IGKV3 Proteins as Candidate Off-the-Shelf Vaccines for ... · Cancer Therapy: Preclinical IGKV3 Proteins as Candidate "Off-the-Shelf" Vaccines for Kappa-Light Chain–Restricted B-Cell

Cancer Therapy: Preclinical

IGKV3 Proteins as Candidate "Off-the-Shelf" Vaccines forKappa-Light Chain–Restricted B-Cell Non-HodgkinLymphomas

Debora Martorelli1, Massimo Guidoboni1, Valli De Re2, Elena Muraro1, Riccardo Turrini7, Anna Merlo7,Elisa Pasini1, Laura Caggiari2, Luca Romagnoli8, Michele Spina3, Roberta Mortarini9, Daniela Gasparotto4,Mario Mazzucato5, Antonino Carbone6, Antonio Rosato7, Andrea Anichini9, and Riccardo Dolcetti1

AbstractPurpose: An increasing set of B-cell non-Hodgkin lymphomas (B-NHL) show a biased usage of IGKV3-

20 and IGKV3-15 immunoglobulin genes, a feature that could be exploited for the development of ready-to-

use, broadly applicable cancer vaccines.

Experimental Design: The immunogenic properties of clonal IGKV3-20 and IGKV3-15 proteins were

analyzed with particular focus on their ability to elicit cross-reactive responses against molecularly related

IGKV proteins expressed by different B-cell lymphoproliferative disorders.

Results: IGKþ lymphoma patients show humoral and T-cell responses to IGKV3-20 and IGKV3-15

proteins and IGKV3-specific cytotoxic T lymphocytes (CTL) can be easily induced ex vivo. IGKV3-20–specific

CTLs cross-react against different IGKV3 proteins, an effect mediated by the presence of 21 shared,

sometimes promiscuous, T-cell epitopes, presented by common HLA class I allele products, thus assuring

abroadHLAcoverageof IGKV3-based vaccines.Manynatural epitope variants are carriedby IGK light chains

expressed by a broad spectrumof B-NHLs andwe show that IGKV3-20–specific CTLs cross-react also against

several of these variant epitopes. Bothhumoral andCTL-specific responseswere inducedbyKLH-conjugated

IGKV3-20 protein in HLA-A2-transgenic mice and coinjection of IGKV3-20–specific CTLs with IGKV3-20þor IGKV3-15þ lymphoma cells into SCIDmice totally prevented tumor growth, thus confirming the ability

of these effectors to mediate efficient and cross-reactive cytotoxic responses also in vivo.

Conclusions: These results provide the rationale to exploit IGKV3 proteins as "off-the-shelf" vaccines for

a large fraction of lymphoma patients. Clin Cancer Res; 18(15); 4080–91. �2012 AACR.

IntroductionTherapeutic vaccines targeting B-cell non-Hodgkin lym-

phoma (NHL) idiotype (Id) represent a promisingapproach to improve the clinical control of these malig-

nancies (1, 2). Indeed, immunization with tumor B-cell Idhas been associated with induction of lymphoma-specifichumoral and cellular responses, molecular remissions, andprolongedprogression-freeordisease-free survival in severalphase I/II studies (1–8). Moreover, 1 of 3 phase III random-ized trials carried out so far has shown prolonged remissionduration in follicular lymphoma patients in first remission(9).Although the reasons for thesediverging results are still amatter of debate, differences in themethods used for vaccineproduction couldhave accounted for thedifferent outcomesobserved. In all these studies, in fact, idiotypic immunoglo-bulins (Ig) were produced in vitro from each patient usingdifferentprotocols,whichusually imply considerable effortsand costs (1, 2). Therefore, there is the need to develop Idvaccines that can be more easily manufactured and appli-cable to a larger numberof patients (10). Indeed, availabilityof broadly usable Id vaccines would considerably boost theclinical application of this therapy and allow the compar-ison of the results of different clinical trials.

Recent evidence suggests that the limitations related tothe individualized production of Id vaccines may be over-come. In fact, distinct sets of stereotyped Igs have beenidentified in various B-cell malignancies, suggesting that a

Authors' Affiliations: 1Cancer Bio-Immunotherapy Unit, 2Clinical andExperimental Pharmacology, 3Medical Oncology A, 4Experimental Oncol-ogy 1, 5Laboratory Medicine, and 6Pathology, C.R.O., IRCCS–NationalCancer Institute, Aviano; 7Department of Oncology and Surgical Sciences,University of Padova/IOV, Padova; 8Areta International, Gerenzano; and9Human Tumors ImmunobiologyUnit, Fondazione IRCCS - INT,Milan, Italy

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address forM.Guidoboni: InfectiousDisease and ImmunogeneticsSection, Clinical Center and Trans-NIH Center for Human Immunology,National Institutes of Health, Bethesda, MD.

D. Martorelli and M. Guidoboni contributed equally to the article.

Corresponding Author: Riccardo Dolcetti, Cancer Bio-ImmunotherapyUnit, Department of Medical Oncology, C.R.O. National Cancer Institute,Via F. Gallini 2, Aviano 33081, Italy. Phone: 39-0-434-659660; Fax: 39-0-434-659196; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-0763

