Generation of Tumor Antigen-Speciﬁc T Cell Lines …...Cancer Therapy: Preclinical Generation of...

Cancer Therapy: Preclinical

Generation of Tumor Antigen-Specific T Cell Lines fromPediatric Patients with Acute Lymphoblastic Leukemia—Implications for Immunotherapy

Gerrit Weber1,2,5,6, Ignazio Caruana1,2,5,6, Rayne H. Rouce2,6,7, A. John Barrett8, Ulrike Gerdemann1,2,5,6,Ann M. Leen1,2,5,6, Karen R. Rabin2,6,7, and Catherine M. Bollard1,2,3,4,5,6

AbstractPurpose: Although modern cure rates for childhood acute lymphoblastic leukemia (ALL) exceed 80%,

the outlook remains poor in patients with high-risk disease and those who relapse, especially when

allogeneic hematopoietic stem cell transplantation is not feasible. Strategies to improve outcome and

prevent relapse are therefore required. Immunotherapy with antigen-specific T cells can have antileukemic

activity without the toxicities seen with intensive chemotherapy, and therefore represents an attractive

strategy to improve the outcome of high-risk patients with ALL. We explored the feasibility of generating

tumor antigen-specific T cells ex vivo from the peripheral blood of 50 patients with ALL [26 National Cancer

Institute (NCI) high-risk and 24 standard-risk] receiving maintenance therapy.

Experimental Design: Peripheral blood mononuclear cells were stimulated with autologous dendritic

cells pulsed with complete peptide libraries of WT1, Survivin, MAGE-A3, and PRAME, antigens frequently

expressed on ALL blasts.

Results: T-cell lines were successfully expanded from all patients, despite low lymphocyte counts and

irrespective ofNCI risk group. Antigen-specificity was observed inmore than 50%of patients after the initial

stimulation and increased to more than 90% after three stimulations as assessed in IFN-g-enzyme-linked

immunospot (ELISpot) and 51Cr-release assays. Moreover, tumor-specific responses were observed by

reduction of autologous leukemia blasts in short- and long-term coculture experiments.

Conclusion: This study supports the use of immunotherapy with adoptively transferred autologous

tumor antigen-specific T cells to prevent relapse and improve the prognosis of patients with high-risk ALL.

Clin Cancer Res; 19(18); 5079–91. �2013 AACR.

IntroductionThe outcome of patients with acute lymphoblastic leu-

kemia (ALL), the most common childhood leukemia, hassignificantly improved with the advent of intensive chemo-therapy regimens that can achieve cure rates of more than90%.However, for patients classified as high-risk, accordingto National Cancer Institute (NCI) criteria, or those whorelapse, the best chance of cure is allogeneic hematopoieticstem cell transplantation (HSCT; ref. 1). Not all patients are

eligible for HSCT and over a third of transplanted patientsstill relapse afterHSCT for ALL (1, 2). Therefore, strategies toprevent relapse in patients with ALL, classified as high-risk,are needed. Immunotherapy is an effective tool for thetreatment of leukemia. First shown in patients with chronicmyelogenous leukemia (CML), the infusion of unselecteddonor lymphocytes (DLI) has been shown to stably erad-icateminimal residual disease (3, 4). In ALL, a high absolutelymphocyte count at the end of induction therapy is asso-ciatedwith improved survival, suggesting the importance ofthe host immune response in controlling disease (5). Inaddition, in the transplant setting, there is strong evidencefor a T cell–mediated graft-versus-leukemia effect (6). How-ever, DLI rarely induces responses in patients relapsing afterHSCT (7–9), suggesting that the antileukemic effects of Tcells may require augmentation to prevent or treat relapsedALL. Recently, T cells genetically modified with chimericantigen receptors (CARs) targeting a CD19 epitope wereshown to induce prolonged remissions in patients withrelapsed ALL (10, 11). However, relapse can occur byselection of a CD19 negative leukemia cell population(12). We have shown that T cells with native tumor-spec-ificity can be activated and expanded using single epitopes

Authors' Affiliations: 1Center for Cell and Gene Therapy, Departments of2Pediatrics, 3Medicine, and 4Immunology,BaylorCollegeofMedicine; 5TheMethodist Hospital; 6Texas Children's Hospital; 7Texas Children's CancerCenter, Houston, Texas; and 8Hematology Branch, National Heart, Lung,and Blood Institute, NIH, Bethesda, Maryland

