Brock

mAbs via antibody signalling may also play acritical role in cytotoxicity [7,8].

Ideally, a therapeutically useful mAb has a hightumour uptake with a rapid blood clearance withlittle normal tissue binding. A heterogeneousdistribution of tumour antigens, paucity andirregularity of the tumour vasculature and a highinterstitial pressure limiting antibody diffusionresult in heterogeneous antibody uptake with poortumour penetration. Preferential uptake to theperipheral regions of tumour nodules is frequentlyseen. The problems and advances in the dosimetryof RIT are well discussed by Humm [9].

A further potential advantage in using mAbs totarget radiotherapy, includes the “crossfire” effectof irradiation. This refers to the fact that not everytumour cells need to have been targeted to sustainirradiation induced damage. Thus, radionuclideemissions extend over several cell diameters (β-particle length of 1mm equates to around 20 celldiameters) and may impinge on antigen-negativecells through crossfire from surrounding antigen-positive tumour cells coated with radiolabelledantibodies. For the radioisotopes, which arecurrently in routine use, such as Iodine–131 andYttrium-90 the β-particles emitted have beenestimated by O’Donoghue to eliminate tumour cellswithin 1-5 mm of their deposition [9]. Antibodieslabelled with gamma emitters also have theadvantage that they can be readily used to imagetumours using standard radioscintigraphictechniques and estimates made of tumour andnormal tissue dosimetry.

In animal models using antibody fragments hasincreased the therapeutic ratio. Monovalentfragments, such as Fab’, show a morehomogeneous distribution and greater intratumouraldose when used for RIT than bivalent mAbs such asIgG and F(ab’)2

.[25]. Antibody fragments, forexample single chain Fv’s are less immunogenicand facilitate recombinant engineering and bacterialexpression. Although such antibody fragments lackFc arms and demonstrate improved tumourpenetration and increased tumour to normal tissueratios, the increased renal doses usually precludeincreasing the overall tumour dose withoutunacceptable renal toxicity [26]. Unfortunately noneof these techniques have yet translated intoimproved efficacy of RIT in clinical trials

Homogeneous distribution and retention of themAb within the tumour, coupled with rapid bloodclearance, have always been thought necessarypharmacokinetic features for effective RIT. Frompre-clinical studies many investigators haveconcluded that non-internalising surface membraneantigens are the best targets forradioimmunotherapy at least when using mAb’slabelled with 131I [10-12]. A number of investigatorshave demonstrated endocytosis of bound antibodyresults in metabolic degradation and loss ofradionuclide from targeted cells with antigens thatinternalise in vitro. Most successful RIT clinicaltrials to date have involved integral surface antigenssuch as CD20 [13-21] and MHC Class II [22,23],which are neither shed into the circulation norinternalised after antibody binding.

Murine antibodies are most widely used asmAbs and are frequently immunogenic, with humananti-mouse antibody (HAMA) being formed againstthe mAb and coupled molecules. More than 80% ofpatients with solid tumours will develop a HAMAresponse after a single injection or a murineantibody [27]. HAMA responses are less frequentlyseen however in lymphoma patients and were seenin just 7% of a heavily chemotherapy treated group.Secondary antibodies may result in rapidelimination of the antibody/conjugate and can beanti-isotypic (anti-murine) or anti-idiotypic. Suchimmunogenicity can be reduced by cleavage of theantibody to form fragments and by recombinantDNA techniques to form constructs such as mouse-human chimeras and minibodies. Meredith et al.have shown that after chimerization of mAb 17-1A,the patients’ HAMA responses are virtually nil[28]. However the incidence of anti-idiotypicHAMA does not appear to be reduced.

LIMITATIONS OF MONOCLONALANTIBODIES

The two major limitations to the use of mAbs intargeting radiotherapy are the low overall tumouruptake, relative to sustained levels in the blood andnormal tissues, and the immunogenicity of murinemAbs. Indeed, reported tumour to normal tissuedoses have varied enormously from between 3-50fold. Both of these factors may severely limit theradiation dose delivered and overall effectiveness.Tumour uptake with whole IgG’s is generally lessthan 0.001% of the injected dose/g of tumourtissue.

Attempts at reducing immunogenicity may allowfor repeated mAb dosing and for giving larger totalamounts of Ab, thereby increasing the effective half-life, with the possibility of increasing the overalltumour dose. Plasmaphoresis has also been shownto allow the repeated use of mAb after thedevelopment of a HAMA response [29].

Radioimmunotherapy of Cancer Current Pharmaceutical Design, 2000, Vol. 6, No. 14 1401

Immunosuppression using Cyclosporin [30] orDeoxysperualin [31] has also reduced the HAMAresponse.

THE RADIOISOTOPE

The selection of the radioisotope for theimmunoglobulin labeling depends on the purposeof immunoglobulin administration. For RIT thereare a number of potential therapeutic radionuclides,some of which are shown in Table 1 along withsome of their physical characteristics. No account istaken here of the availability or convenience.Despite this apparent choice there are relatively fewradioisotopes which are in routine use for thedelivery of RIT and their characteristics are shownin Table 2.

The targeting and pharmacokinetics ofradiolabelled mAbs may be affected by an excess ofantigen in easily accessible normal tissues. In theclinical RIT of lymphoma, this can be the spleen,which acts as an “antigen sink” [18]. Thebiodistribution is often “unfavourable”, with poortumour to normal tissue ratios in such patients whohave a large tumour burden and/ or splenomegaly[18]. Circulating antigens shed by the tumour cellsmay also interact with the mAb and altered Abclearance has been seen in patients with very highcirculating antigen levels [32]. However, clinical andpreclinical studies which have attempted to correlatetumour targeting and dosimetry with circulatingantigen levels have generally not shown any greatdifferences [33, 34,35].

Table 1.

Radionuclide OptimalDiameter (mm)

Optimal CureBounds (mm)

32P 22.0 18.0 – 30.0

67Cu 2.0 1.6 – 2.8Pre-infusion or co-infusion of unlabelled mAbprior to RIT has been shown to improve thedistribution of the radiolabelled mAb[13,16,17,36,37]. This type of approach where theunlabelled anti-tumour antibody binds to or pre-targets the tumour has become increasinglycommon practice. Non-specific uptake byreticuloendothelial cells via Fc receptor binding andbinding to normal B cells in the lymphoid tissue isthus reduced. After allowing sufficient time for thefree antibody to clear from the circulation theradionuclide is administered as a low molecularweight ligand which localises rapidly within thetumour. The unbound radionuclide is thus quicklyexcreted by the kidneys.

77As 5.0 3.6 – 6.0

90Y 34.0 28.0 – 42.0

105Rh 2.8 2.0 – 3.6

109Pd 7.0 6.0 – 9.0

111Ag 9.0 7.0 – 13.0

121Sn 1.6 1.0 – 2.0

131I 3.4 2.6 – 5.0

166Ho 21.0 18.0 – 25.0

177Lu 2.0 1.2 – 3.0

An alternative “pre-targeting” two-stage strategyinvolves pre-targeting with biotinylated antibodyfollowed by the administration of radiolabelledavidin, whereby antibody and the radionuclide caninteract via the biotin-avidin interaction [38].Multistep techniques giving three subsequentinjections may improve yet further on this technique[38]. This technique has been used against pan-carcinoma antigens and for the targeting of gliomas[38,39]. Bispecific antibodies have also beendeveloped and used in attempts to improve tumourtargeting. Using such a two step pretargetingapproach in nude mice bearing renal cell xenografts,Boerman demonstrated increased tumour : bloodratios, which were as high as 3500:1, 72 hours postinfusion [37]. Other approaches to improvetargeting have included improving tumour bloodflow or endothelial permeability by combining RITwith vasoactive drugs [41].