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(15) August 1, 20124080

on April 16, 2021. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 15, 2012; DOI: 10.1158/1078-0432.CCR-12-0763

http://clincancerres.aacrjournals.org/

much higher frequency of Id sharing exists among patientsthan appreciated previously. In particular, the molecularcharacterization of Hepatitis C Virus (HCV)-related lym-phomas showed that >70% of these cases preferentially useeither IGKV3-20 or IGKV3-15 light chains, with a highdegree of homology across different lymphomas (11–14). Similar molecular features have been also found in aproportion of non-HCV–related B-NHLs, including auto-immunity-associated lymphoproliferations (13), diffuselarge B-cell lymphomas (15), marginal zone B-cell lympho-mas (16, 17), follicular lymphomas (18), multiple myelo-ma (18), and chronic lymphocytic leukemias (18–20). Onthese grounds, IGKV3-based vaccines could be used in aconsiderable fraction of patients with different B-celllymphoproliferations.This study aimed at characterizing the immunogenic

properties of IGKV3-20 and IGKV3-15 proteins with thefinal goal to provide the rationale for the use of thesemolecules as "shared" vaccines. We herein show thatthese proteins are immunogenic ex vivo and capable ofeliciting humoral and T-cell responses in patients withNHL. Notably, these recombinant proteins induce specificcytotoxic T lymphocyte (CTL) responses that cross-reactagainst related IGVK proteins expressed by different lym-phomas/leukemias. The IGKV3-20 protein, in particular,can be regarded as a "string of beads" carrying multiplepromiscuous class I-restricted T-cell epitopes that arenaturally recognized in patients with IGKV3þ lympho-mas. In addition, some of these epitopes are frequentlyshared across a broad spectrum of IGK light chain familiesused by different B-cell lymphomas/leukemias, thussupporting a broad application of these candidatevaccines.

Materials and MethodsPatients and healthy donors

Peripheral blood was collected from healthy donors and13 patients with HCV-NHL (Supplementary Table S1). Allparticipants provided signed informed consent, and thestudywas approved by local IRB.HLA typingwas conductedby standard high-resolution typing.

Production of recombinant Id proteinsSequences from 1 IGKV3-15 and 2 IGKV3-20 rearranged

genes were PCR amplified from NHLs using family-specificprimers (21). IGKV3-specific fragments were subclonedintopFLAG-MACand transformed into JM109 strainEscher-ichia coli (21). The expressed proteins were purified byaffinity column and their sequence verified by fingerprint-ing analysis of trypsin-digested extracts (22). Endotoxinlevel was <1.0 EU/mg of protein (LAL method).

Cell linesLymphoblastoid cell lines (LCL) were generated by

in vitro transformation of B cells with the B.95.8 Epstein–Barr virus isolate. The SH9 LCL was established fromperipheral blood mononucleated cells (PBMC) of a patientwith Hodgkin lymphoma. The DG75 Burkitt’s lymphomacell line was obtained from DSMZ and confirmed by fin-gerprinting (Power Plex 1.2; Promega) in April 2011. Celllines were cultured in RPMI-1640, containing 10% heat-inactivated FBS (Gibco), 2mmol/L L-glutamine, 100 mg/mLstreptomycin, and 100 IU/mL penicillin (Sigma-Aldrich)and maintained at 37�C in a 5% CO2 atmosphere.

Generation and characterization of IGKV3-specificCTLs

Generation of IGKV3- and peptide-specific CTLs wasconducted as described (23, 24). After 3 stimulations withprotein- or peptide-loaded DCs, CTL cultures were charac-terized with the following FITC-, PE-, ECD-, PC5-, PC7-labeled antibodies used in a 5-color combination: anti-CD3(UCHT1, IgG1), anti-CD45RA (2H4LDH11LDB9, IgG1),anti-CD4 (13B8.2, IgG1), anti-CD8 (SFCI21ThyD3, IgG1;Beckman Coulter), anti-CCR7 (3D12, Rat, IgG2b; BD Bios-ciences PharMingen), anti-CD11a (HI111, IgG1), anti-CD44 (IM7, Rat IgG2b), anti-CD49d (9F10, IgG1, k) andanti-CD62L (DREG56, IgG1, k; eBioscience). Mouse FITC-and PE-IgG1 (679.1Mc; Beckman Coulter) and Rat PE-IgG2a (eBRa; eBioscience) were used as isotypic controls.Cytofluorimetric analysis was conducted with a CytomicsFC500 and the data analyzed with CXP software (BeckmanCoulter). To generate peptide-specific CTL microcultures,dendritic cells (DC) were differentiated from peripheralblood monocytes of HLA-A�0201þ donors and loadedovernight with 25 mg/mL of each of 10 different IGKV3-20–derived peptides selected for HLA-A�0201 binding.The HLA-A�0201–restricted cyclin D1–derived peptide(LLNDRVLRA) and the Flu-matrix58–66 peptide served asnegative and positive controls, respectively. For each pep-tide, 30 replicate microcultures were set-up and fed with IL-

Translational RelevanceCancer vaccines for B-cell lymphomas targeting

idiotypic proteins constitute a promising therapeuticstrategy. Nevertheless, the individualized production ofidiotypic vaccines is still complex, costly, and poorlyapplicable on a large scale. Moreover, the differentmanufacturing protocols currently used to produceidiotypic vaccines make it difficult to compare theresults of different clinical trials. These limitations maybe overcome by the exploitation of the immunogenicproperties of distinct clonal immunoglobulin lightchains, such as IGKV3-15 and IGKV3-20, which arebroadly expressed by different B-cell lymphoprolifera-tions. The ability of IGKV3-based recombinant vaccinesto elicit cross-reactive responses against molecularlyrelated light chains provides the rationale to activatea phase I/II study aimed at assessing the safety andefficacy of these vaccines. Achievement of these goalswould make available a ready-to-use recombinant vac-cine for improved consolidation therapy of low-gradeB-cell lymphomas.

