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e1 CancerBiology&Therapy 2007;Vol.6Issue2

[CancerBiology&Therapy6:2,e1-e6,EPUBAheadofPrint:http://www.landesbioscience.com/journals/cbt/abstract.php?id=3574;February2007];©2007LandesBioscience

Elia Neninger1,*Rosa M. Díaz2

Ana de la Torre3

Rolando Rives4

Alain Díaz2 Giselle Saurez2

Mariano R. Gabri5

Daniel F. Alonso5

Barbara Wilkinson2 Angel M. Alfonso2

Tania Combet2 Rolando Pérez2

Ana M. Vázquez2

1Hermanos Amejeiras Hospital; Havana, Cuba

2Center of Molecular Immunology; Havana, Cuba

3Celestino Hernández Hospital; Villa Clara, Cuba

4Neumology Hospital; Havana, Cuba

5Laboratory of Molecular Oncology, Quilmes National University; Buenos Aires, Argentina

*Correspondence to: Elia Neninger; Department of Medical Oncology, Hermanos Ameijeiras Hospital; Havana City, Cuba 10300; Tel.: 53.7.876.1434; Fax: 53.7.272.0644; Email: nenin@infomed.sld.cu

Original manuscript submitted: 08/02/06Manuscript accepted: 11/05/06

This manuscript has been published online, prior to printing for Cancer Biology & Therapy, Volume 6, Issue 2. Definitive page numbers have not been assigned. The current citation is: Cancer Biol Ther 2007; 6(2):http://www.landesbioscience.com/journals/cc/abstract.php?id=3574Once the issue is complete and page numbers have been assigned, the citation will change accordingly.

KEy WoRDS

Anti-idiotypeantibody;clinicalstudy;immu-notherapy;cancervaccines

ABBREViATioNS

MAb monoclonalantibodyAb2 anti-idiotypeSCLC smallcelllungcancer

ACKNoWlEDGEMENTS

Seepagee6.

ABSTRACT1E10 is an anti‑idiotype murine monoclonal antibody (Ab2 MAb) specific to an

Ab1 MAb which reacts with NeuGc‑containing gangliosides, sulfatides and with antigens expressed in some human tumors. Preparations containing this Ab2 were capable to induce a strong anti‑metastatic effect in tumor‑bearing mice. We conducted a Phase I clinical trial to evaluate the toxicity and humoral immune response elicited by 1E10 vaccine in patients with small cell lung cancer (SCLC). Eligible patients were those who after received chemotherapy and/or radiotherapy had partial or complete response to treatment. Patients received four biweekly injections with 2 mg of aluminum hydroxide‑precipitated 1E10 MAb, then other six doses at 28‑day intervals, and later the patients who maintained a good performance status were reimmunized. Six patients with limited‑stage disease and three with extensive‑stage disease were enrolled in the study. Most of the patients who received at least four doses of 1E10 vaccine developed strong specific antibody responses against 1E10 MAb and NeuGc‑GM3 ganglioside. Antibodies able to react with lung carcinoma tissue sections were detected in sera from vaccinated patients. A prolonged survival was observed in several patients treated with the anti‑idiotype vaccine. No evidence of serious adverse effects was found.

iNTRoDuCTioNSmallcelllungcancer(SCLC)representsbetween15–20%ofalltypesoflungcancer,

anditisdistinguishedfromnonsmall-celllungcancer(NSCLC)byitsrapidgrowthanddevelopmentofdistantmetastases.1Withouttherapy,lifeexpectancyofthepatientsfromdiagnosisislessthanfourmonths.SCLCisresponsivetochemotherapyandirradiation,but these initial objective responses do not translate in significant increase in survival.Withtheincorporationofchemotherapy,themediansurvivalforpatientswithlimited-orextensive-stagediseaseisapproximately18and10months,respectively.2-5

So far, the benefits of an optimized chemotherapy have been limited, a variety oftreatmentstrategieswhichincludestheuseofanti-idiotypeMAbsasvaccineshavebeenevaluatedinrecentyearsinanattempttoimproveoutcomesforpatientswithSCLC.

ThisapproacharosefromtheJerne’s idiotypenetworktheory6whichpostulatesthatduetotheenormouspotentialityfordiversityoftheimmunoglobulinvariablegenes,theidiotyperepertoirehastomimicthehugeuniverseofselfandforeignepitopes.