186Re 9.0 7.0 – 12.0

188Re 26.0 23.0 – 32.0

194Ir 28.0 24.0 – 34.0

199Au 0.8 0.4 – 1.2

Central estimates of optimal tumour diameters for curability derived from thebaseline parameter estimates together with the upper and lower bounds on optimaldiameter generated by varying the numerical parameters up to limits shown(modified from O’Donoghue [9]).

The objective is a durable, localised dosedistribution pattern in the target site with rapidelimination of the radioisotope from normal tissue[42]. The type of energy emitted (alpha, beta orgamma), the ionization path length and the half-life(both physical and biological) determine, not onlythe tumouricidal effect, but also the toxicologicalprofile. An understanding of the toxicology of these


agents is of major importance in determining thetherapeutic index.

sufficiently long to permit theradioimmunoconjugate to reach the tumour before itdecays and to let non-specific radioactivity wash outof normal tissue background. On the other hand, aRIS reagent should have a short half-life isotope inorder to limit radiation exposure of normal tissues.

For diagnosis, RadioimmunoglobulinScintigraphy (RIS) with photon emissions over 100KeV have a range in tissue that is sufficient to reachthe camera. Photon emissions over 250 KeV willdecrease spatial resolution because the amount ofmaterial needed to collimate emissions cannot beaccommodated in the arrays of pinhole cameras.The half-life of the radioisotope for RIS should be

Table 2 lists the most commonly utilisedisotopes for RIS. The half-life of technetium (Tc)(0.25 days) is too short for the labeling of intact

Table 2.

Radio i so topeHalf-Life

ParticleEnergy ( MeV )

PathLength

( MM ) ϕAdvantages Disadvantages

β- e m i t t e r s

Iodine-131

8.1 days 0.6 0.8Simple radiochemistry;

Gamma emissions allow imaging;

Inexpensive

Abundant gamma emissionsand of sufficient energy to

increase non-specificirradiation;

Dehalogenates in vivo

Yttrium-90 2.5 days 2.2 5.3 No gamma emissions;

High-energy Beta emissions

Requires chelate chemistry;leaches off chelate and

deposits in bone;

Cannot be used for imaging;

Requires surrogate (i.e.,Indium-111 ) for imaging

Rhenium-186 3.7 days 1.1 1.8 Both beta and Gamma emissions

Can be imaged

Leaches of chelate anddeposits in bone;

Availability

Rhenium-188 17.0 hr 2.1 4.4 Same as for Rhenium-186 Same as for Rhenium-186

Copper-67 61.5 hr 0.4-0.6 0.6Same as for Rhenium-186,

But does deposit In bone whenReleased from Chelate

Availability

α- e m i t t e r s

Bismuth-212

Astatine-211

1.0 hr 6.1

5.9

0.04-0.08

0.04-0.08

High energy

High energy

Very short half-life

Availability;

No imaging

Electron Capture

Iodine-125 60.1 days 7.5 0.001-0.02 High energy;

Simple Radiochemistry

Short path length

Requires deposition in or nearcell nucleus for cell killing

ϕ The path length is defined as the radius of the sphere within which 90% of the energy emitted by a radionuclide is absorbed (X90 ).


immunoglobulin (m.w. 150 000) for RISprocedures. Most of the activity will decay prior tothe arrival of the immunoglobulin to the tumour,which if estimated to be approximately 24 hours,would be four times greater than the half-life oftechnetium-99m. Iodine-123 has a similar problemand in addition is not as readily available astechnetium-99m. Both can however be utilised forlabeling smaller immunoglobulin fragments.Indium-111 is often the preferred isotope forlabeling of high-molecular weight wholeimmunoglobulin molecules.

cell transplant has allowed dose escalation to at least3 fold over haematopoietic tolerance. With bonemarrow replacement, hepatic and cardiopulmonarytoxicity become dose-limiting [18,19,20].Supportive measures such as adjuvant cytokinesand blood product transfusion enable minor doseescalation, but a substantial dose escalation does notappear to be possible without autologoustransplantation.

Fractionated RIT has also only resulted in asmall reduction in haematological toxicity, and hasnot allowed dose escalation to the same extent aswith autologous transplantation [21,22].Fractionation is often limited by HAMA responsesdeveloping against the therapeutic antibody, aspreviously discussed [21,22].

Animal studies have consistently indicated thatthe major dose-limiting organ for RIT is the bonemarrow [41]. With the advent of bone marrow andperipheral blood stem cell transplantation, it nowseems likely that cardiopulmonary, hepatic and renaltoxicity will assume far greater importance [20].Indeed, for the high dose Iodine-131 labeled anti-CD20 antibodies used in the treatment ofchemotherapy refractory NHL the dose limitingnormal tissue toxicity was found to becardiopulmonary [18,19]. The enormous influenceof normal tissue toxicity on the therapeutic indexshould ensure that considerations of efficacy andnormal tissue toxicity, rather than those ofconvenience or expense, dictate the choice ofradioisotope.

Myeloablative RIT using 131I-labelled antibodieshas been shown to frequently result in the latetoxicity of abnormal thyroid function tests. TheSeattle group found that 59% of patients treatedwith myeloablative doses of 131I labelled anti-CD20developed an elevated TSH, but currently there areno reported cases of clinical hypothyroidism [20].This finding is indicative of the inadequacy ofcurrent strategies to block the uptake of radioactiveIodine-131 by the thyroid.

Renal toxicity is a major concern when usingradiolabelled antibody fragments or peptideschelated with metals, due to increased renal uptakeand extended retention [44,45]. Doses greater than100 Gy are likely to result in acute renal failure,with acute tubular necrosis developing within 1 to 2weeks after treatment. Lower doses (60 - 100 Gy)can lead to chronic renal failure, with progressiveglomerular sclerosis and tubular atrophy occurringduring the months and years following treatment.Such renal toxicity may be overcome by theadministration of basic amino acids, such as lysine[45].

NORMAL TISSUE TOXICITY

Acute symptoms following RIT appearinfrequent and mild and are usually related to theadministration of antibody products. Suchsymptoms include fever, chills and hypotension.These adverse reactions are usually controlled bypre-medication with anti-histamines andParacetamol/Acetoaminophen and/or Cortico-steroids. It is rare for more severe allergic reactionsto occur and they respond quickly to steroids andepinephrine. A transient serum-sickness typephenomenon, including joint aches and fever, isseen in approximately one third of patientsfollowing intraperitoneal therapy about two weeksafter administration, but appears to be rare aftersystemic RIT [42].

DOSIMETRY

Dosimetry studies are routinely carried out priorto the therapeutic administration and involvefollowing the administration of trace labeled mAb.These studies allow for an estimation of theabsorbed dose to the tumour and to critical normaltissues from the subsequent therapeutic dose andcan estimate the level of for example myelotoxicity[13-16]. Such dosimetric studies enableidentification of those patients who have“favourable” biodistributions, whereby the dose totumour is predicted to be substantially greater than

For systemic and intraperitoneal RIT,myelotoxicity appears to be the dose limitingtoxicity. The extent and duration of themyelosuppression, as might be expected, dependson bone marrow reserve and is reduced by priorcytotoxic chemotherapy, total tumour burden as wellas the radioconjugate stability. The use ofautologous bone marrow or peripheral blood stem


that to normal tissues [18,19]. There is oftenconsiderable inter-patient variation in both thetumour and normal organ doses and an accurateestimate of the dose is difficult to obtain due toassumptions and inaccuracies inherent within themethodology and measurement of aninhomogeneous dose distribution. Dosimetry fornon-gamma emitting radionuclides is difficult, asattempts to image Bremsstrahlung electrons havebeen of limited success. Estimates are obtainedusing a gamma emitter with similar chemicalproperties to the therapeutic nuclide. For example,111 In labeled mAbs are used prior to 90Y therapy[17].