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

Corresponding Author: Catherine M. Bollard, Center for Cell and GeneTherapy, Baylor College of Medicine, 1102 Bates St, Suite 1770.01,Houston, TX 77030. Phone: 832-824-4781; Fax: 832-825-4732; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-0955

�2013 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 5079

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Published OnlineFirst July 9, 2013; DOI: 10.1158/1078-0432.CCR-13-0955

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of tumor antigens as stimulus in vitro for adoptive celltransfer or in vivo in response to vaccines (13, 14). Further-more, we have shown that, in healthy donors, it is possibleto induce T cells specific for multiple tumor antigens whichcan target and kill acute myeloid leukemia cells (15).

We therefore chose to evaluate the feasibility of generat-ing and expanding tumor antigen-specific T-cell lines frompatients with ALL as a potential adoptive immunothera-peutic strategy to prevent relapse in high-risk patients orpatients not eligible for allogeneic HSCT. ALL cells express anumber of tumor-associated antigens (TAAs). We selectedWT1 (16), Survivin (17, 18), MAGE-A3 (19), and PRAME(6, 20) as target antigens for the generation of tumorantigen-specific T cells with the aim of broadening theapplicability of T-cell therapy to the majority of patientswith ALL, and reducing immune escape by the leukemiathrough emergence of clones deficient in TAA.

In this study, we have developed a novel strategy toactivate autologous T cells targeting multiple tumor anti-gens. We showed that by using both autologous dendriticcells and phytohemagglutinin (PHA)-blasts as antigen-pre-senting cells (APC),we can successfully expandTAA-specificT-cell lines from 50 patients with ALL during maintenancetherapy, irrespective ofNCI risk status or lymphocyte count.

Materials and MethodsPatient samples

Peripheral bloodwas obtained from50 pediatric patientswith ALL, receivingmaintenance chemotherapy at the TexasChildren’s Cancer Center (Houston, TX). All families had

provided written informed consent on treatment proto-cols approved by the Baylor College of Medicine Institu-tional Review Board in accordance with the Declarationof Helsinki. Approximately, 40 mL of blood was collect-ed from 50 patients with ALL, 24 standard-risk and 26high-risk patients, according to criteria (ref. 21; Supple-mentary Table S1). Peripheral blood mononuclear cells(PBMC) were isolated by density gradient centrifugationand cryopreserved.

Generation of antigen-presenting cellsMonocyte-derived dendritic cells were generated by plate

adherence of PBMC. PBMC were incubated for 2 hours indendritic cell media (CellGro DC media; CellGenix) sup-plemented with 2 mmol/L GlutaMax (Invitrogen). Non-adherent cells were collected and washed. Adherent cellswere cultured in dendritic cell media in the presence ofinterleukin (IL)-4 (1,000 U/mL) and granulocyte macro-phage colony-stimulating factor (GM-CSF; 800U/mL; bothR&D). On day 5, immature dendritic cells were matured indendritic cell media with a cytokine cocktail consisting ofIL-4 (1,000 U/mL), GM-CSF (800 U/mL), IL-6 (10 ng/mL),TNF-a (10ng/mL), IL-1b (10ng/mL; all R&D), andPGE2 (1mg/mL; Sigma-Aldrich), and were harvested after 48 hoursof maturation for use as APC.

For PHA-blast generation, PBMC were stimulated with themitogen PHA-P (5 mg/mL; Sigma-Aldrich) in presence ofIL-2 to promote blast formation (PHA-blasts). PHA-blastswere cultured in RPMI-1640 supplemented with 5% humanserum (GemCell; Gemini Bio-Products), 2 mmol/L Gluta-Max, and IL-2 (100 U/mL; Teceleukin; Chiron Therapeutics).

Generation of TAA-specific T-cell linesTAA-specific T-cell lines were generated from total PBMC.