InasmallpilotclinicaltrialinpatientswithSCLCusingananti-idiotypeMAbintheGD3gangliosidemodel,promisingresultsofimmunologicalandclinicalresponseswereobtained.7However,whenaphaseIIIclinicaltrialwasperformedinpatientswithSCLCwith limiteddisease, treatmentwith this anti-idiotypeMAbdidnot impactonpatientsurvival. Nevertheless, a trend to prolong survival was observed in those patients thatdevelopedahumoralresponse.8

Wehavepreviouslyreportedavaccinepreparationthatcontainsamurineanti-idiotypeMAb,denominated1E10,9 thatwasobtained fromthe immunizationofBALB/cmicewith the P3 MAb, an antibody that recognizes gangliosides having the N-glycolylatedsialicacid(NeuGc),sulphatedglycolipidsandantigenspresentinmelanomaandbreastcarcinomas,10,11aswellasinlungtumors(unpublishedobservations,seealsoFig.1).

Inchickens,wherelikeinhumansNeuGc-containinggangliosidesarenotexpressedinthenormaltissues,the1E10MAbwascapabletoinducespecificAb3antibodyresponseagainst these gangliosides.12 Additionally, the 1E10 MAb showed an anti-tumor effectagainst lungmetastasesproducedbybreastcarcinomaandmelanomacells insyngeneicandallogenicmice.13Experimentswhichevaluatedtheeffectofthetreatmentwiththis

Clinical Trial

Active Immunotherapy with 1E10 Anti-Idiotype Vaccine in Patients with Small Cell Lung CancerReport of a Phase I Trial

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antiidiotypeMAbinspontaneousmetastasesproducedbyamurinelung carcinoma tumor confirmed the results previously obtained.The anti-metastatic activity was related to an increase of tumorapoptosis and the reduction in thenumberof tumorbloodvessels(articleinpreparation).

Three phase I clinical trials were carried out in patients withadvanced melanoma and breast cancer treated with aluminumhydroxide- precipitated 1E10 anti-idiotype MAb. The results oftheseclinicaltrialsevidencedthatthevaccinewaswelltoleratedandimmunologicallyactive.11,14,15

Thebiologicalpropertiesof1E10MAb, the resultsobtained inthe prior clinical trials and the poor prognosis of SCLC patientsmotivatedustoperformaPhaseIclinicaltrialinpatientswiththisdisease. The results of this clinical study confirmed the immuno-genicity and safety of this vaccine given in an extended regimentoSCLCpatients. Inaddition, the inductionofantibodyresponseagainstlungtumorscouldalsobeobserved.Aprolongedsurvivalwasobservedinseveralpatientstreatedwiththeanti-idiotypevaccine.

MATERiAlS AND METhoDSPreparation of 1E10 MAb for the clinical trial.1E10MAbwas

purified from mouse ascites in the Good Manufacturing Practice(GMP)facilitiesoftheCenterofMolecularImmunology.Purificationof1E10MAbwasperformedbyDEAE-exchangechromatographyfollowedbyaffinitychromatographyonProteinA-CLSepharose4B column and size exclusion chromatography on Sephadex G-25column (Amersham Pharmacia Biotech, Uppsala, Sweden). Thepurity of the isolated immunoglobulin was more than 97% asdeterminedbySDS-PAGE,high-pressureliquidchromatography,andisoelectricfocusing.ThevaccinewasproducedinaccordancewiththeGoodManufacturingPracticeguidelinesandcertifiedbytheQualityControl Department of the Center of Molecular Immunology.Briefly, sterile purified 1E10 MAb was aseptically mixed at a finalconcentrationof2mg/mLwith5mg/mLofaluminumhydroxideasadjuvant(SuperfosBiosector,Frederikssund,Denmark).Themixturewasgentlystirredfor3hoursatroomtemperature.Thealuminumhydroxide-precipitatedMAbwasaliquotedintopyrogen-free,sterileglassvialsandstoredat4˚Cuntiluse.Thefinalproductwastestedforsterility,pyrogenicity,andgeneralsafetyinmiceandguineapigsbefore use according to United States Pharmacopeia16,17 and toBritishPharmacopeia.18