NON-MYELOABLATIVE THERAPY INLYMPHOMA

Initial trials using unlabeled mouse mAb weredisappointing and studies incorporating mousemonoclonal pan-B-Cell reactive antibody (anti-CD20 antibodies) showed only temporary andpartial responses [49]. Although tumour-specificanti-idiotypic monoclonal antibodies have produceddurable responses in 50-70% of patients withrelapsed non-Hodgkin’s lymphomas [50, 51]broader application of this technique has beenlimited by the logistical difficulties inherent increating custom anti-idiotypic antibodies forindividual patients. Chimeric anti- CD20 mAb(IDEC C2B8 - Rituximab) gained FDA approval in1997 and became the first mAb licensed for use asan anti-cancer agent [52]. It has become establishedas a safe and effective treatment and is being testedin several clinical trials with combinationchemotherapy [53]. Clinical studies havedemonstrated the persistence of antibody bound totumour cells two weeks after administration andonly minor side effects [54]. In the Phase II pivotaltrial using Rituximab as a single agent given as four375mg/m2 weekly infusions the overall responserate was 48 % with a complete response (CR) rateof only 6% [54].

Sequential gamma-camera imaging, plasma andurine measurements and occasionally tumour andbone marrow biopsies have all been used to estimatedose. The dose to the tumour and normal organs iscalculated knowing the quality of radiation, theequilibrium constant and the specific absorbedfraction to the target using the MIRD (MedicalInternal Radiation Dose) Committee guidelines.Imaging-based dosimetry appears to correlatereasonably well with biopsy measurement of activity[46]. Bone marrow dosimetry is based on theassumption that marrow activity is proportional tothat in the blood and the blood activity is evenlydistributed. Marrow dose is therefore estimatedusing serial blood measurements and the MIRDequations. The development of increasinglyadvanced computer programs will allow the dose tobe estimated to a greater number of organs,including those such as the lungs, bladder andkidney which do not accumulate sufficient antibodyto be detected by a gamma camera.

There have been several clinical trials of RITperformed in patients with chemotherapy refractoryB-cell lymphomas. Impressive responses have beenreported using a variety of antibodies, deliveryschedules, radioisotopes and doses of radioactivity(Table 3). Target antigens that have been utilisedinclude CD20 [13-21], CD21 [55], CD22 [56-58],CD37 [13,18, 59, 60], MHC class II allele HLA-DR10 [22,23,61], immunoglobulin idiotype [62].The main clinical criteria used in the selection ofantibodies were that the target antigen be wellexpressed on the tumour, but not on critical, non-renewable, normal tissue such as the nervoussystem.

CLINICAL RESULTS OF RADIO-IMMUNOTHERAPY

RIT has been studied in many types of humanmalignancies, usually as part of phase I / II studiesby intravenous administration but for localiseddisease regional administration may beadvantageous and intraperitoneal, intrathecal,intratumoural and rarely intraarterial have all beenused. Results from some studies using regional RITappear promising with increased tumour doses andlower systemic toxicity reported than with systemicRIT [47,48]. However these regional approaches arenot as well developed or the reported responses asimpressive as with RIT of haematologicalmalignancies and therefore this review will largelybe devoted to the treatment of lymphomas andleukaemias.

The first clinical results of RIT in B celllymphoma and Chronic Lymphocytic Leukaemiawere reported by DeNardo et al. [61]. Using 131I-labeled Lym-1 antibody directed against the HLA-DR variant antigen low dose fractionatedradiotherapy, between 30 and 60 mCi every 2 to 6weeks was delivered, until maximal tumourresponse, dose-limiting toxicity, development ofHuman anti-mouse antibody response (HAMA), orthe delivery of 300 mCi was reached. Ten out of 18patients responded with 2 complete responses(CR). The complete responses were seen aftermultiple administrations and more than 260mCi


Table 3. Non- Myeloablative Radioimmunotherapy Trials for B-Cell Lymphomas

Antibody(cumulative mg)

TargetAnt igen

Radionuclide(cumulative mCi)

No o fTreatments

Evaluablepat ients

R e s p o n s e sResponse rateCR + PR % ( )

References

B1 (15-1565) CD20 131I (38-161) 1 113 60 CR, 27 PR ,(77 %)

67

B1 (2-110) CD20 90Y (13.5-21.6) 1 4 1CR, 1PR(50 %)

17

Y2B8 (55-294) CD20 90Y (19.9-53.4) 1-2 14 5 CR, 6 PR(78 %)

17

Rituximab (100-250mg/m2) prior to

90Y- IDEC-Y2B8

CD20 90Y (0.2-0.4 mCi/kg) 1 51 13 CR, 21 PR,(67 %)

21

OKB7 (25) CD21 131I (90-200) 3-4 18 1PR, 12MR(6 %)

55

LL2 CD22 131I 1-7 12 2CR, 2PR, 2MR(33 %)

56

Humanised LL2 CD22 131I 1 10 1CR, 2PR, 1MR(33 %)

124

MB-1 (40) CD37 131I (25-161) 1 10 1CR, 2PR, 1MR(33 %)

60

Lym-1 HLA-DR10 131I (37-1044) 1-20 30 3 CR, 14 PR(57%)

22

Lym-1 (52-290) HLA-DR10 131I (102-809) 1-4 21 7 CR, 4 PR,(52 %)

23

Lym-1 (30-67) HLA-DR10 131I (26-1044) 1-16 57 11 CR, 20 PR

(54.3 %)

125

Lym-1 (135-288) HLA-DR10 67Cu (131-388) 1-4 12 1 CR, 6 PR(58%)

126

Anti-Idiotype (1000-4050)

Idiotype 131I (10-54) 1-4 9 2 CR, 1PR, 1MR(33 %)

127

(9.6GBq) of Iodine-131. The results were updatedin 1998 when DeNardo and co-workers reported on21 patients (6 low grade, 12 intermediate grade, 3high grade NHL), 14 of whom received more thanone dose of RIT [23]. The dose limiting toxicitywas grade 3 to 4 thrombocytopenia seen in 10patients and grade 3 to 4 neutropenia in 6 patients.The Maximum Tolerated Dose (MTD) was foundto be 100 mCi/m2 for each of the first two dosesgiven 4 weeks apart. Eleven responses were seenwith 4 PR’s and 7 CR’s. The mean responseduration appeared to depend upon the histology andwas 16.1 months (3-35.5) for low grade; 8.1months (2-24) for intermediate, and just 5 months(1.5-8.8) for high-grade disease.

Trials using the target antigens CD21 and CD22have been largely disappointing [55-58] and havetended to confirm, in vitro studies which suggestthat such antigens modulate and internalize, and aretherefore unlikely to be therapeutically useful, atleast with conjugated radioiodine [10-12].Goldenberg et al. reported a small study of 7patients treated with 131I-LL2 antibody (anti-CD22)given as an intact or F (ab’)2 protein [56]. Patientsreceived a dose of 30 mCi followed by a dose of 20mCi a week later. Two partial responses of aboutthree months duration were seen. A high proportionof the patients (5 out of the 7) experienced severemyelosuppression despite the small radiation doses.In 1993, Czuczman and associates reportedsimilarly low response rates using the 131I-OKB7


(anti-CD21) mAb. Eighteen patients received 90 to200 mCi in 30 to 50 mCi fractions spaced 3 daysapart in a dose escalation trial [55]. One patientachieved a PR and 12 a mixed or minor response.The median duration of response was 4 months.Myelosuppression was once again the dose-limitingfactor.

responses and two partial responses in 9 patients[13.] One of the complete remissions lasted 8months, while the other 3 were continuing 8 to 11months after RIT at the time of the report. Of notethese results were achieved with only one small RITdose and were accompanied by negligiblemyelosuppression, which permitted further doseescalation.

Immunoglobulin idiotype has also beeninvestigated as a potential target for RIT. Parker etal. (1990) reported one complete remission and twopartial responses lasting 2 to 12 months among sixpatients treated with 90Y-anti-idiotype antibodies[62]. Administration of unlabeled anti-idiotypeantibody before the infusion of radiolabeledantibody to clear circulating free idiotypeimmunoglobulin was found to improve tumourtargeting. Myelosuppression was very modest at thedose range given (10 to 54 mCi in one to fourcycles). There are additional difficulties ininterpreting results using radiolabeled anti-idiotypeowing to the well-documented efficacy of “cold”anti-idiotype antibodies [8, 63]. The seriouspractical limitations in customizing antibodies forindividual patients’ tumours according to theirparticular idiotype make this approach difficult toapply widely.