Matured dendritic cells were harvested and used as APC andpeptide-pulsedwith amix of four peptide libraries (PepMix;JPT Peptide Technologies): WT1, Survivin, MAGE-A3, andPRAME.Dendritic cells were used at a stimulator-to-effectorratio of 1:10. T cells were cultured in RPMI-1640 supple-mented with 45% Clicks media (Irvine Scientific), 5%human AB serum, and 2 mmol/L GlutaMax. For initialstimulation, a cytokine mix containing IL-7 (10 ng/mL),IL-12 (10 ng/mL), IL-15 (5 ng/mL) (all Peprotech), and IL-6(100 ng/mL) was added (15, 22). T cells were restimulatedwith peptide-pulsed autologous irradiated (30 Gy) PHA-blasts at a ratio of 1:1 on day 10 to 12 and cultures weremaintained in IL-2 (50 U/mL)–supplemented media andrestimulated every 7 days as described previously for aminimum of four stimulation cycles, but could be kept inculture for up to eight restimulations without loss of spec-ificity. No further selection or enrichment of T cells wascarried out at any point throughout the sensitization andexpansion period.

IFN-g enzyme-linked immunospot assayPeptide recognition was tested in an IFN-g-enzyme-

linked immunospot (ELISpot) assay (23). Recognition ofthe pooled TAAs as well as single antigens was tested as

Translational RelevanceAlthough the majority of pediatric patients with acute

lymphoblastic leukemia (ALL) have excellent outcomes,the prognosis remains poor in patients with high-riskdisease and thosewhorelapse, especiallywhenallogeneichematopoeietic stem cell transplantation is not feasible.Several new treatments are being investigated, includingimmune-based therapies. Immunotherapy approacheshave included vaccines, which generally target a singleHLA-restricted epitope. More recently, CD19-specificantibodies and CD19CAR gene-modified T cells haveshown remarkable successes for CD19þ malignancies,including pediatric ALL. Targeting single antigens caneliminate cells expressing the targeted epitope, but canlead to outgrowth of antigen-negative populations. Thisarticle validates a novel approach expanding T cells frompediatric patients with ALL, targeting multiple tumor-associated antigens. Simultaneous targeting of multipleantigens may decrease the risk of tumor immune escapewhen T cells are administered in vivo. Hence, this inno-vative immunotherapeutic strategy has the potential toincrease the potency of vaccines and CD19-directed ther-apies to prevent relapse and improve the prognosis ofpatients with high-risk ALL.

Weber et al.

Clin Cancer Res; 19(18) September 15, 2013 Clinical Cancer Research5080




compared with no-peptide (media) control (labeled ascontrol in all graphs) and irrelevant peptide (NY-Eso-1),which was not used for T-cell generation.Millipore Multi Screen HTS filter plates (Millipore) were

coated with IFN-g capture antibody (Mabtech) at a con-centration of 10 mg/mL over night at 4�C. Plates werewashed with PBS and blocked for 1 hour at 37�C to ruleout nonspecific protein binding. T cells were washed andresuspended and stimulated with PepMix or single pep-tides at a concentration of 0.1 mg/mL. The plates wereincubated for 16 to 20 hours at 37�C. For development,plates were washed in PBS/0.05% Tween 20 and incubatedwith biotinylated IFN-g detection antibody (0.5 mg/mL;Mabtech) for 2 hours at 37�C, followed by incubationwith streptavidin-coupled alkaline phosphatase complex(Vectastain; Vector Laboratories) for 1 hour at roomtemperature and spots were developed by incubation with3-amino-9-ethylcarbazole substrate solution. Spot-form-ing cells (SFC) were counted and evaluated by ZellnetConsulting using an automated plate reader system (KarlZeiss).

HLA-blocking experimentsHLA-restriction of antigen recognition was tested in IFN-

g–ELISpot using autologous PHA-blasts pulsed with therelevant peptide (positive control) or without peptide (neg-ative control) and blocking antibodies against HLA class Iand II (bothBDBiosciences). ELISpot plateswere incubatedand developed as described earlier.