Eligibility criteria.To be eligible for the study, patients had tohave histological or cytological proven SCLC, limited- (LD) orextensive-stagedisease(ED),acompleteorpartial(≥75%)remissionafter completion of conventional treatment. Criteria for eligibilityalsoincludedWHOperformancestatus≤2,age≥18years,measur-abledisease,normalhematopoietic,hepaticandrenalfunctions,andlifeexpectancyhigherthansixmonths.Priorchemotherapyand/orradiationtreatmenthadtobecompletedbetween4to8weeksbeforeentry in the study. All patients signed written informed consentform.Themainexclusioncriteriawerepregnancyorlactation,brainmetastases or a second malignancy, previous history of enceph-alopathy, acute and severe allergic events, chronic or acuteinfectiousdisease,andautoimmunedisease.Patientsthathavereceivedtreatmentwithmonoclonalantibodiesorotherbiologicalmodifieroftheimmuneresponsewerenotincluded.

TheprotocolwasapprovedbytheInstitutionalReviewBoardsofthehospitalswherethestudywasdeveloped,andauthorizedbytheNationalRegulatoryAuthorityforDrugQualityControl.

Study design and patient evaluation. This was a multicentricphase I clinical trial in which after completion of chemotherapyandcurative intent thoracic irradiationallpatientshada completehistoryandphysicalexamination,completebloodcellcount,chem-istry profile, urinalysis, evaluation of the performance status, andtumor measurements by chest X-ray and thoracic and abdominalcomputedtomography(CT)scan.Clinicalandradiologicalevalua-tionswereperformedbeforeinclusionandtheneverythreemonths.TheadversereactionswereevaluatedaccordingtotheNCICommonToxicity Criteria (version 3). Patients who received one or morevaccinedoseswereevaluablefortoxicityandclinicalresults.Patientswhoreceivedatleastfourvaccinedoseswereevaluableforhumoralresponse.

Treatment schedule. Patients were injected intradermally withten doses of 2 mg of Aluminum hydroxide 1E10 MAb as basetreatment: the first fourevery14daysand the remaining sixevery28days.Reimmunizationswereadministeredquarterlyifthepatientshada favourableclinical status.Progressivediseasewasnotconsid-eredatreatmentinterruptioncriterion.

Serum samples were collected at baseline and at each fourteendaysduringthefirstfivedoses.Thereafter,serawerecollectedeverythreemonths,untiloneyear.

Measurement of antibody response. Anti-1E10 idiotype anti-bodies were determined by ELISA, as previously reported11; usingmicrotiterMaxiSorpplates(Nunc,Roskilde,Denmark)coatedwith500 ng/well of purified 1E10 MAb or its F(ab')2 fragments. Asisotype-matchedcontrols,iorcea-1(anti-carcinoembrionicantigen)and ior C5 (against a glycoprotein expressed on human colorectalcells) MAbs and their F(ab')2 fragments were used.19,20 Alkalinephosphatase-conjugatedantibodiesagainsthumanIgMorIgGwereusedassecondaryantibodies.Absorbancewasmeasuredat405nminanELISAreader(OrganonTeknika,Salsburg,Austria).

Anti-NeuGc-GM3IgMandIgGantibodieswereassessedbyanELISApreviouslyreported,11usingPoliSorpplates(Nunc,Roskilde,Denmark) coated with NeuGc-GM3 (200 ng/well). Plates coatedwithNeuAc-GM3oronlywithmethanolwereusedtodeterminethespecificityofserumreactivity.Biotinylatedsecondaryantibodieswereused and reaction was developed as described.The highest serumdilutiongivingopticaldensityvalues≥0.2after substractionof the

Figure 1. Immunohistochemical staining of SCLC by P3 MAb, healthy patient serum and autologous pre‑ and postimmune sera from a patient immunized with aluminum hydroxide‑precipitated 1E10 MAb. (A) Healthy donor serum (B) P3 MAb (20 μg/ml) (C) Preimmune serum (Patient 3) (D) Postimmune serum (Patient 3). Serum dilution (1/500).

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valuesobtainedforeachserumdilutioninwellstowhichnoantigenwasaddedandbeingatleastthreetimesthevaluecorrespondingtothepreimmuneserumatthesamedilutionwasconsideredastiter.Apoolof3AB-serafromnormalhealthydonorswasusedasacontrol.Assays were performed in triplicate for each sample and the coef-ficient of variation (CV) was less than 15%.The optical densities(OD)oftheblankswerelessthan0.1.