Thirty-four patients with relapsed low orintermediate grade NHL who had receivedtherapeutic doses of 131I-B1 were reported byKaminski et al. [14]. Preloading techniques wereagain used to optimize the radiolabelled antibodydistribution. To study the effect of preloading ondose distribution, the first 25 patients receivedintravenous doses of tracer antibody (5mCi [185MBq] on 20mg B1) on successive weeksimmediately preceded by an infusion of 35mg ofunlabeled anti-B1 one week and 450 mg the next.The unlabelled antibody dose resulting in theoptimal tracer biodistribution was also given beforethe radioimmunotherapeutic dose. A 450 mg doseof unlabeled antibody appeared best for thedosimetry, and subsequent patients received only asingle tracer dose and radioimmunotherapeutic dosepreceded by an infusion of unlabeled anti-B1. Doseescalation was based on the tracer predicted wholebody radiation dose and proceeded in 10 cGyincrements from 25 cGy through to 85 cGy. Themaximum tolerated whole body dose was 75cGy,with myelosuppression as the dose limiting toxicity.Twenty-eight patients receivedradioimmunotherapeutic doses of between 1.258GBq - 5.957 GBq (34 to 161 mCi), resulting incomplete remission in 14 patients and a partialresponse in 8. All 13 patients with low-gradelymphoma responded, and 10 achieved a completeremission. All patients with chemotherapy sensitivedisease responded. Of the eight patients withtransformed lymphoma and the 19 patients withchemo-resistant disease responses were seen in 6and 13 respectively. Complete remissions were seenin 5 patients with large tumour burdens. The medianduration of complete remission exceeded 16.5months and 6 patients remained in completeremission 16-31 months after treatment. Althoughthe great majority of the response occurred after theRIT two additional patients who received only tracerdoses had partial responses [14].

Kaminiski et al. and Press et al. in their initialphase I trials used 131I labeled-MB1 (anti-CD37)antibodies [13,18, 59, 60]. Kaminiski reported oneCR and one PR lasting 2 and 6 months respectivelyamong ten patients treated following successfultumour imaging with trace labeled anti-CD37. Fortyof trace labeled (3 to 7 mCi) anti-CD37 wasadministered followed by serial gamma cameraimaging. Dose escalation was based on tracerestimates of delivered whole-body dose.

The CD20 molecule is a B cell specific plasmamembrane protein, expressed on 90% of B celllymphomas and chronic lymphocytic leukaemias[64,65], is not expressed on haematopoietic stemcells and is not shed from the plasma membranebefore or after treatment [49]. In vitro studies havedemonstrated the advantages of targeting CD20 inRIT over a range of other potential B cell targets[10,11,12]. For these reasons it has been the focusfor much of the recent clinical work. Predosing withunlabelled antibody has been shown to improve thesubsequent delivery of radiolabelled anti-CD20mAb in a xenograft model [66]. At the American Society Clinical Oncology

(ASCO) meeting in 1998 an update of the overallclinical experience was presented. This included113 patients treated at two UK and 2 US centres, 88patients with low grade lymphoma and 25 withtransformed disease. The overall response rate was

The use of non-myeloablative doses of 131I-labeled anti-CD20 (B1) has been investigated byKaminiski et al, in Michigan. The initial study,published in 1993, reported four complete


77.0% with a CR rate of 45.1%. Thirty-fourpatients (67%) were in continuous completeremission with the longest duration >4 years [67].Later that year in December of 1998, Kaminski etal, presented the results from a phase III trial of131I-labeled B1 in patients with advanced (98%stage III/IV) stage chemo-refractory low gradeNHL at the American Society of Hematologymeeting [16]. Sixty patients (median age 60 years),who had all received at least two chemotherapyregimens (median 4) and had either no response ora response duration of less than 6 months to themost recent, received the non myeloablative regimenof 131I-labeled anti-B1 described above. A CR wasseen in 17% and a PR in 65%. The results comparevery favorably to the response rates seen usingchemotherapy in the same patients, where responsesto the most recent chemotherapy regimen were 3%CR and 25% PR. This data formed the basis of theFDA license submission in 1999.

PCR negative from the blood or bone marrow [68].It is therefore difficult to be certain of thesignificance of the PCR results. It appears that mAbmay effectively clear “compartments”, such asblood and bone marrow, without necessarilyproducing a complete resolution at all sites ofinvolvement.

Yttrium-90 labelled murine anti-CD20monoclonal antibody has been used in Stanfordagainst relapsed low or intermediate grade B-celllymphoma [17] as part of a phase I/II doseescalation study. Once again, unlabelled antibodygiven prior to the therapy improved thebiodistribution. The dose was escalated from 13.5-50 mCi (0.5- 1.85 GBq) 90Y-anti-CD20 mAb with 3or 4 patients being treated at each dose level. Threepatients were retreated at the 40 mCi dose level. Thefirst four patients were imaged and then treated withthe B1 antibody (IgG2a anti-CD20), the remaining14 patients received the murine IDEC-Y2B8 (IgG1anti-CD20) antibody. The overall response ratefollowing a single dose of 90Y-anti-CD20 mAbtherapy was 72%, with 6 complete responses and 7partial responses and freedom from progression of3-29+ months. The only significant toxicity wasmyelosuppression.

Given these impressive response rates to a singletreatment with 131I-labeled murine anti-CD20antibody and the incurability of advanced low gradelymphoma with conventional therapy, the use ofRIT has been extended to first-line treatment.131I-anti-B1 therapy has used in previously untreatedpatients and the results of the first phase II wereupdated at ASCO 1998 with data available from 21evaluable patients. A dosimetric dose of 35 mgantibody radiolabeled with 5 mCi (185 MBq) of 131Iwas followed one week later with a quantity of 131Ithat delivered 75 cGy to the whole body based onprevious clearance of whole-body radioactivity.Both doses were preceded by a 450 mg infusion ofunlabelled anti-B1. The response rate (CR + PR)was 100 % and 15 of 21 patients (71%) achievedcomplete response, including 8 of 13 (62%) withbulky disease. The median duration of response forpatients with complete responses had not beenreached (range 3-17 months). All 7 patients whodeveloped progressive disease had high tumourburdens initially. Using PCR the presence of thet(14;18) translocation in blood and bone marrowwas determined prospectively in all patients beforeand again after therapy. Among twelve patients withpositive PCR results in the blood at baseline and inwhom peripheral counts had recovered, 9 wereclinically progression free, 7 in complete remission,and 4 have been persistently PCR negative from 6to 15 + months. The 3 patients with progressivedisease never achieved PCR negative status [15].The difficulty in interpreting these results iscompounded by early clinical data in patients treatedwith Rituximab who have been found to haveresidual nodal disease even when

Recent data have confirmed the efficacy ofYttrium-90 chelated ibritumomab tiuxetan (IDEC-Y2B8) [21]. A multicentre phase I/II trial wasconducted to compare two doses of unlabeledrituximab given before radiolabeled antibody, todetermine the maximum-tolerated single dose ofIDEC-Y2B8 that could be administered withoutstem-cell support, and to evaluate safety andefficacy. Eligible patients had relapsed or refractory(two prior regimens or anthracycline if low-gradedisease) CD20(+) B-cell low-grade, intermediate-grade, or mantle-cell non-Hodgkin's lymphoma(NHL). There was no limit on bulky disease, and59% had at least one mass greater than or equal to 5cm. The maximum-tolerated dose was found to be0.4 mCi/kg IDEC-Y2B8. The overall response ratefor the intent-to-treat population (n = 51) was 67%with 26% achieving a CR and 41% a PR.Responses occurred in patients with bulky disease,defined as greater than or equal to 7 cm (41%) andsplenomegaly (50%). Kaplan-Meier estimate oftime to disease progression in responders andduration of response is 12.9+ months and 11.7+months, respectively. Adverse events were primarilyhaematologic and correlated with baseline extent ofmarrow involvement with NHL and baseline plateletcount. One patient (2%) developed HAMA and aphase III study is currently ongoing.