51Chromium-release cytotoxicity assayCytolytic activity of T cells was assessed in a 51Chro-

mium (51Cr)-release cytotoxicity assay. Autologous PHA-blasts were peptide-pulsed and labeled with 51Cr for 1hour at 37�C, washed, and resuspended. Effector cellswere used at effector-to-target (E:T) ratios of 40:1 to1.25:1 and tested against peptide-pulsed target cells[specific and irrelevant peptide (NY-Eso-1) not used forT-cell generation] and unpulsed target cells and incubat-ed for 4 to 6 hours. Spontaneous lysis was determined bymeasuring 51Cr-release into the supernatant on a gam-ma-counter. Spontaneous release was assessed by incu-bating target cells alone, maximum lysis by addition of1% Triton X-100 (Sigma-Aldrich). Specific lysis was cal-culated as follows:

Specific lysis ð%Þ ¼ 100� ½ðexperimental release

� spontaneous releaseÞ/ðmaximum release

� spontaneous releaseÞ�:

Phenotyping of T-cell linesT-cell lines were phenotyped by extracellular antibody

staining with anti-CD3, CD4, CD8, CD45RA and RO,CCR7, CD62L, CD56, and CD19 (all BD Biosciences) andanalyzed on a BD FACSCalibur Flow Cytometer. Controlsamples labeled with appropriate isotype-matched antibo-dies were included in each experiment. Data were analyzedusing FlowJo Flow Cytometry software (TreeStar).

Coculture with autologous blastsTo test recognition of autologous blasts, TAA-specific T-

cell lines were cocultured with each patient’s autologousleukemic bone marrow sample, which had been cryopre-served at diagnosis. Coculture experiments were carried outat ratios of 10:1 or 5:1 (T cells:leukemic blasts) for 3 daysin presence of IL-2 (50 U/mL). Nonspecific autologous Tcells were used as negative controls. Leukemia cells werequantified by costaining with anti-CD10 and CD19 anti-bodies and samples were acquired on a BD FACSCaliburFlow Cytometer using Count Bright absolute countingbeads (Molecular Probes) for quantification of absolutecell counts.

ELISADetection of cytokine release in the supernatants of

cocultured T cells was analyzed by an ELISA specific forIFN-g and IL-4 (both R&D). For detection of IFN-g , a 96-well microplate was coated with anti-IFN-g capture anti-body and incubated overnight. Cell culture supernatantswere incubated and specific binding of IFN-g was detectedby incubationwith a biotinylated detection antibody, strep-tavidin-coupled horseradish peroxidase and developed byincubationwith substrate solution.Optical density of plateswas read at 450 nmwith awavelength correction at 570 nm.For IL-4, the same principle was applied and optical densitywas determined at 490 nm. Cytokine release was quantifiedin comparison with a specific standard; the assay was run induplicates.

Colony-forming unit assayA colony-forming unit (CFU) assay was carried out to

showblast elimination in a long-term coculture. T cells werecoculturedwith autologous leukemic blasts at a 10:1 ratio inIscove’s modified Dulbecco’s medium (IMDM; Invitrogen)substituted with 10% FBS and 2 mmol/L GlutaMax for 6hours. After this preincubation, wells were collected andincubated in MethoCult methylcellulose media (STEM-CELL Technologies) in duplicates. Colonies were countedon day 14 and relative inhibition of colony formationcalculated in comparisonwith nonspecific T cells as follows:

Relative inhibition ð%Þ ¼ 100� ½ðCFUspecific T cells/

CFUnonspecific T cellsÞ � 100�:

Statistical analysisData are summarized as mean � SD or mean � SE, as

noted in the text or figure legends. Student t test was used todetermine whether there was a statistically significant dif-ference between samples, with two-tailed P values less than0.05 indicating a significant difference.

ResultsTAA-specific T-cell lines can be reliably expanded frompatients with ALL onmaintenance therapy, irrespectiveof their NCI risk status or absolute lymphocyte count

TAA-specific T-cell lines were generated from peripheralblood obtained from50 patients with ALL, 26 designated as

Tumor Antigen-Specific T Cells in Children with ALL

www.aacrjournals.org Clin Cancer Res; 19(18) September 15, 2013 5081




high-risk and as 24 standard-risk according to NCI criteria,during the maintenance phase of chemotherapy. Bloodsamples were collected at least 3 months after the start ofmaintenance therapy to minimize effects of prior, moreintensive chemotherapy andwere used for the generation ofdendritic cells, PHA-blasts, and T-cell lines. Stimulation ofPBMC was carried out with antigen–pulsed dendritic cellsusing four complete peptide libraries spanning the entireamino acid sequence of the TAAsWT1, Survivin, MAGE-A3,and PRAME.