The presence of Ab3 antibodies specific to gangliosides wasalso detected by immunostaining on HPTLC plates, as previouslydescribed.11

The presence of antibodies reacting with lung tumors wasperformed by an immunohistochemical study of the pre and postvaccination serum in fixed and processed lung cancer material.Serumwasuseddiluted1:500andabiotinylatedhumananti-IgM+IgGantiserum(Vector),wasusedassecondantibody.Developmentof the reaction was performed using peroxidase/DAB complexand a counterstainingwasdonewithmildhematoxyline.P3MAb(20μg/mL)andhealthydonorserumwereusedaspositiveandnega-tivecontrols,respectively.Theresultswereclassifiedwithrespecttobackground reactivity, as follows: (-) negative, (+) mildly positive,(++)moderatelypositive,and(+++)intensivelypositive.

Statistical analysis.Themeanvalueandthestandarddeviationofthevaluesobtainedinthetriplicatesofeachsamplewerecalculated.Eachexperimentwasrepeatedatleasttwice.ThemeanvaluesandthestandarddeviationswereplottedusingtheMicrocalOriginprogram.SurvivaltimeswereestimatedusingtheKaplan-MeiermethodusingSPSSProgram(version10).

RESulTSPatient characteristics. Between 2000 and 2003, nine patients

withhistologicalconfirmedSCLCwereincludedinthetrialinthreedifferent hospitals. Patient characteristics are presented inTable 1.The average age was 59 years (range 45–70). As prior therapy, sixpatients had received chemotherapy and radiotherapy, and threeof them, only chemotherapy according to the standard of care.Chemotherapyconsistedmainlyonplatinum/etoposideasfirst lineandcyclophosphamide/doxorubicin/vincristin(CAV)assecondline.After finished standard therapy, two patients reached a completeresponseand sevenapartial response (≥75%),according toWHOclassification. At the time of entering the trial, three patients had

metastases:contralaterallungmetastasis(twopatients)andsubcuta-neousmetastasis(onepatient).

Treatment administration.Oftheninepatients includedinthestudy, four concluded the treatment schemewith the anti-idiotypevaccine (ten doses) and later they were reimmunized quarterly,receivinga totalof12 to17dosesof thevaccinepreparation.Theremainingfivepatientsreceivedbetween4to9dosesofthevaccine.

Toxicity. All patients were evaluated for toxicity and theadverseeventsobservedinthevaccinatedpatientsareshowninTable2. The toxicity due to the treatment with aluminum hydroxide-precipitated1E10MAbwasclassifiedasgrade1and2,accordingtoCTCclassification.

Toxicity consisted mainly of local reaction at the injection sitewith erythemaand indurationoccasionally associated tomildpainthat disappeared in a few days (2–4). Only one patient presentedanabscessattheinjectionsitethatrequiredmedicaltreatmentwithantibiotics and local cures, without hospitalization, recovering in15days.Thiseventwasclassifiedasgrade2.Onepatienthadgradefever 1, arthralgias (1) and cephalea grade 1 (2). An increase inbloodpressurewas reported in onepatient, but this patient had aprevioushistoryofhypertension.Twopatientspresentedparestheticalterations of inferior and superior limbs that were related withthe platinum-based therapy regimen that patients received as part

Table 2. Adverseevents

Event Grade 1 2Injection site pain 2Local erythema 3 1Injection site induration 3 1Injection site abscess 1Cephalea 2Fever 1Chill 1Arthralgia 1Paresthesis 1 1High blood pressure 1

Table 3 Antibodytitersandisotypeofhumoralresponse ofpatientstreatedwithaluminum‑precipitated 1E10MAb

Patient Anti‑1E10a Anti‑NeuGc‑GM3b No igM igG igM igG1 ‑ 1:5000 1:800 ‑2 ‑ 1:100 000 1:800 ‑3 ‑ 1:10 000 1 :800 1:16004 ‑ 1:10 000 ‑ 1:4005 ‑ 1:1000 ‑ ‑6 ‑ 1:3200 1:400 ‑7 ‑ 1:100 000 1:800 ‑9 ‑ 1:1000 1:400 ‑

Different serum dilutions from vaccinated patients were added to microplates coated with a1E10 MAb F(ab’)2 fragments (500 ng/well) or bNeuGc-GM3 (200 ng/well), and ELISA assays were performed as described in Material and Methods.