MYELOABLATIVE RADIOIMMUNO-THERAPY IN NHL

of tumour) in one. Ten of the 12 patients treatedwith anti-B1 went into complete remission, 7 ofwhich remained in remission at the time ofpublication (3-36 months). All patients treated at thetwo highest dose levels had marked asthenia,nausea, diarrhoea and anorexia. Diffuse interstitialpneumonitis and congestive cardiomyopathyoccurred in one patient and severe hypotensionrequiring vasopressors in another, with both ofthese patients recovering.

This approach has the theoretical advantage thathigher doses of radiation may allow maximal andpossibly curative therapy from targeted mAbtherapy [69]. The published results are shown inTable 4. On first inspection response rates and theduration of response would appear to be superior tothose seen with most of the non-myeloablative RITregimens described. However direct comparison isdifficult as the studies are small, non-randomizedand involve a highly selected patient population.

A favourable distribution was more likely inpatients with small volume disease, (tumour burdensless than 500 mls) and without massivesplenomegaly, than in those who had grosslyenlarged spleens and large tumour burdens. Forpatients with a favourable biodistribution the CRrate was 84% with a PR rate of 11%. Theseremissions appeared durable and the medianduration of response exceeded 11 months at thetime of publication. Finally, the anti-CD20 antibody(B1) was considered to be superior to the anti-CD37 (MB-1) because favourable biodistributionwas achieved with 2.5mg/Kg as compared with10mg/Kg for the anti-CD37.

Press and colleagues in the Seattle group havepioneered the myeloablative approach,demonstrating the feasibility of using high doses of131I- labeled anti-B cell antibodies with eitherautologous bone marrow (ABMT) or peripheralblood stem cell transplantation (PBSCT). Theyinitially assessed the biodistribution, toxicity andefficacy of high doses of radiolabeled anti-CD20mAb’s (anti-B1). Sequential biodistribution studieswere performed with escalating doses of antibody(0.5-10 mg per kilogram of body weight) tracelabeled with 185-370 MBq (5-10 mCi) of 131I. Thedoses of radiation absorbed by tumours and normalorgans were estimated by serial gamma cameraimaging and tumour biopsies. Patients consideredhaving “favourable biodistribution” by virtue of thefact that their tumours were estimated to receivegreater doses of radiation than the liver, lungs, orkidneys were eligible for therapeutic infusions of131I-labeled antibodies.

The subsequent phase II trial used 2.5mg/Kganti-B1 with a maximally tolerated dose of 27 Gy tonormal organs as estimated from the biodistributionstudies [19]. Twenty-five patients with relapsed Bcell NHL were recruited. The median age was 47(24-60). Two patients had diffuse large celllymphomas arising by transformation fromfollicular lymphomas, the other pathologies werefollicular small cell, mixed and follicular large cell.Tumour burden varied from 4 to 3610 ml (median176 ml). Fifteen patients had stage IV disease, 9patients had stage III disease, and one had stage IIdisease. Eleven had increased serum lactatedehydrogenase concentrations. All patients hadbeen previously heavily treated with a mean of threedifferent chemotherapy regimens.

The initial published study reported 19 patientswith a favourable distribution who receivedtreatment with either MB-1 (anti-CD37) or anti-B1(anti-CD20) [18]. Nineteen patients received an RITdose, 12 with anti-B1 (58 to 1168 mg) of between10.36 GBq-28.75 GBq (280-777 mCi) of 131I. Allbut one of the 12 patients treated with anti-B1 RITrequired marrow re-infusion. Overall, there were 16complete remissions, a partial response in 2 and aminor response (25 to 50 percent regression

All of the patients with a tumour volume of lessthan 500 mls (n= 22, 88% of the patients) had

Table 4. Myeloablative Radioimmunotherapy Trials for B-Cell Lymphomas

Antibody(cumulative mg)

Antigen target Radionuclide(cumulative mCi)

No. EvaluablePat ients

R e s p o n s e s Reference

Anti-Idiotype (1000) Idiotype 131I (232) 1 1CR 127

B1(58-1168) CD20 131I (280-785) 29 23 CR, 4PR 18-20

1F5 (274) CD20 131I (608) 1 1 18

MB-1 (275-970) CD37 131I (234-628) 6 6 CR 18


favourable biodistributions. None of the 3 patientspresenting with tumour burdens above 500 mls(1259-3610 mls) met this criterion until they hadreceived further chemotherapy and their tumourburdens were reduced (440-660 mls). Twenty-onepatients received therapeutic infusions consisting of2.5mg/Kg (153-288 mg) of B1 protein conjugatedto 12.765-29.045 GBq (median 19.906 GBq) of131I. One patient did not receive a therapeuticinfusion of 131I because of antibody responseagainst murine immunoglobulins, which appearedafter the trace-labeled infusion. Sixteen of the 21patients achieved complete remission; 2 partialremissions and one a minor response. Two patientsdid not show any response; one had transformedimmunoblastic diffuse lymphoma and died ofprogressive disease 1.5 months later, the other diedof sepsis.

evaluating clinical data with anti-CD20 mAb’s inNHL, for example, is that these mAb’s are clearlytherapeutic in their own right [49,52,54]. Themechanisms may include the recruitment of naturaleffectors, such as complement and ADCC, or directcytotoxic signalling via the CD20 antigen [7].Therefore in any RIT study it is difficult to establishhow much of the therapeutic effect is due toantibody effectors, antigen target specificity,targeted radiation or whole body irradiation.

Further work is required to define the roles ofthe radiation and the antibody specificity. Forexample, it might be expected that the higher dosesof radiation delivered to tumour (range 10-90 Gy)in the Seattle studies would be more effective thanthe lower doses (1.5 Gy-30 Gy) delivered in theMichigan study. However not all mAbs should beconsidered as inactive delivery vehicles andradiation dose response considered in isolationwithout the potential therapeutic effects of theantibody being considered. Some mAbs do appearto have little therapeutic effect in vivo and the Lym-1mAb is one such example [20]. It is however apotent means of delivering what has been describedas “systemic radiotherapy” [23]. With such mAbsdelivering “systemic radiotherapy” thefundamental radiobiological considerations revolvearound whether low doses of low dose-rateirradiation are more tumouricidal than equivalentdoses of high dose-rate external beam irradiation.This question has been addressed by Knox et al.[69] who found significantly increased efficacy ofRIT when compared with dose equivalent X-irradiation in a murine B cell lymphoma model.

All patients required bone marrow (n=19) orperipheral blood stem cell infusion (n=2). Recentlythe long-term follow-up of the 29 patients treated inPhase I and Phase II trials was reported [20].Twenty-four of the 29 patients are alive and 15progression-free after a median follow-up of 37months. At five years, overall survival andprogression-free survival are projected at 68% and42% respectively. None of the surviving patientshas objective impairment of performance status orcardiac function. Late toxicities have beenuncommon, except for elevation of the thyroidstimulating hormone level in 60% of the subjects[20].

Only the results from Kaminski et al. [13-16]and Knox et al. [17] and more recently Witzig et al.[21] using non–myeloablative doses of anti-CD20mAb’s compare favourably with the frequency,quality, and duration of disease responses seen inthe myeloablative studies described above. Theresults achieved by the Seattle group were howeverusing doses of radioactivity approximately seventimes those used by the Michigan group in the 131Istudy and approximately ten times those deliveredby the Stanford group in the 90Y-anti-CD20antibody study. Consequently as might be expectedthe toxicity profile is more severe. Practicalconsiderations of myelo-ablative therapy includeprolonged patient isolation, to limit both theradiation exposure to staff and the public, prolongedhospitalisation for the supportive management ofmyelosuppression opportunistic infections and theconsiderable extra cost.