IGKV3 Proteins as "Off-the-Shelf" Vaccines

www.aacrjournals.org Clin Cancer Res; 18(15) August 1, 2012 4081




2 (60 IU/mL) and IL-7 (10 IU/mL) at day 2. All cultureswererestimulated at days 7 and 14 with autologous peptide-loaded and irradiated (7 Gy) PBLs (1 � 105 cells/well) andat day 21 analyzed for cytotoxicity by a calcein-AM releaseassay (23). All tests were conducted in triplicate. HLA class Iw6/32 mAb (10 mg/mL; Abcam) or HLA-A�0201–specificcr11.351 mAb (10 mg/mL; 25) were used to assess HLArestriction, and the anti-Epstein–Barr VirusNuclear Antigen1 (EBNA1) mAb (1:50; Millipore) was used as irrelevantantibody. Fluorescence intensity wasmeasured with a Spec-traFluorPlus fluorimeter (Tecan).

IFN-g ELISPOT assaysIFN-g-ELISPOT assay was conducted with a commercial

kit (Endogen) according to manufacturer’s instructionsand using as stimulators (�105 cells/well) autologousmonocytes pulsed with either 10 mg/mL of Id protein or20 mg/mL peptide, and immunomagnetically purifiedCD8þ T lymphocytes as responders (105 cells/well).Plates were evaluated by a computer-assisted ELISPOTreader (Eli.Expert, A.EL.VIS). The number of spots innegative controls never exceeded 5 spots/well. Responseswere considered significant if >5 IFN-g–producing cellswere detected.

Detection of humoral responses to IGKV3-20 inlymphoma patients

The analysis was carried out using a chemiluminescentELISA test. Briefly, 96-well microtiter plates were coatedovernight with either recombinant IGKV3-20 protein orTetanus Toxoid (TT, Calbiochem) antigens (2 mg/mL). Serafrom lymphoma patients or donors were added to the wellsin triplicate and incubated for 2 hours at room temperature(RT). Plates were washed, and horseradish peroxidase-conjugated goat antihuman IgG (Sigma-Aldrich) wasadded. After incubation for 1 hour at RT, plates werewashed, developed using chemiluminescence substrateLiteAblot (CELBIO), and read with a GENios Elisa-reader(Tecan).

T-cell epitope mapping and characterizationIdentification of IGKV3-20–derived epitopes was car-

ried out with the iTopia Epitope Discovery System (Beck-man Coulter; ref. 26). Briefly, 100 9-mer peptides over-lapping by 8 residues spanning the entire length of theprotein (108 aa) were screened for their ability to bind 8HLA class I alleles (A�0101, A�0201, A�0301, A�1101,A�2402, B�0702, B�0801, and B�1501) under optimalconditions (27). The percentage of binding was assessedusing a fluorimeter and peptides which bound MHCmolecules >20% compared with the reference positivecontrol were selected for analysis of affinity, expressed asED50 (the concentration at which the peptide is bound tothe MHC at 50% maximum binding efficiency). Peptideswere also analyzed for off-rate (complex stability) whereT1/2 represents the time taken for a reduction fromoptimal binding of 50% after a shift to dissociatingconditions (27).

IGKV3-20–specific humoral and T-cell responses inanimal models

Six- to 8-week-old female SCID or HHD mice were usedaccording to the national guidelines (28). HHD mice wereimmunized by subcutaneous (s.c.) injections of KLH-con-jugated IGKV3-20 (100 mg) emulsified in complete Freundadjuvant (Sigma-Aldrich). Subsequent injections were con-ducted at day 14, 21, 30, and 45 with 100 mg of KLH-conjugated IGKV3-20 emulsified in incomplete Freundadjuvant. HHD sera were analyzed at day 0 and 30 byELISA. Ten mg/mL of nonconjugated IGKV3-20 were coatedonto a 96-well Maxisorp NUNC-immunoplate (ThermoScientific) in 0.05M sodium carbonate/bicarbonate bufferpH 9.6 (Sigma-Aldrich), overnight at 4�C. After incubationfor 1 hour at RTwith 100mL perwell of PBS-10%FBS, plateswere incubated for 1 hour at RTwith sequential dilutions ofmouse sera in PBS-10%FBS (from1:50 to1:6400).Negativecontrol was PBS-10% FBS alone and sera at day 0 (1:50 inPBS-10% FBS). Then, plates were incubated for 1 hour at RTwith secondary HRP-conjugated goat antimouse antibody(Charles River Laboratories; 0.02 mg/mL in PBS-0.05%Tween). Finally, 100 mL per well of substrate solution(prepared using SIGMA-Fast OPD tablets) were added for5 minutes at RT in the dark. Reaction was stopped with 50mL/well of HCl 3N (Carlo Erba), and plates analyzed at 490nm using a VictorX4 instrument (Perkin-Elmer). Data wereused to generate a titration curve. The same sera used inELISA test were analyzed for their ability to stain DG75 cellsin flow cytometry.

IGKV3-20–specific CTLs were generated from HHDmiceshowing humoral responses capable of staining DG75 cellscytofluorimetrically. Candidate mice were sacrificed, theirsplenocytes isolated and cultured (25� 106) with 1 mmol/LIGKV3-20 protein or g-irradiated (60 Gy) DG75 cells (1 �105) for 4 days. Splenocytes from nonimmunized HHDmice were used as negative controls. At day 4, cells wereharvested and CTL cultures positive at the cytotoxicityassay were maintained with appropriate stimulus (either1 mmol/L protein or 1� 105 g-irradiated DG75), and testedevery 2 weeks to assess their lytic ability. Winn assay wasconducted by injecting s.c. 5 � 106 DG75 or SH9 cells/animal mixed with either RPMI or donor-derived IGKV3-20–specific CTLs (5 � 106/animal), testing 6 animals/group. Mixing was conducted by incubating cells for 5minutes at 4�C immediately before injection, to rule outT-cell/target interaction before in vivo transfer. Tumorgrowth was evaluated every 2 to 3 days using a caliper (29).