Table 1 Characteristicsofpatientsincludedinthestudy

Entered 9 (100%)Median age (range) 59.4 (45–70%)Sex Male 4 (44%) Female 5 (56%)Who performance status 0 1 (11) 1 7 (78%) 2 1 (11%)Stage LD 6 (67%) ED 3 (33%)Previous therapy CHT‑RT 6 (67%) CHT 3 (33%)Metastatic sites Lung 2 (22%) Subcutaneous 1 (11%)Response to initial therapy Complete 2 (22%) Partial 7 (78%)

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of the standard treatment. Adverse events did not exacerbate withincreasingnumberofvaccinedoses.Noothersevereorunexpectedadverseeventswereobserved.Neitherbiochemicalnorhematologicalabnormalitieswerereported.

Immunological responses. Eight of the nine patients wereevaluatedforhumoralimmuneresponses.Onepatientwasexcludedfor immunological studies because the serum after receiving thefourthdoseofthevaccinecouldnotbeobtained.

All eight evaluated patients developed an IgG antibody responseagainst1E10wholeimmunoglobulinmoleculeandagainstitsF(ab')2fragments. The titers of this response ranged from 1:1000 to 1:100000, as measured by ELISA (Table 3). The maximum values ofthis response were generally obtained after administrating the 6thdose of the vaccine. There was a greater recognition of 1E10 MAbby immune patients’ serum than of the rest of the MAbs tested,suggesting the presence of response against the idiotype of 1E10MAbinadditiontotheresponseagainsttheisotype(Fig.2A).NospecificIgMorIgGantibodiesweredetectedagainst1E10MAbatthelowest

Figure 2. Kinetics and specificity of serological antibodies to 1E10 MAb and NeuGc‑GM3. Sera from a representative vaccinated SCLC patient were added to ELISA plates coated with 1E10 or and isotype‑matched MAb F(ab´)2 fragments (A) and to wells coated with NeuGc‑ or NeuAc‑GM3 (B), and ELISA assays were assessed as described in Material and Methods. Arrows indicate the time of vaccinations. (C) Serum reactivity against gangliosides determined by HPTLC immunostaining (a) Gangliosides were chromatographed with choloroform:methanol:0.2% CaCl2 in 2.5 M NH3 (5:4:1) and visualized with orcinol (A). (B) Binding patient’s immune serum.

Figure 3. Sequential computerized tomography (CT) of the lungs from a patient treated with aluminum hydroxide‑precipitated 1E10 MAb. (A) After finished second line of chemotherapy, it was detected a residual tumor and the presence of fibrosis. (B) Nine months after starting re‑immunizations, there was a complete tumor response together with fibrosis and pleural thickness, only fibrosis and pleural thickness. White arrows indicate tumor mass and black arrow indicate pleural thickness.

serum dilution tested (1:100), when preimmune sera from patients(Fig.2A)orserafromhealthydonors(datanotshown)wereused.

AstrongandspecificresponseagainstNeuGc-GM3wasdetectedinsevenoftheeightevaluablepatients(Titers1:400-1:1600).MostofthemdevelopedanIgMresponse,andintheseraoftwopatients,the vaccine induced the production of both IgM and IgG anti-bodies(Table3).SpecificantibodyresponsestoNeuGc-GM3werefoundinallresponderpatients,andnoreactivitywithNeuAc-GM3was observed, neither by ELISA nor by HPTLC-immunostaining(Fig.2BandC).Asitwasreportedbeforeinbreastcancerpatientsimmunized with aluminum-hydroxide-precipitated 1E10 MAb, along lasting anti-1E10 and anti-NeuGc-GM3 serological responsewere detected in patients before starting reimmunizations (≥3monthsafterreceivingtendosesofthevaccine)(Fig.2AandB).NospecificreactionwasobservedagainstNeuGc-GM3whenpreimmunesera frompatients (Fig.2B)or sera fromhealthydonors (datanotshown)wereused(lowestdilutiontested1:100).