Generation of dendritic cells as well as T-cell lines waspossible from all samples obtained, despite 76% of patientshaving low absolute lymphocyte counts of less than 1,000/mL(Supplementary Table S1). At least a minimal expansion of1.2-fold could be achieved in all patients with a maximum31.4-fold expansion (mean 6.4-fold) after three stimulati-ons (Fig. 1A). Phenotyping of the ex vivo–expanded T-celllines showed a mean CD3þ content of 97.2% (range,80.3%–99.9%) and varying distribution of CD4þ (mean,38.4%; range, 8.3%–89.4%) andCD8þ (mean, 42.6%; range,7.9%–82.1%) T cells, few natural killer cells (mean, 1.3%;range, 0%–10.9%) and rare residual B cells (mean, 0.2%;range, 0%–5.8%; Fig. 1B). Themajority of bothCD4þ (mean,51.2%; range, 12.0%–93.0%) and CD8þ (mean, 49.3%;range, 4.0%–91.0%) T cells were composed of CD45ROþ

/CD62L�/CCR7� T cells in accordance with an effector-mem-ory phenotype. Very few na€�ve CD45RAþ/CD62Lþ/CCR7þ

cells (CD4þ:mean, 1.0%, range, 0%–8%;CD8þ:mean, 0.2%,range, 0%–3.0%) and central-memory CD45ROþ/CD62Lþ/CCR7þ cells (CD4þ: mean, 5.2%, range, 0.1%–33.0%;CD8þ: mean, 2.9%, range, 0%–13.0%) were present afterthree restimulations (Fig. 1C and D).

Ex vivo–expanded TAA-specific T cells recognizemultiple target antigens with broad epitope specificity

Antigen specificity of the ex vivo–expandedT-cell lineswasevaluated weekly in response to the mix of TAAs andindividual antigens in IFN-g-ELISpot assay. Specific reactiv-ity against the pooled TAAs after the initial stimulationcould be seen in 54% of standard-risk and 65% of high-risk patients (data not shown). After three stimulations,92% of standard-risk and 96% of high-risk patients showedspecific responses against at least one antigen. There wasalmost equal distribution of standard-risk versus high-riskpatients showing reactivity against one (21% standard-risk;19% high-risk), two (21% standard-risk; 23% high-risk),three (21% standard-risk; 23% high-risk), or all four anti-gens (29% standard-risk; 19% high-risk; Fig. 2A). Themajority of the expanded TAA-specific T-cell lines elicitedresponses against PRAME (79% standard-risk vs. 69%high-risk), followed by WT1 (71% standard-risk vs. 54%high-risk) and MAGE-A3 (58% standard-risk vs. 57% high-risk). In contrast, T-cell responses to Survivin were seen in aminority of patients (42% standard-risk vs. 27% high-risk; Fig. 2B). Similarly, highest specificity as measuredby spot counts in IFN-g-ELISpot assays,was seen in responseto the pooled TAAs (standard-risk: mean, 312, range, 31–970; high-risk: mean, 258, range, 17–777) and PRAME(standard-risk: mean, 230, range, 0–851; high-risk: mean,173, range, 2–681), followed by MAGE-A3 (standard-risk:mean, 168, range, 0–1,324; high-risk: mean, 127, range,0–562), WT1 (standard-risk: mean, 121, range, 2–534;high-risk: mean, 145, range, 2–642), and least againstSurvivin (standard-risk:mean, 109, range, 0–541; high-risk:mean, 84, range, 2–498; Fig. 2C).

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Figure 1. Expansion and phenotypeof T-cell lines. A, n-fold (mean �SD) expansion of antigen-specificT-cell lines generated frompatientswith ALL during maintenancetherapy. Cell counts wereassessed at the end of eachrestimulation cycle on days 10, 18,and 25 of culture (n ¼ 50). B,phenotype of T-cell lines after thethird stimulation; gated on livelymphocytes. C, T-cell subsetswithin the CD3þ/CD4þ population.D, T-cell subsets within the CD3þ/CD8þ population.

Weber et al.