There are several lines of evidence to suggest thatantibody effects are an important component ofsuccessful of RIT for lymphomas and that weshould not consider successful RIT as simply being“systemic irradiation”. Evidence of a major role forantibody activity may be inferred from theresponses that have been observed with a number ofdifferent naked anti-CD20 mAb’s [49,52,54]; andthe preliminary data from the randomised trial ofunlabeled B1 versus 131I-labelled B1 [71].Impressive responses have also been seen with“tracer” doses of radiolabeled anti-CD20antibodies even before the “therapeutic” doses ofRIT have been administered [14].

Recent results in syngenic B-cell lymphomaanimal models of RIT have helped to clarify the roleof antibody by demonstrating the relativecontribution of mAb and of targeted irradiation tothe therapeutic effects in vivo. An additivetherapeutic effect between some mAb’s and targeted

Despite the promising clinical results we remainlargely ignorant about the mechanisms operatingbehind these responses. One of the difficulties in


irradiation has been demonstrated [72]. In thismodel system treating animals with 131I-anti-Idiotype (Id) early in the disease gave only partialprotection. Intriguingly however when treating laterin the disease course, the therapeutic effect of RITwas much more pronounced, with >90% of animalsdisease-free beyond 350 days. This effect wasshown not to depend upon the dose of irradiationdelivered to the tumour but on host responses andantibody signalling effects [8,62]. The resultsparallel those seen with naked antibodies to Id andCD40, where a better therapeutic effect was seen inthe setting of a greater tumour burden [73].Conversely, 131I-anti-MHC II was highly effectivewhen used early in the disease providing >85%long-term cures, but was much less effective whenused against advanced tumour.

Following on from this a study was performedwith 131I-M195 (50 or 70 mCi/m2) in 7 patients withacute promyelocytic leukaemia (APL) in secondremission following treatment with all-trans retinoicacid [77]. Two of 6 patients with detectablePML/RAR-µ mRNA after all-trans retinoic acidtherapy had negative RT-PCR determinationsfollowing 131I-M195. The median disease freesurvival was 8 months, but HAMA developed in 5of the 7 patients. In view of the immunogenicity ofthe murine M195 antibody, a homodimerichumanised IgG1 antibody was developed that hadimproved effector function as well as the ability tointernalise and retain radioisotope in targetleukaemia cells [78]. This humanised M195 (Hu-M195) was subsequently used in a phase 1B trial inwhich patients with relapsed or refractory myeloidleukaemia were treated at dose levels of 0.5-10.0mg/m2 in six doses over eight days [78]. Theoptimal biodistribution was achieved at 3mg/m2 andno HAMA responses were observed. More recentlyin an attempt to increase the efficacy of RIT thealpha emitter 213Bi has been conjugated to the Hu-M195. Bismuth-213 has a t1/2 of just 45 minutesand emits high Linear Energy Transfer (LET) alphaparticles (8 MeV) with a pathlength of 50-80 µm[80]. Theoretically 213Bi-labeled mAb can killleukaemia cells with 1-2 alpha particles withminimal bystander effect [81]. A phase I doseescalation study has been initiated using this agentin patients with refractory and relapsed myeloidleukaemias. It appears to be stable in vivo andrapidly targets disease sites within minutes ofintravenous injection, with a target to backgroundratio of >10 000 [82].

Interestingly these results in animal models canbe related to those seen in the clinic, using forexample the 131I-Lym-1 directed against the HLA-DR10 antigen [23]. The eradication of tumourappears to stem from a combination of radio-targeting activity and the inherent cytotoxic activityof the carrier mAb. This idea is supported by theclinical results emerging, which suggest that simplymeasuring dose delivered to tumour may not predicttumour response [74].

RIT IN LEUKAEMIAS

Studies in leukaemia patients using RIT haveconcentrated upon the CD33 antigen, principally incombination with marrow transplantation. In a doseescalation trial of 131I-murine anti-CD33 (M195) inpatients with relapsed or refractory myeloidleukaemias [75]. Eight out of 24 patients treatedwith 50-210 mCi/m2 in divided doses had sufficientmarrow cytoreduction to proceed to BMT. Three ofthese patients achieved marrow remission, but 37%of assessable patients developed human anti-mouseantibody (HAMA). In an effort to intensify therapyprior to first or second BMT 131I-M195 (120-230mCi/m2) was combined with Busulphan andCyclophosphamide [76]. All patients engrafted and18 achieved complete response with three patientsfollowing first BMT remaining in unmaintainedremission for 18+ and 29+ months. Six patientsrelapsed and 10 patients died in remission oftransplant-related complications. Despite thesecomplications, these studies provided data tosuggest that leukaemic cytoreduction could beachieved with 131I-anti-CD33 even in multiplyrelapsed or chemotherapy-refractory leukaemia andthat RIT might be useful as part of a preparatoryregimen for BMT.

There have been other very encouraging resultsusing RIT targeting the CD45 antigen with 131I-anti-CD45 (BC8 antibody) in combination withCyclophosphamide (CY) and total body irradiation(TBI) as a marrow transplant conditioning regimenfor acute leukaemia [83]. Twenty patients weretreated with a dose of 131I estimated to deliver 3.5Gy (level 1) to 7 Gy (level 3) to liver, with marrowdoses of 4 to 30 Gy and spleen doses of 7 to 60Gy, followed by 120 mg/kg CY and 12 Gy TBI.Toxicity was not measurably greater than that ofCY/TBI alone and the maximum tolerated dose wasnot reached. Results from a phase I study of 131Ianti-CD45 combined with 120 mg/kg CY and 12Gy total body irradiation in HLA-matched relatedtransplants for AML in first remission were recentlyreported [84]. Forty-four patients with advancedacute leukemia or myelodysplasia received abiodistribution dose of 0.5 mg/kg 131I-BC8 (murineanti-CD45) antibody. The mean +/- SEM estimatedradiation absorbed dose (centigray per millicurie of


131I) delivered to bone marrow and spleen was 6.5+/- 0.5 and 13.5 +/- 1.3, respectively, with liver,lung, kidney, and total body receiving loweramounts of 2.8 +/- 0.2, 1.8 +/- 0.1, 0.6 +/- 0.04,and 0.4 +/- 0.02, respectively. Thirty-seven patients(84%) had favorable biodistribution of antibody,with a higher estimated radiation absorbed dose tomarrow and spleen than to normal organs. Thirty-four patients received a therapeutic dose of 131I-antibody labeled with 76 to 612 mCi 131I to deliverestimated radiation absorbed doses to liver (normalorgan receiving the highest dose) of 3.5 Gy (level 1)to 12.25 Gy (level 6) in addition to CY and TBI.The maximum tolerated dose was level 5 (delivering10.5 Gy to liver), with grade III/IV mucositis in 2 of2 patients treated at level 6. Of 25 treated patientswith acute myeloid leukemia(AML)/myelodysplastic syndrome (MDS), 7survive disease-free 15 to 89 months (median, 65months) post-transplant. Of 9 treated patients withacute lymphoblastic leukemia (ALL), 3 survivedisease-free 19, 54, and 66 months post-transplant.The authors concluded that 131I-anti-CD45 antibodycan safely deliver substantial supplemental doses ofradiation to bone marrow (approximately 24 Gy)and spleen (approximately 50 Gy) when combinedwith conventional CY/TBI [84].

extensively pre-treated patients with metastaticchemo-refractory disease, usually involving a singleintravenous injection of a non-myeloablative dose ofRIT. Generally response rates have beendisappointing and short lived, with often noobjective responses [32]. Many cases of symptompalliation and minor objective responses have beenreported. Promising results have however been seenin the treatment of breast cancer, where 131I-chimericL6 resulted in a 40% partial response [86]. There issome evidence suggesting that high dose therapy(requiring bone marrow/stem cell reinfusion) mayresult in improved response rates. IntraperitonealRIT has been primarily used for patients withrecurrent or persistent ovarian cancer, more recentlycombined with chemotherapy as part of amultimodality approach. Intralesional RIT hasprimarily been used for the treatment ofparenchymal brain tumours.