Duetotheimportanceofevaluateiftheanti-idiotypevaccinewasabletoinduceinthepatientsantibodiesagainstnotonly1E10MAbandNeuGc-GM3,butalsoagainstSCLCtumors, twopatientseraobtained before and after vaccination, and a healthy donor serumand a healthy donor serumwere tested against paraffin-embedded tumor tissues. As shown inTable4andFigure1,anintensestainingoftumortissuewasevidentforthepatientwiththehighestanti-NeuGc-GM3post-vaccinationtiter.Although theprevaccination sample showedcertain reactivityagainst the cancer cells, there was a clear increase in the reactivityafterimmunizationonSCLCtissues.Interestingly,serumreactivitywas evident against both autologous and heterologous lung tumortissue. Normal serum did not show reactivity against the tissueNormal serum did not show reactivity against the tissuesample.

Clinical outcome.Clinicalresultsweremonitored,althoughthisstudy was not designed to evaluate the therapeutic efficacy of the

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vaccinepreparation.AscanbeseeninTable5,twopatientsbearingextensive-stage disease survived more than 20 months from thediagnose.Ontheotherhand,threeofsixpatientswithlimited-stagediseasewerealive36monthsafterthetumordetection.

Oneofthepatientswithlimitedstagedisease,whowasincludedin this studyafter reachingapartial response (75%)with first lineof chemotherapy, presented a local progression after completingtheinductionimmunizationtreatment(60%increaseofthetumorlargestdiameter).Then,thepatientreceivedCAVregimen,assecondline treatment for five months. Later, the patient was evaluatedand a reduction of tumor mass with remarkable fibrosis (Fig. 3A)was observed. A month after finished the second line of chemo-therapy, thepatientwas reimmunizedevery threemonthswith theanti-idiotypevaccine,andafterthethirdreimmunizationdose(ninemonths after finished the second line chemotherapy treatment), acompletedisappearanceofthetumorandanincrementofthefibrosiswasobserved(Fig.3B).Thepatientremainsdiseasefreeuntilatthemomentofthiswriting.

Asecondpatienthadanextensive-stagediseasewhichwasevalu-atedasstablediseaseaftercompletionofchemotherapy.Sheachievedprolongeddiseasestabilizationfor20months.Thepatientreceivedatotalof12immunizationswith1E10MAbvaccine.Sheisaliveatthemomentofthisreport.

Thesmallnumberofpatientsenrolledinthisclinicaltrialdidnotallowmakingacorrelationbetweentheimmunologicalandclinicaloutcomes.

DiSCuSSioNThe present report describes the

results of a Phase I clinical trialin which patients with SCLC weretreated with the anti-idiotype 1E10MAb vaccine. Our main goals wereto evaluate safety and the immuneresponses generated by the vaccinepreparationinthesepatients.

It has been reported that thedevelopment of human anti-mouseantibodies (HAMA) in patients,following injections of murineMAbs, resulted in adverse reactionsand lack of treatment efficacy insome clinical trials.21 In contrast,othergroupshaveshownresultsthatindicate that the development ofHAMA may in fact prove to bebeneficial in immunotherapy andthis effect seems to be due to thecapacity of some MAbs to inducethe generation of cascades of anti-idiotypeantibodiesthathavesurvivaladvantageintumorpatients.22-24

Inourstudy,theantibodiesgener-ated in SCLC patients are directedpredominantly against 1E10’s idio-typeandnotagainstitsisotype.Theimmunodominance of 1E10 MAbidiotypehasbeenobservedinalltheanimal species immunized with this

MAb,12includingcancerpatients.11,14,15

A specific Ab3 response against 1E10 MAb was induced in89% of the patients after repeated administrations of the anti-id-iotype vaccine.The existence of specific antibody response againstNeuGc-containing gangliosides (Ab1') was demonstrated in 78%of the vaccinated patients. This response rate was very similar tothose obtained in previous Phase I clinical trials using 1E10 MAbvaccine in patients with melanoma and breast cancer.11,14,15 It isimportant to emphasize that the treatment with this anti-idiotypevaccinewascapabletoinducetheproductionofantibodiesnotonlyspecificagainstN-glycolylatedgangliosides,butalsoagainstantigenspresented in the lung tumor sections.The capacity of 1E10 MAbvaccinetoinducetheproductionofantibodiesinthepatientswiththesamespecificityofP3MAb(Ab1)confirmsthebehaviorof1E10MAbas“internalimage”inhumans.