Cytolytic activity of T-cell lines was tested in a standard51Cr-release assay against peptide-pulsed autologous PHA-blasts. Mean lysis of target cells pulsed with the pooled TAAsat anE:T ratioof 20:1was 27% for T-cell lines generated fromthe standard-risk patients (range, 0%–77%) versus 31% forthe high-risk patients (range, 0%–90%). Killing of targetspulsed with the individual antigens for the standard-riskpatients showed: mean, 13% (range, 0%–66%) for WT1;mean, 10% (range, 0%–58%) for Survivin; mean, 12%(range, 0%–70%) for MAGE-A3; and mean, 26% (range,0%–74%) for PRAME. No appreciable differences in cyto-lytic responses were seen in T-cell lines generated fromhigh-risk patients:mean, 15% (range, 0%–100%) forWT1;mean,8% (range, 0%–58%) for Survivin; mean, 16% (range, 0%–68%) for MAGE-A3; and mean, 21% (range, 0%–55%) forPRAME (Fig. 2D). No killing (<6%) of unpulsed target cellswas observed.

Polyclonal MHC class I- and II–mediated T-cellresponses can be elicited in vitro from patients ontreatment of ALLTo compare the epitope-specific responses in T-cell lines

generated from patients with ALL to those previouslydescribed in healthy donors (15, 24–28), we conductedepitope mapping for WT1 in 11 T-cell lines. Reactivityagainst minipools of 15mer peptides overlapping by 11amino acids spanning the whole sequence of WT1 wastested, as previously described (29). Subsequently, confir-mation of single-peptide recognition and HLA-restrictionwas conducted (15).Results of the epitope-mapping experiments of a repre-

sentative TAA-specific T-cell line are shown in Fig. 3. As seenin Fig. 3A, IFN-g-ELISpot assay showed 70 SFC/105 cells inresponse to pooled TAAs after the initial stimulation, which

increased to 200 SFC/105 cells after three in vitro stimula-tions. The expanded T-cell line recognized both WT1 andPRAME (Fig. 3B). Epitopemapping was then conducted forWT1, which showed reactivity against two regions withinWT1, peptides #9/10 (QWAPVLDFAPPGASAYGSL) andpeptides #71/72 (LCGAQYRIHTHGVFRGIQD). UsingHLAclass I- and class II–blocking antibodies, the overlappingsequence of peptides #71/72 was confirmed to contain anHLA class I–restricted epitope previously published byWolfl and colleagues (28; Fig. 3C; Table 1). In contrast,recognition of peptides #9/10wasHLA class II–restricted, asconfirmed by blocking experiments. This 15mer peptide isknown to contain both HLA class I- and II–restricted epi-topes, however, different from the HLA-restrictions seen inour patient (ref. 24; Fig. 3D; Table 1). In all, analysis of theWT1-specific T-cell responses in 11 patient-derived T-celllines identified a total of 11 15mer class I- and II–restrictedpeptides, as shown in Table 1. The majority of these pep-tides have previously been described and identified to beimmunogenic in healthy volunteers (15, 24–28). Hence,these results show that we can successfully generate antigen-specific T-cell lines from patients with ALL, and the spec-trum of the specificity in these lines is similar to that ofhealthy donors with a range of class I- and II–restrictedepitopes (15, 24). Furthermore, the polyfunctionality ofthe expanded tumor antigen-specific T cells is shown in Fig.3E where a T-cell line with specificity against all the targetedantigens released both IFN-g and TNF-a in response tostimulation.

Antigen recognition and antileukemic activity againstautologous bone marrow blast samples

We next investigated whether TAA-specific T cells gener-ated from patients with ALL were able to recognize and kill

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Figure 2. Summary of antigen-specific responses in IFN-g-ELISpot and cytotoxicity assays.A, number of antigens recognizedin IFN-g-ELISpot by antigen-specific T-cell lines generated frompatients classified as standard-risk(SR; n¼ 24; left) or high-risk (HR; n¼ 26; right). B, percentage ofpatients recognizing each of thefour antigens or the mix of allantigens (TAA) used for T-cellgeneration in IFN-g-ELISpot assay(standard-risk, white bars; high-risk, black bars). C, number of SFC/105 cells (mean � SE) in standard-risk (n ¼ 24; white bars) and high-risk (n¼ 26; black bars) patients foreach of the antigens and TAA.Responses were assessed afterthe third stimulation. D, cytolyticactivity in a standard 51Cr-releaseassay against peptide-pulsedautologous target cells (PHA-blasts) at an E:T ratio of 20:1 (mean� SE; standard-risk, n ¼ 24, whitebars; high-risk, n¼ 26, black bars).