CNS TUMOURS

Both primary brain tumours and leptomeningealdisease have been treated with RIT. For primarytumours, RIT has been studied as therapy for smallvolume residual disease and in patients withrecurrent disease after conventional treatment.Various antibodies, radionuclides and routes ofadministration have been utilised [87-91]. Tumourresponse can be difficult to assess radiographically;disease free survival, disease free interval andquality of life assessments are commonly used asmarkers of disease response.

Waldmann et al. have investigated the use ofanti-Tac mAb (anti-interleukin-2 receptor) in AdultT-cell leukemia (ATL) caused by the retrovirushuman T-cell lymphotropic virus-I. Anti- Taclabeled with Yttrium-90 was given to 18 patientswith ATL, the first 9 patients in a phase I dose-escalation trial and the second group of 9 in a phaseII trial involving a uniform 10-mCi dose [85].Patients in whom a response was observed wereable to receive up to eight additional doses. At the 5to 15 mCi doses used, 9 of 16 evaluable patientshad responses (7 PR and 2 CR). The responsesobserved appear better than those with unmodifiedanti-Tac. Severe toxicity was largely limited to thehaematopoietic system.

GLIOMAS AND LEPTOMENINGEALTUMOURS

Gliomas account for 70 % of all primary braintumours in adults. The high-grade tumours,especially glioblastoma multiforme (GBM) areaggressive tumours with a poor median survival ofjust 12 months. Gliomas are initially wellcircumscribed with metastases occuring rarely andlate in the disease course. Standard treatmentinvolves surgery and external beam radiotherapyand occasionally chemotherapy. The treatment ofgliomas with systemic administration of RIT,although feasible, is fraught with difficulties, asdescribed by Hopkins and colleagues [92].Radioimmunoconjugates penetrate the blood/brainbarrier poorly, and even in situations where theblood-brain barrier is disrupted, accumulation ofradioisotope is low. The natural history and locationof the tumours has led to the development ofintrathecal administration with direct tumour

SOLID TUMOURS

The results for RIT in solid tumours arecurrently some way short of the highly promisingdurable response rates seen for the haematologicalmalignancies. This is likely to be due to tumourheterogeneity/poor mAb penetration and theinherent radioresistance of solid tumours relative tothe exquisite radiosensitivity of lymphomas. Thereare wide variations in the antibodies andradionuclides used as well as the tumour dosesdelivered. Most of the trials have been in


injection or injection into the surgical bed. Thisapproach has proved to be possible and has shownpromising results, especially for patients with lowergrade tumours and small volume disease. Bonemarrow toxicity is not dose limiting. Cerebraloedema is seen frequently and is usually transient.

therapy for GBM, and survival appears to beprolonged [91].

Diffuse leptomeningeal deposits theoreticallyrepresent an ideal target for RIT, being low volumedisease and with the possibility of delivering localintrathecal treatment, thereby reducing systemictoxicity. Kemshead et al. describe well thefeasibility of this approach with preliminary results[94 and references therein]

Riva et al. recently reported the results of a phaseI and a phase II trial, administering 131I labelledmAbs to the tumour bed of 111 patients, with 20 inthe phase I and 91 in the phase II studies [89].Murine IgG mAbs BC-2 and BC-4 were usedwhich react with 2 separate epitopes on theintracellular and stromal glycoprotein tenascin.Tenascin is expressed at high levels in gliomas,especially GBM. Ninety-one patients had GBM, theother pathologies were 8 oligodenrogliomas (7anaplastic oligodendroglioma), 2 grade IIastrocytoma and 10 anaplastic grade IIIastrocytomas. All patients received conventionalsurgery and radiotherapy; 54 also receivedchemotherapy. Patients with both a new diagnosisand relapsed disease were recruited. Toxicity wasminimal and repeated treatments were possible. TheMTD was 2590 MBq with larger doses causingmarked cerebral oedema. The mean dose per cyclewas 300 Gy. Scintigraphy was used to demonstratetumour uptake, which was high and sustainable.Response varied with histology and inversely withtumour grade. Tumour volume also affectedresponse rates in the patients with GBM. Of the 74phase II GBM patients 1 patient showed a CR, 9 aPR, 10 stable disease (SD) and 23 no evidence ofdisease (NED - patients who had RIT when thetumour mass was very small and not detectable butwho remained free of disease from the start oftreatment). The overall response rate(CR+PR+NED+SD) was 66.6% in those with atumour volume of less than 2 cm2 with a mediansurvival of 25 months. For those with bulky lesionsthe overall response rate was 17.8% with a mediansurvival of 17 months. Quality of life was notformally assessed in this study On the basis ofthese results, further studies are being performedusing 90Y-labeled mAbs [93].

GASTROINTESTINAL MALIGNANCY

The clinical use of RIT in GI malignancies hasgenerally been disappointing with poor responserates [95,96]. However there may be a role for RITin elimination of small volume disease and recentpre-clinical trials have provided encouraging results.Behr et al. compared responses to unlabeled mAbs(one directed against a membrane glycoprotein andthe other to CEA - FO23C5), the same mAbs 131I-labelled and 5 FU/Folinic acid chemotherapy innude mice bearing colon cancer xenografts. Therewas no reduction in tumour growth with either theunlabelled mAbs or chemotherapy. Responses wereseen in the RIT group, with cures reported in 35-55% of mice [97]. A pilot clinical study of patientswith small volume (< 3cm) metastatic colon cancerwho are receiving escalating doses of 131I-FO23C5is continuing and so far 10 patients have beenreported with durable anti-tumour effects beyond 12months. Bone marrow toxicity appears to be thedose limiting effect.

Anti-CEA mAbs have shown good targeting in avariety of CEA producing tumours includingovarian, breast, colorectal, pancreas and medullarythyroid cancer [98-102]. Juweid et al. have recentlyreported results from a Phase I/II study using 131I-MN-14 F(ab)2 anti-CEA MAb for patients withmetastatic medullary thyroid carcinoma [102].Fifteen patients were enrolled in this dose escalationstudy. The dose escalation was based on estimatesof radiation dose to the bone marrow, and theradioactive dose given was determined by apretherapy diagnostic study in which 8 mCi (0.6-20mg) of 131I-MN-14 F(ab)2 was administered 1 weekprior to therapy. Three patients received an initialdose of 140 centigray (cGy) to bone marrow, 11received 180 cGy, and 1 received 220 cGy.Myelosuppression was the only significanttreatment-related dose-limiting toxicity and theMTD appeared to be 180 cGy to the bone marrow.Therapy with 131I-MN-14 F(ab)2 appeared welltolerated and showed evidence of biochemical and

Gliomas have also been treated with systemicRIT including intravenous or intraarterialapproaches. The intraarterial route is associated withan increased risk of complications and has not beenshown consistently to give improved tumour uptake[88]. An initial pilot study by Miyamoto andcolleagues used 125I-anti-epidermal growth factorreceptor antibody 425 in 15 patients with recurrentgliomas. An objective response was seen in 20% ofpatients with 40% showed SD. Subsequently, thesame group has given RIT as primary adjuvant


radiologic anti-tumor activity. Seven patients had amedian of 55% reduction of tumor markers and 11of 12 assessable patients continued to haveradiologically stable disease for periods rangingfrom 3+ to 26+ months at the time of publication.HAMA developed in 8 of 11 patients and it wouldtherefore appear that humanized anti-CEA MAbswith dose escalation, alone or in combination withother therapy modalities, is indicated for futuretrials.

especially in the treatment of small volume disease[109]. Some trials have used laparotomy to assessresponse to RIT, as low volume peritoneal diseasecan be difficult to assess accurately usingconventional imaging techniques. Crippa et al.reported CR’s in 5 of 16 patients with microscopicresidual disease or positive washings treated withintraperitoneal 131I-MOV-18 [110]. Only one ofthese CR’s remained disease free beyond 12months (34 months) with the median disease freesurvival for the other 4 CR’s being just 10.5months. Other studies have assessed the efficacy ofthis approach using actuarial survival.Intraperitoneal RIT is currently being investigatedas part of multi-modality therapy. Meredith et al.reported 27 patients who received intrapeitoneal 177

Lu labelled CC49 (anti TAG -72) with or withoutinterferon-alpha and with or without Paclitaxel,within a phase I trial. All patients had TAG-72expressing tumour limited to the abdomen aftersurgery/chemotherapy. Three of these patients withsmall volume disease achieved a CR and remaindisease free at 3-5 years [111].