ResultsEx vivo immunogenicity of IGKV3-20 and IGKV3-15recombinant proteins

The IGKV3-15 and IGKV3-20 proteins are expressed by asizeable fraction of B-cell malignancies (Table 1). In par-ticular, IGKV3-20 is used 2 to 3 times more frequently thanIGKV3-15. Two representative IGKV3-15 and IGKV3-20recombinant proteins were used to induce CTL cultures bystimulating PBMCs of healthy donors with protein-pulsed

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Clin Cancer Res; 18(15) August 1, 2012 Clinical Cancer Research4082




autologousDC. SpecificCTL lineswere easily obtained from14 donors with either IGKV3-20 (n¼ 12) or IGKV3-15 (n¼4) proteins (Fig. 1A and Supplementary Table S2). Overall,almost 30% of the cells in the cultures (29.5� 5.9%, mean� SD; n ¼ 10) were CD8þ T cells, most of which (>90%)were effector memory (CCR7�CD45RA�) or terminally-differentiated (CCR7�CD45RAþ) T cells (SupplementaryTable S3). Notably, these cultures were also composed of asignificant proportion of CD4þ T cells (62.76� 6.20%, n¼10). All CTL cultures generatedwith IGKV3-20 or IGKV3-15showed class I-restricted cytotoxic activity against autolo-gous LCLs pulsed with the immunizing protein (Fig. 1A).Considering the derivation from antigen-experienced B

cells of most lymphomas expressing IGKV3-20 or IGKV3-15, theseproteinsmayhaveundergone theprocessof somatichypermutation and may thus show a certain degree of inter-patient variation in the amino acid sequence. This does notapparently constitute a major limitation because CTLsinduced by a prototypic IGKV3-20 light chain (VK3-20a)were also able to specifically kill autologous targets loadedwith idiotypic VK proteins of the same subfamily (VK3-20b, Figs. 1B and C) derived from unrelated NHL. Theseresults were obtained in 4 donors with different HLA class Ihaplotypes (Fig. 1B), thus showing that the CTL responsesinduced by a "prototypic" IGKV3-20 protein may have apotential immunotherapeuticvaluealsoinunrelatedpatientscarrying lymphomas showing the same VK restriction.

IGKV3 proteins induce cross-reactive CTL responsesThe IGKV3-15 and IGKV3-20 proteins show a sequence

homology >80% (Fig. 1D), a biochemical basis for theinduction of cross-reactive responses. To address this issuedirectly, we verified whether IGKV3-15–specific CTLs couldkill autologous targets presenting IGKV3-20, and vice versa.As shown in Fig. 1E, IGKV3-15- and IGKV3-20–specificcultures obtained from 3 donors significantly killed, in aclass I–restricted fashion, also mismatched targets. Intrigu-ingly, IGKV3-20–induced CTLs killed IGKV3-15–loadedtargets with an efficiency comparable to that of matchedIGKV3-15-CTLs; only in a single case, IGKV3-20-CTLsshowed about a 2-fold higher killing activity againstIGKV3-15 targets than matched CTLs (Fig. 1E).

Sequencing of IGKV genes expressed by a large panel of B-cell lines allowed the identification of the DG75 BL cell line(HLA-A�0201/6601, B�4101/5001) naturally expressingIGKV3-20, and the SH9 LCL (HLA-A�0201/0101,B�0702/0801) expressing the IGKV3-15 protein. Notably,both these cell lines were killed in an antigen-specific andHLA-A�02–restricted manner by IGKV3-20–specific CTLsobtained from donors single-matched for HLA-A�0201with target cell lines (Fig. 2). These findings indicate thatIGKV3-20 and IGKV3-15 are naturally processed in lym-phoid cells and produce "cross-reactive" epitopes.

IGKV3-specific humoral and CD8þ T-cell responses inlymphoma patients

The presence of memory T-cell responses specific forIGKV3-20 and IGKV3-15 proteins was investigated in 13patients with IGKþ HCV-NHLs. A 48-hour ELISPOT assaydisclosed low-to-moderate amounts of IFN-g–producing Tcells in themajority of patients (3-98 SFC/100.000 CD8þ Tcells), whereas all donors (n ¼ 4) showed low-levelresponses (<7 SFC/100.000 CD8þ T cells; Fig. 3A). More-over, specific humoral responses to IGKV3-20 protein werealso observed inmost of the patients fromwhom serumwasavailable (n ¼ 11; Supplementary Fig. S1).

IGKV3-20 CTL epitope mapping and validationUsing the iTopia assay (26), we identified 21 IGKV3-20

peptides able to bind at least 1 of 8 MHC allele products.Interestingly, although most peptides (n¼ 18) bound onlyone allele, the P5, P28, and P97 peptides bound 2 to 3different alleles (Supplementary Table S4). All peptideswere low-to-medium binders, with ED50 ranging from110 to 255 mmol/L (Supplementary Table S4) and half-times varying from 18 to 135minutes, similarly to other Id-derived peptides (30).

The ability of HLA-A�0201–binder peptides to elicit spe-cific T-cell responses was assessed by IFN-g-ELISPOT withpurified CD8þ T cells obtained from 3 IGKþ HCV-NHLpatients and 2 healthy donors. IFN-g–producing T cellswere found in the majority of patients with lymphoma,with higher responses for most epitopes as compared withhealthy donors (15–20 vs. 5–10 SFC/100,000 CD8þ T

Table 1. Distribution of IGKV3-15 and IGKV3-20 proteins among B-cell malignancies

IGKV3-15 IGKV3-20 IGKV3-15 or IGKV3-20 IGKV3 family

B-cell malignancy N (%) N (%) N (%) N (%) Ref.