autologous leukemia cells in vitro. Coculture experimentswere set up for 8 patient-derived TAA-specific T-cell linesusing, as targets, autologous ALL blasts obtained from bonemarrow aspirates that had been cryopreserved at diagnosis.Antigen-specificity aswell as short- and long-term cocultures

were evaluated in IFN-g-ELISpot, flow-based, and CFUassays. In addition, the coculture supernatants were evalu-ated by IFN-g and IL-4 ELISA assays. Nonspecific T cellsderived from the same patient were used as controls. InELISpot assays at a 1:1 ratio of leukemia cells to T cells, the

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Figure 3. HLA-restriction of antigenrecognition. IFN-g-ELISpotresponses during the course ofthree restimulation cycles againstTAA after the initial stimulation (A)and against the four individualantigens after the third stimulation(B). Epitopemapping forWT1usinga 15mer peptide libraryoverlapping by 11 amino acidsspanning the whole sequence ofWT1 indicated recognition of tworegions within the WT1 sequence:WT1 peptides #71 and #9. C, IFN-gsecretion in response to peptide#71 can be inhibited by HLAclass I–blocking antibody inELISpot assay, which implies aCD8þ-restricted recognition ofthis epitope. Autologous peptide-pulsed APCs were used forstimulation or preincubated withblocking antibodies. D, peptide #9recognition is reduced by HLAclass II–blocking antibodyindicating a CD4þ-restrictedrecognition of this peptide (A–D,mean � SD). E, intracellulardetection of INF-g and TNF-a afterantigen stimulation in presence ofcostimulatory antibodies anti-CD28 and CD49d, Brefeldin, andMonensin followed by intra- andextracellular antibody stainingshows cytokine production atvarying levels restricted to theCD8þ population. Plots shownare gated on live lymphocytes andCD3þ cells.

Weber et al.





Tab

le1.

Immun

ogen

icWT1

pep

tides

iden

tified

byep

itopemap

pingof

WT1

-spec

ificTce

llsderived

from

patientswith

ALL

Patient

ID15

mer

no.

AA#

Pep

tidese

que

nce

HLA

-restriction

HLA

-A

HLA

-B

HLA

-DRB1

HLA

-DQB1

Referen

ce(AA#,

HLA

-res

triction)

948

9/10

33–47

QWAPVLD

FAPPGASAYGSL

Class

II02

/33

51/53

03/13

02/06

AA#3

7-45

(HLA

-A� 020

1;ref.27

)AA#3

8-46

(HLA

-A� 020

1;ref.24

)AA#3

7-48

(HLA

-DRB1�04

02;ref.24

)58

229–

243

MTS

QLE

CMTW

NQMNL

Class

IIAA#2

35-243

(HLA

-A24

;ref.26

)AA#2

35-243

(HLA

-A� 020

1;ref.24

)10

249/10

33–47

QWAPVLD

FAPPGASAYGSL

n.d.

02/03

07/15

09/11

02/03

AA#3

7-45

(HLA

-A� 020

1;ref.27

)AA#3

8-46

(HLA

-A� 020

1;ref.24

)AA#3

8-48

(HLA

-DRB1�04

02;ref.24

)11

0632

125–

139

ARMFP

NAPYLP

SCLE

Class

I24

/68

08/35

11/13

03AA#1

26-134

(HLA

-A� 020

1;ref.49

)AA#1

24-138

(HLA

-DR53

;ref.25

)11

293

9–23

NALL

PAVPSLG

GGGG

n.d.

02/33

65/35

04/13

03/06

AA#6

-15(HLA

-A� 020

1,B� 570

1;ref.24

)29

113–

127

GPPPPSQASSGQARM

n.d.

1137

1255

–69

ASAYGSLG

GPAPPPA

n.d.

02/26

14/18

01/11

03/05

AA#5

8-66

(HLA

-A� 020

1;ref.24

)59

/60/61

233–

255

LECMTW

NQMNLG

ATLK

GVAAGSS

n.d.