The titers of Ab3 and Ab1' antibodies reached in the SCLCpatientsarelowerthantheonesobtainedintheprevioustrialsalreadymentioned. A possible explanation to this result could be thatthepatientswho enter in this studyhad received various cycles ofoncoespecific treatmentbetween fourandeightweeksbefore enterin the trial. It is well demonstrated that previous chemotherapyand radiotherapy treatments could affect the immunological statusof the patients.25 Another explanation could be that in thismalignantdiseasetheimmunesystemofthepatientsmightbemoredeteriorated.26

Nevertheless, in contrast from reports of the use of other anti-idiotypeMAbinadifferentgangliosidemodelinSCLCpatients(7

Table 4 AntibodyresponseagainstSCLCtumors

Patient Antibody Titers Against Serum Reactivity Against SClC Tissue Section No NeuGc‑GM3 Autologous Tumor Tissue heterologous Tumor Tissue* igG igM Preimmune Postimmune Preimmune Postimmune HD 0 0 NA NA ‑ 3 1:1600 1:800 ++ +++ + ++ 4 1:400 0 ‑ ‑ ++ ++

*From archival specimens. HD, healthy donor; NA, not applicable.

Table 5 Timetoprogressionandsurvivalofallpatientsincludedintheclinicaltrial

Patient Age Disease Prior oncospecific 1E10 MAb Survival from Survival No Stage Therapy Therapy Treatment inclusion from Response No Doses the Trial Diagnosed (Months) 1 63 LD CHT PR (75%) 7 6 17 2 70 LD CHT CR 17 37 45 3 45 ED CHT CR 14 25 37 4 68 LD CHT /RT PR (75%) 9 12 18 5 57 ED CHT /RT PR (75%) 6 5 12 6* 58 LD CHT /RT PR (75%) 12 42 52 7* 54 LD CHT /RT PR (75%) 12 40 46 8 55 LD CHT /RT PR (>75%) 4 2 10 9 46 ED CHT /RT PR (>75%) 5 9 20

CHT, chemotherapy; RT, radiotherapy; LD, limited-stage disease; ED, extensive-stage disease; CR, complete response; PR, partial response. *Still alive.

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and8),evidentantibodyresponsesagainstboth,themurineimmu-noglobulinandNeuGc-GM3ganglioside,were reached inmostofour patients due to the immunization protocol with 1E10 MAbvaccine.

Although thepatientswere repeatedly injectedwith1E10MAbvaccineandthatthisvaccinationregimengeneratedstrongantibodyresponses to the murine immunoglobulin, only a low rate of sideeffectswasobserved,confirmingpreviousreportsaboutthesafetyofthetreatmentwiththisanti-idiotypevaccine.11,14,15

A characteristic of SCLC is that although is sensitive to initialchemotherapy, the tumorrapidly recurredandpatientsdie.Recenttrials confirm no impact in patient’s survival of the addition ofnewchemo-therapeutics agents to standardcisplatinandetoposidefirst-linetreatment.5,27Thus,newapproachestoimprovesurvivalofSCLCareneeded.

Infact,thereisaconsensusthatmorethanasingleaberrantpathwayneedstobeblockedtohaveaneffectinthemalignanttransformationprocessanddifferentgroupsarecarryingonproof-of-conceptstudiestovalidatenewtargetsforimmunotherapyinSCLC.28-30

The study we described here was not designed to evaluate thetherapeutic efficacy of 1E10 MAb vaccine, but nevertheless, theclinical responses were monitored. The treatment with this anti-idiotypevaccine seems toprolongsurvival in someof thepatients.ThesefigurescompareveryfavorablywiththehistoricalsurvivaldataforpatientswithlimitedorextensivestageSCLC.2-5

Phase II randomized trial should be performed to evaluate theclinicaleffectof1E10MAbvaccineinSCLCandtodefineifthereis any correlation between the immune response induced by theanti-idiotypevaccinewithclinicalbenefitforpatients.

AcknowledgementsWethankDr.L.E.Fernández(VaccineDept,CenterofMolecular

Immunology)forgenerouslyprovidinggangliosides.ThisstudywassupportedbyRecom-BioS.L.andbytheCubanGovernment

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