HCV-related NHL 3/21 (14.3) 12/21 (57.1) 15/21 (71.4) 16/21 (76.2) 10, 13Diffuse large B-cell lymphoma 4/20 (20) 1/20 (5) 5/20 (25) 7/20 (35) 10, 14Splenic marginal-zone lymphomas 1/31 (3.2) 3/31 (9.7) 4/31 (12.9) 5/31 (16.1) 17Sj€ogren syndrome–associatedlymphoproliferations

2/7 (28.6) 4/7 (57.1) 6/7 (85.7) 6/7 (85.7) 12

MALT lymphomas 2/18 (11.1) 7/18 (38.9) 9/18 (50) 9/18 (50) 15, 16Follicular lymphomas — 3/47 (6.4) — 6/47 (12.8) 17Multiple myeloma 4/71 (5.6) 7/71 (9.9) 11/71 (15.5) 11/71 (15.5) 17Chronic lymphocytic leukemias 28/614 (4.6) 72/614 (11.7) 100/614 (16.3) 139/614 (22.6) 17–19






cells; Fig. 3B). In addition, epitope-specific CTLs weresuccessfully induced ex vivo from 6 different donors usingeach of 10 A�0201-restricted, IGKV3-20–derived peptides(Fig. 3C).Notably,CTLs inducedwithP20andP33peptideswere able to kill lymphoma cells expressing either IGKV3-20(DG75) or IGKV3-15 (SH9) in aHLA-A2–restricted fashion(Fig. 3D).

Cross-reactivity of CTL responses elicited by IGKV3-20–derived epitopes

The ability of IGKV3-20–specific CTLs to "cross kill"targets presenting or naturally expressing IGKV3-15prompted us to verify whether the IGKV3-20–derived epi-topes are also shared by other IGK proteins from the IGKV3as well as from unrelated IGKV families, to better define the

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Figure 1. A, CTL cultures generated with IGKV3-15 or IGKV3-20 showed class I–restricted cytotoxicity against autologous LCLs pulsed with the immunizingprotein. Recombinant p29 BARF1 protein was used as unrelated control. B, CTLs induced by the NHL patient-derived IGKV3-20a protein specificallykill also autologous targets loadedwith an unrelated IGKV3-20b. E:T ratio¼ 20:1 (P < 0.02, Student t test). C, amino acid sequence alignment of the prototypicand 2 variant IGKV3-20 proteins, IGKV3-20a and IGKV3-20b. D, sequence homology of DG75 and SH9 IGKV3 proteins. E, IGKV3-20- and IGKV3-15–specificCTL cultures derived from 3 donors killed, in a class I–restricted fashion, both matched (same IGKV3) and mismatched (IGKV3-20 vs. IGKV3-15 andvice versa) targets. The anti-EBNA1 mAb was used as irrelevant antibody.

Martorelli et al.





extent of lymphoid tumors potentially treatable withIGKV3-20–based vaccines. Sequences of T-cell epitopeswere then aligned against all germ-line IGKV sequences

retrieved from the ImMunoGeneTics information system�

(31). As a conservative rule, only peptides showing only oneamino acid change compared with the IGKV3-20 epitopeswere considered. The analysis showed that most of theIGKV3-20 epitopes are conserved in several light chains ofthe IGKV3 family (Supplementary Table S5). Interestingly,2 of these epitopes (P32 and P33) are shared also by severalother IGKV families extensively used among B-cell tumors(Supplementary Table S5). Moreover, the HLA-A allelesrestricting these peptides (A�1101 and A�2402 for P32and A�0201 for P33) would ensure a broad populationcoverage.To assess whether the cross-reactivity of IGKV3-20–spe-

cific CTLs against IGKV3-15–loaded targets was mediatedby the recognition of identical or variant epitopes, 30independent CTL microcultures, for each of 13 differentpeptide/donor conditions, were generated either using P20,P32, P33, P46, P47, and P48 peptides. Each peptide-specificculturewas tested using both the native and the correspond-ing IGKV3-15–derived variant peptide epitope as target.Part of peptide-specific microcultures were able to efficient-ly recognize and kill T2 cells loaded not only with the samenative peptide, but also with the variant epitope peptides(Supplementary Fig. S2A and S2B). More interestingly, fewcultures showed cell lysis selectively to variant peptides,although no cytotoxicity against the native epitope wasobserved (Supplementary Fig. S2). Thus, IGKV3-20-CTLsrecognize both similar and variant epitopes in IGKV3-15,further emphasizing the cross-reactivity of IGKV3-inducedresponses.We also analyzed the ability of 2 representative IGKV3-

20–derived A�0201 peptides to generate CTLs able to cross-react against 9 epitope variants (2 for P20 and 7 for P33)

differing only for 1 amino acid and carried by IGK chainsexpressed by different B-cell tumors (Supplementary TableS5). Four of these "variant" peptides had a single aminoacid substitution at the MHC anchor sites. As shown in Fig.4A, T-cell lines generated from A�0201 donors with theP20 and P33 peptides specifically recognized and efficient-ly killed autologous LCLs pulsed with all the epitopevariants as well as IGKV3-15þ and IGKV3-20þ lymphomacell lines (Fig. 4B). Notably, in some instance, the killing ofLCLs pulsed with the variant peptides was even higher thanthat induced by the "native" peptides (Fig. 4A and B). Theextent of cytotoxic responses against variant peptides wasretained at comparable levels also at low effector:targetratios (1:1) and when LCLs were pulsed with peptideconcentrations as low as 1 mg/mL (not shown), thusassuring about the specificity of recognition. All the variantpeptides investigated showed a similar or, more frequently,lower predicted binding affinity to A�0201 as comparedwith the "native" epitopes (Supplementary Table S5).These findings suggest that the IGKV3-20 protein carriesnatural heteroclytic versions of several CTL epitopes inthe IGK proteins broadly expressed among lymphoidmalignancies.