OVARY

The prognosis for epithelial cancers is poor, with5-year survival rates reported of between 29-40%for patients treated with conventional therapyconsisting of cyto-reductive surgery andchemotherapy. Unlabelled mAbs have been used inan attempt to improve of these rates and Wagnerdescribed the induction of anti-idiotypic antibodiesto the CA125 antigen by repeated administration ofthe F(Ab)2 fragment of the anti-CA125 mAb OC125 [103]. CA125 is a glycoprotein expressed on80% of ovarian cancer cells. Weiner et al. reportedgrowth inhibition in a human ovarian cancerxenografts using bispecific antibody against theHER-2/neu antigen [104] .

BREAST

Clinical trials have demonstrated responses toRIT in 30-60% of heavily pre-treated patients withmetastatic breast cancer [112]. Antigens includeHER-2/neu (c-erb B2), a transmembrane tyrosinekinase receptor for a polypeptide growth regulatorymolecule which is overexpressed in 20-30% ofbreast adenocarcinomas [113, 114]. Phase IIIstudies using unlabelled antibody to HER-2/neuhave shown tumour responses in extensively treatedpatients. Good results have also been seen usingradiolabeled chimeric (Ch) L6 mAb. DeNardo et al.reported a partial response in 4 out of 10 patientstreated with 131I-Ch L6 and a minor response in 7patients [115]. Wilder et al. reported a 40% partialresponse rate in breast cancer patients treated with131I-ChL6 [116].

Intraperitoneal RIT has predominately beenstudied for ovarian carcinoma where peritonealseedlings are commonly seen. Response ratesdepend on tumour volume and dose; there is ageneral trend of increasing chance of tumourcontrol with reducing tumour volume, but this trendis not uniformly reported [105]. The largest single-institution study comes from the HammersmithHospital, London [106,107]. Yttrium-90 labelledmurine mAb HMF-G1 was administered to 25patients with stage 1c-IV epithelial ovarian cancer,as a single intraperitoneal dose followingcompletion of conventional chemotherapy [108].No objective responses were seen in tumournodules >2cm. 9 out of 16 patients with <2cmnodules responded, as did 50% patients withmicroscopic disease. Compared to historicalcontrols matched for age, stage, histological typeand grade, survival at 5 years was increased in theRIT group to 80% compared to 55% in the controls(p=0.003). Acute and late bowel toxicity was alsomuch lower for the RIT group compared to thosewho received whole abdominal irradiation [108]. Amulticentre phase III randomised trial is currentlyongoing.

Using PBSC support, 2000-4000 cGy/ cycle oftherapy can be delivered to sites of metastaticdisease [112]. Cagnoni et al. reported a phase 1study of high dose RIT with the radiolabeledantibody 90Y-hu-BrE-3 (targeted against the MUC 1antigen) and autologous PBSC support. Four of 9patients showed a response with 2 partial responsesand 2 minor responses were seen [117]. Combinedmodality therapy incorporating RIT is highly likelyto be more effective than RIT alone. DeNardo et al.have observed a synergistic effect betweenPaclitaxel chemotherapy and RIT with 90Y-ChL6 ina xenografted mouse model, which gives further

Two phase I studies performed in the UnitedStates have also shown promising results,


credence to this approach. Further studies haveshown that the optimal time of Paclitaxeladministration is 48 hours after the RIT, withmaximal deposition of the radiolabeled mAb andPaclitaxel in the tumour and no Paclitaxel in thebone marrow during the time of maximum marrowirradiation [118]. A clinical trial using Paclitaxel andRIT in patients with metastatic breast cancer isunderway.

and occurred at dose levels of 4.5-12 mCi/m2. Themaximum tolerated dose of 90Y-CYT-356 was 9mCi/m2. Only one patient developed a human anti-mouse antibody 4 weeks after treatment. No patientachieved a complete or partial response based onprostate-specific antigen and/or radiological criteria.Three patients had transient subjective improvementin the symptomatology of their disease. In addition,patients treated with 12 mCi/m2 of 90Y-CYT-356had a slightly longer freedom from diseaseprogression than patients treated with doses of 90Y-CYT-356 less than or equal to 9 mCi/m2.RENAL

Recent results in metastatic renal cell cancer(RCC) have been promising. Divgi et al. treated 33patients with 131I-labelled mouse mAb anti-G250 aspart of a phase I/II trial [119]. The G250 antigen isa transmembrane phosphoprotein expressed on themajority of RCC. Targeting of radioactivity to allsites of disease of 2cm or greater was demonstratedand 17 patients showed stable disease, while 2 had aminimal response. The maximum tolerated dosewas 90mCi/m2 131I. The majority of patientsdeveloped reversible abnormal liver function tests;the major problem however was of HAMA, with allpatients developing anti-mouse antibodies by 4weeks after treatment [120]. In an attempt toovercome this problem, chimeric G250 has beenengineered and the excellent mAb localisationrepeated with an impressive tumour uptake of0.5233 % injected dose/g [120].

There are likely to be potential roles of RIT inthe treatment strategies of other solid tumours in thefuture and one such interesting novel approach is inthe treatment of Kaposi’s sarcoma (KS). KS is awell vascularised, radioresponsive tumour andtherefore theoretically an ideal candidate for RIT.Leigh et al. have demonstrated the presence of theL6 antigen in KS biopsies and have shown goodtumour targeting to KS by Ch-L6 in xenograftedmouse models [123]. Further clinical results areeagerly awaited.

CONCLUSION

Although the concept behindradioimmunotherapy is not new, the techniqueremains in its infancy as regards clinicaldevelopment. Several variables have been foundwhich affect delivery, but it is likely that more needto be studied. The clinical data suggest thatradioimmunotherapy will soon play a significantrole in the treatment of NHL, especially low-gradelymphoma. Knowledge of the mechanisms ofeffective treatment remains scarce but it appears thatthe induction of apoptosis by radiation and/orantibody effector mechanisms may be critical tosuccessful therapy. Further work will define theseprocesses and allow us to optimise this approach inthe clinic.

PROSTATE

A Phase II trial of 75 mCi/m2 131I-anti-TAG-72high-affinity antibody CC49 has been studied in 15patients with hormone-resistant metastatic prostatecancer [121]. No acute adverse reactions occurred,but unfortunately all patients had developedevidence of an immune response to CC49 by 4weeks. Six of 10 symptomatic patients had bonepain relief, but no patients met the radiographic orPSA criteria for objective response. Positiveimaging of bone and/or soft-tissue lesions washowever noted for 13 of the 15 patients. REFERENCES

More recently a Phase I dose-escalation studyusing 90Y-CYT-356 monoclonal antibody wasperformed in 12 patients with hormone-refractoryprostate carcinoma by Deb et al. [122]. Of the 12patients, 58% had at least one site of diseaseimaged after administration of 111In-CYT-356. Thedose of 90Y ranged from 1.83-12 mCi/m2. Both111In and 90Y-CYT-356 were tolerated well, withoutsignificant non-hematological toxicity.Myelosuppression was the dose-limiting toxicity

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