Humoral and cellular responses to IGKV3-20 in miceImmunization of HHD mice with KLH-conjugated

IGKV3-20 yielded high titers of specific antibodies (Fig.5A). Sera were also tested for the capacity of stainingcytofluorimetrically the IGKV3-20þDG75 cells. After a firstround of 3 immunizations, DG75 cells were not recognizedand additional immunizations were required to obtain adetectable staining signal (Fig. 5B). Interestingly, althoughELISA Ig titers remained almost constant (not shown),indicating that antibodies to IGKV3-20 had already reacheda plateau, repeated immunizations boosted detectablelevels of Ig capable of recognizing naturally folded, publicepitopes physiologically present on the membrane-mounted protein.

Immunization-induced CTL generation was alsoexploited in the HHD mouse model, which allows thepresentation of epitopes in the HLA-A�0201 context, andhence the generation of mouse CTLs potentially able torecognize human A�0201þ target cells. Splenocytes fromIGKV3-20–immunized mice were restimulated in vitrowithirradiatedDG75 cells and tested 4days later for lytic activity.As shown in Fig. 5C, these effectors showed a strong IGKV3-20–specificCTL activity. To assesswhether the in vitro abilityof CTL lines to target and lyse IGKV3þ cells could find an invivo counterpart in the capacity of restraining tumor growth,SCIDmicewere injected inoneflankwithDG75 tumor cellsadmixed with human IGKV3-20-specific CTLs (ratio 1:1),although the opposite flankwas inoculatedwith tumor cellsalone. ControlDG75 tumors developed in100%of injectedanimals 8 to 9 days after inoculation, and reached theallowed maximal tumor volume in about 15 days. Con-versely, at the site where DG75 cells and IGKV3-20–specificCTLs were coinjected, no growth, nor formation of tumormasses occurred over the entire observation period, thus

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Figure 2. DG75 (IGKV3-20þ) and SH9 (IGKV3-15þ) cells were efficientlykilled in a HLA-A�0201–restricted manner (cr11.351 mAb) by IGKV3-20–specific CTLs obtained from donors single-matched for HLA-A�0201with target cells. E:T ratio ¼ 20:1. IGKV3� LCLs and BARF1 p29 pulsedLCL were used as negative controls (P < 0.02, Student t test).






showing the tumor growth inhibitory activity of IGKV3-20–specific CTLs (P < 0.001, n ¼ 3; Fig. 5D). Similar findingswere observed with the IGKV3-15þ SH9 cell line, thusconfirming the cross-reactivity of IGKV3-20 CTL responsesalso in vivo (P ¼ 0.001, n ¼ 3; Fig. 5E).

DiscussionIn this study, we have extensively characterized the

immunogenic properties of the homologous IGKV3-20and IGKV3-15 proteins, which can be considered as pro-totypic "off-the-shelf" vaccines. In fact, these proteins are

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Figure 3. A, IFN-g-ELISPOT showing IGKV3-15- and IGKV3-20–specificCD8þ T-cell responses in IGKþ lymphoma patients and in 4 donors.Mean values of T-cell responses to TT were used as positive control. B, CD8þ T-cell responses against HLA-A�0201–restricted IGKV3-20 epitopes in 3 patients with NHL (top)and 2 donors (bottom). FLU M158–66 peptide was used as positive control and background responses were evaluated on empty monocytes. C, induction ofIGKV3-20 peptide-specific T-cell responses. Lysis of peptide-loaded T2 cells by day þ21 microcultures from 6 HLA-A�0201þ donors (D1–D6). For eachpeptide, and each donor, 30 independent microcultures (*) were set up and tested at 20:1 E:T ratio. Microcultures generated with the Flu-M158–66 peptidewere used aspositive control. Results expressed as specific lysis of peptide-loadedT2 cells after subtraction of lysis of empty T2cells. Gray area,mean�3SDof the spontaneous release of T2 cells. D, cytotoxic activity of CTLs induced from PBMCs of 3 donors with the P20 and P33 IGKV3-20 peptides against DG75(IGKV3-20þ) and SH9 (IGKV3-15þ) cells (E:T ratio ¼ 20:1). Lysis of an autologous LCL pulsed with IGKV3-20 is shown as a positive control. HLA-A�02restriction was confirmed with the cr11.351 antibody.

Martorelli et al.





collectively expressed by a large fraction of HCV-relatedNHL (>70%) and by a sizeable proportion of HCV-unre-lated lymphoid malignancies (Table 1). Both IGKV3 pro-teins were shown to easily elicit HLA class I–restrictedspecific CTL responses ex vivo from different donors, thusexcluding the existence of "holes" in the TCR repertoirebecause of central tolerance. These proteins are also natu-rally immunogenic in vivo, as shown by the detection ofspecific humoral and CD8þ T-cell responses in patientswith lymphoma, ruling out also the occurrence of periph-eral tolerance/ignorance. Notably, the presence a significantproportion of CD4þ T lymphocytes within the culturesgenerated with IGKV3-20 and IGKV3-15 is of relevance inthe light of the critical role of CD4þ T-cell responses inmediating the vaccine effect.An ideal "shared" IGKV3 vaccine should elicit CTL

responses effective against unrelated lymphomas expressingIGKV3 proteins. This is a relevant issue if we consider thatmost of NHLs potentially treatable with these vaccines

derive from antigen-experienced B cells, whose Id proteinsmay thus carry somatic mutations possibly affectingimmune recognition. Our findings indicate that CTLsinduced with a prototypic IGKV3-20 may be effective alsoagainst IGKV3-20 proteins derived from different NHLs,supporting the potential immunotherapeutic value of thisvaccine for unrelated patients with IGKV3-20þ lympho-mas. Furthermore, we also show that CTLs induced byIGKV3-20 or IGKV3-15 elicit mutually cross-reactiveresponses, being able to efficiently kill target cells presentingor naturally expressing the mismatched IGKV3 protein.Considering the relatively frequent usage of IGKV3-20 andIGKV3-15 among lymphoid malignancies, these findingssupport a broad applicability of these vaccines. The higherprevalence of IGKV3-20 expression and its slightly higherefficiency in eliciting "cross-reactive"CTL responses indicatethat IGKV3-20 is the preferred candidate for vaccination.

The immunogenicity of IGKV3-20 is supported by theidentification of 21 different CTL epitopes presented by

Figure 4. A, cross-reactive CTLresponses induced by CTL culturesgenerated from 4 donors with theHLA-A�0201 P20 and P33 IGKV3-20peptides against 2 and 7 variants ofthe P20 and P33 epitopes,respectively. Lysis of autologousLCLs loaded with either nativepeptides (N) or recombinant IGKV3-20 is shown as well as the HLA-A�0201 restriction (cr11.351 mAb).IGKV3-20- LCLs and cyclin D1–derived peptide (Cy D1) loaded LCLsserved as negative control (P < 0.02,Student t test). B, lytic activity ofCTLs generated from 3 donors withnative or variant HLA-A�0201 P20and P33 peptides against lymphomacells naturally expressing IGKV3-20(DG75) or IGKV3-15 (SH9).

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commonHLA class I alleles. All these peptides were low-to-medium binders, consistently with their derivation from aself-protein and with the features of other Id-derived epi-topes (30). Memory CD8þ T-cell responses to the majorityofHLA-A�0201–restricted IGKV3-20 epitopeswere detectedin patients with HCV-NHL, indicating that these epitopesare immunogenic in vivo. Moreover, we also showed thatCTLs specific for these epitopes can be generated fromdifferent A�0201þ donors. Notably, these effectors wereable to kill in a HLA-A2–restricted fashion lymphoma cellsexpressing either IGKV3-20 or IGKV3-15, showing thatthese shared epitopes are efficiently generated and pre-sented by tumor cells.

Notably, IGKV3-20 carries 3 promiscuous epitopes ableto bind 2 or 3 differentHLA class I allele products. Given therelatively well-established class I binding motifs for mostalleles (32, 33), binding of epitopes across a broad spectrum

of HLA class I alleles has been considered the exceptionrather than the rule (34, 35). Indeed, several CTL epitopescan be presented in the context of more than 1 HLA class Iallele product, although this is largely limited to the sameHLA supertype (34–37). The 3 IGKV3-20 promiscuous CTLepitopes show binding properties that extend beyond asingle HLA supertype, being in this respect similar to viralepitopes (38). Therefore, a broad HLA coverage of theIGKV3-20 vaccine is assured not only by a relatively highnumber of CTL epitopes presented by common HLA mole-cules, but also by the promiscuity of distinct epitopes.Notably, IGKV3-20 promiscuous peptides are able toinduce specific effectors with similar cytotoxic activity fromdonors with unrelatedHLA alleles (Supplementary Fig. S2Cand S2D), a relevant prerequisite for the induction ofcomparable CTL responses across different HLAbackgrounds.

Figure 5. A, ELISA assay carried outwith unconjugated IGKV3-20protein showing the absorbancevalues of sera from 4 mice (blackand white circles and triangles)immunized with IGKV3-20-KLH(day 31). Black squares, meanvalues of absorbance of sera fromthe same mice on day 0. B, flowcytometry of DG-75 cells stainedwith different mouse sera obtainedafter 31 (left) and 60 days (right)from immunization. C, cytotoxicactivity ofCTLsobtained fromHHDmice immunized with IGKV3-20and restimulated in vitro withg-irradiated DG75 cells against theIGKV3-20þ DG-75 cell line (blackcircles) or an IGKV3-20–negativeB-cell line (white circles). As acontrol, splenocytes fromnonimmunized HHD mice weresimilarly restimulated and used aseffectors against the same targetcells (black and white squares,respectively). D and E, Winn assay.SCID mice (n ¼ 3) were injectedwith the DG-75 (D) or SH9 (E) celllines alone (white circles) andpremixed with IGKV3-20–specificCTLs (black circles) at day 0. Thepanel shows the tumor massgrowth. Statistical analysis wasconducted with ANOVA (repeatedmeasures of variance analysis).Both Winn assays were repeatedtwice using 2 different donors forCTL generation.

Martorelli et al.





One of the most intriguing results of this study is thedemonstration that 2 IGKV3-20–derived epitopes are fullyconserved and shared by various IGKV families broadlyexpressed among lymphomas/leukemias. Moreover, sever-al "variants" of the IGKV3-20–derived epitopes are carriedby clonotypic IGKV proteins expressed by different lym-phoid tumors. Notably, IGKV3-20–specific CTLs efficientlyrecognize and kill autologous targets presenting differentA�0201-restricted IGKV3-20 "variant" epitopes, oftenwith ahigher efficiency as compared with that elicited against the"native" epitopes. This is consistent with the lower pre-dicted affinity showed by some of the "variant" peptides,which carry single amino-acid substitutions at the MHCanchor sites. These findings indicate that the IGKV3-20protein carries "natural" heteroclitic versions of several CTLepitopes shared byVK/VJ proteins broadly expressed amonglymphoid malignancies. Available evidence indicates thatheteroclitic peptide modifications can increase the immu-nogenicity of low-binding peptides, thus leading toimproved ability to generate CTL responses against primarytumors (39–41). The enhanced immunogenicity of heteroc-litic peptides has been elegantly shown also using analogpeptides derived from native epitopes of Ig heavy chains(42). Moreover, heteroclitic peptides from various tumorantigens are now widely applied in clinical studies, both assingle peptides and included inmultiepitope vaccines (43).The demonstration that the IGKV3-20 protein naturallycarries multiple CTL epitopes shared among various IGKVfamilies, and that may behave as heteroclitic peptidesfurther supports the applicability and the potential efficacyof this vaccine in a discrete proportion of IGK-expressingleukemias/lymphomas.We also show that the IGKV3-20 protein is immunogenic

in mice, being able to elicit both humoral and CTL-specificresponses. Although the relevance of this model may beconsidered somehow modest because of the foreign natureof the IGKV3-20 protein into a mouse environment, none-theless HHDmice proved capable of generating IGKV3-20–specific, HLA-A2–restricted CTLs that, in turn, correctlyrecognized and lysed the relevant human targets, thussupporting the potential role of vaccination to elicit anti-lymphoma therapeutic T-cell responses. In this regard,coinjection of human IGKV3-20–specific CTLs withIGKV3-20þ or IGKV3-15þ lymphoma cells into SCIDmicetotally prevented the growth of tumor cells, thus confirmingthat these effectors mediate efficient and cross-reactivecytotoxic responses also in vivo.One possible limitation of the use of single-chain, recom-

binant IGKV3 proteins as "shared" vaccines is related to theloss of the unique B-cell epitopes generated by the associ-ation of the variable portions of the heavy and light chains.Although this may prevent the induction of therapeuticallyrelevant anti-Id antibodies (44), single-chain Id proteinsmay also elicit the generation of antibodies to public deter-minants conserved in the native Ig. Our results support thispossibility, even though repeated immunizations are prob-ably needed to obtain antibodies able to detect the nativeIGKV3-20 and thus potentially able to mediate clinically

relevant responses. Another potential limitation of our "off-the-shelf" vaccine is the possible deletion of a fraction ofnormal IGKV3þ B lymphocytes. Available evidence, how-ever, indicate that it is unlikely that this could constitute amajor concern, as phase II and III trials have convincinglyshown that Id vaccines including both heavy and lightchains are safe andassociatedwithonlymodest toxic effects,with no evidence of immune compromise related to deple-tion of part of B-cell repertoire.

Our findings are consistent with the recent identificationof CTL epitopes in clonal Ig light chains, mainly belongingto different IGV lambda gene families (45). Notably, thesingle IGKV epitope identified was a somatically mutatedversion of our IGKV3-20 p48peptide (Supplementary TableS2). Thedemonstration that severalCTL epitopes are carriedby a single Id light chain such as IGKV3-20 supports a rolefor this protein as "off-the-shelf" vaccine for extended sub-sets of B-cell tumors. Besides overcoming the need forcustomized vaccine production, the IGKV3-20 vaccine mayhave the additional immunological advantages to be acarrier of a set of peptides naturally selected for heterocliticsubstitutions associated with nearly optimal immunogenicfeatures. This set of T-cell epitopes potentially covers a highproportion of IGKV-restricted B-cell tumors, as at least 1IGKV3-20 epitope can be found in several light chains fromdifferent IGKV families. Moreover, IGKV3-20 also containspromiscuous epitopes, thus assuring a broadHLA coverage.On the whole, these data provide the rationale to activate aphase I/II study aimed at assessing the safety and efficacy ofthe IGKV3-20 vaccine for patients with lymphomas expres-sing molecularly correlated Id proteins. Achievement ofthese goals would make available a ready-to-use recombi-nant vaccine for improved consolidation therapy of low-grade B-cell lymphomas.

Disclosure of Potential Conflicts of InterestR. Dolcetti is a consultant/advisory board member of Areta Int. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design:M.Guidoboni, V.D. Re, R. Mortarini, A. Rosato, A.Anichini, R. DolcettiDevelopment of methodology: D. Martorelli, M. Guidoboni, V.D. Re, L.Romagnoli, R. Mortarini, D. Gasparotto, M. Mazzucato, A. Rosato, A.AnichiniAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): D. Martorelli, V.D. Re, E. Muraro, R. Turrini, A.Merlo, L. Romagnoli, M. Spina, M. Mazzucato, A. RosatoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):D. Martorelli, M. Guidoboni, V.D. Re, R.Turrini, A. Merlo, R. Mortarini, A. Rosato, A. AnichiniWriting, review, and/or revision of the manuscript: D. Martorelli, M.Guidoboni, V.D. Re, R. Turrini, A.Merlo, M. Spina, R.Mortarini, A. Carbone,A. Rosato, A. Anichini, R. DolcettiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. Martorelli, V.D. Re, E. Pasini, L.Caggiari, A. Rosato, R. DolcettiStudy supervision: V.D. Re, A. Rosato, R. Dolcetti

AcknowledgmentsThe authors thank T. Ooka (Universit�e Claude Bernard Lyon-1, France)

for the kind gift of recombinant p29 BARF1 protein, A. Molla and C. Vegetti(Fondazione IRCCS, INT Milan) for technical support, and Miss S. Colussi(CRO-IRCCS, Aviano) for the editing.






Grant Support

The study was supported by grants from the European Community(FP6 037874), the Italian Ministry of Health (ACC-4), the AssociazioneItaliana per la Ricerca sul Cancro (to R. Dolcetti, A. Anichini, R. Mortar-ini), and the Fondazione Federica per la Vita. Elisa Pasini is a FIRCfellow.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 5, 2012; revised May 22, 2012; accepted May 31, 2012;published OnlineFirst June 15, 2012.

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2012;18:4080-4091. Published OnlineFirst June 15, 2012.Clin Cancer Res Debora Martorelli, Massimo Guidoboni, Valli De Re, et al.

Restricted B-Cell Non-Hodgkin Lymphomas−Kappa-Light Chain IGKV3 Proteins as Candidate ''Off-the-Shelf'' Vaccines for

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IGKV3 Proteins as Candidate Off-the-Shelf Vaccines for ... · Cancer Therapy: Preclinical IGKV3 Proteins as Candidate "Off-the-Shelf" Vaccines for Kappa-Light Chain–Restricted B-Cell