AA#2

35-243

(HLA

-A24

;ref.26

)AA#2

35-242

(HLA

-A� 020

1;ref.24

)AA#2

38-246

(HLA

-A� 010

1,A� 020

1,B� 350

8,C

� 170

1;ref.24

)AA#2

39-249

(HLA

-�24

02;ref.2

4)AA#2

35-249

(HLA

-DRB1�11

04;ref.2

4)AA#2

42-250

(HLA

-A� 010

1,A� 020

1;ref.24

)AA#2

43-252

(HLA

-A� 020

3;ref.24

)AA#2

46-253

(HLA

-A� 690

1;ref.24

)76

/77

301–

319

RRVPGVAPTL

VRSASETSE

n.d.

1221

71/72

271–

289

LCGAQYRIHTH

GVFR

GIQ

Dn.d.

03/24

07/48

04/16

03/05

AA#2

86-293

(HLA

-Cw7;

ref.28

)AA#2

69-278

(HLA

-A� 010

1,B� 350

1;ref.24

)12

8910

9/11

043

3–44

9RHHNMHQRNMTK

LQLA

LClass

I24

35/40

08/11

03/04

AA#4

36-445

(HLA

-A� 020

1,A� 240

2,B� 400

1;ref.24

)13

319/10

33–47

QWAPVLD

FAPPGASAYGSL

Class

II02

/31

0715

06AA#3

7-45

(HLA

-A� 020

1;ref.27

)AA#3

8-46

(HLA

-A� 020

1;ref.24

)AA#3

8-48

(HLA

-DRB1�04

02;ref.24

)71

/72

271–

289

LCGAQYRIHTH

GVFR

GIQ

DClass

IAA#2

86-293

(HLA

-Cw7;

ref.28

)AA#2

69-278

(HLA

-A� 010

1,B� 350

1;ref.24

)13

4960

237–

251

TWNQMNLG

ATL

KGVA

n.d.

02/24

18/40

04/11

03/03

AA#2

35-242

(HLA

-A24

;ref.26

)AA#2

35-242

(HLA

-A� 020

1;ref.24

)AA#2

38-246

(HLA

-A� 010

1,A� 020

1,B� 350

8,C

� 170

1;ref.24

)AA#2

39-249

(HLA

-A� 240

2;ref.24

)AA#2

35-249

(HLA

-DRB1�11

04;ref.2

4)AA#2

42-250

(HLA

-A� 010

1,A� 020

1;ref.24

)AA#2

43-252

(HLA

-A� 020

3;ref.24

)AA#2

46-253

(HLA

-A� 690

1;ref.24

)14

859/10

33–47

QWAPVLD

FAPPGASAYGSL

Class

II01

/02

37/40

07/14

02/03

AA#3

7-45

(HLA

-A� 020

1;ref.27

)AA#3

8-46

(HLA

-A� 020

1;ref.24

)AA#3

8-48

(HLA

-DRB1�04

02;ref.24

)78

309–

323

TLVRSASETS

EKRPF

AA#3

17-327

(HLA

-A1;

ref.50

)15

1671

/72

271–

289

LCGAQYRIHTH

GVFR

GIQ

D02

49/51

11/14

03AA#2

86-293

(HLA

-Cw7;

ref.28

)AA#2

69-278

(HLA

-A� 010

1,B� 350

1;ref.24

)

Abbreviations

:n.d.,no

tdetermined

;AA,a

minoac

id.






TAA-specific T cells showed increased IFN-g-release (mean,300 SFC/105 cells; range, 8–1,701) compared with control Tcells (<10 SFC/105 cells; Fig. 4A). No cytokine secretion byleukemia cells alone was observed. Coculture of T cells at anE:T ratio of 10:1 in the presence of IL-2 (50U/mL) showed avariable elimination of autologous leukemia blasts in vitro.After 3 days of coculture with TAA-specific T cells there were13.6% residual blasts remaining compared with 46.8% inthe controls (calculated relative to the initial leukemia cellcounts onday0;P¼0.04; Fig. 4B).Analysis of IFN-g and IL-4levels in the coculture supernatants showed high concentra-tions of IFN-g by TAA-specific T cells after 3 days of coculturewith autologous blasts (mean, 630 � 364 pg/106 cells/mL)comparedwith control T cells (mean, 0 pg/106 cells/mL; P¼0.04). Lower levels of IL-4 were secreted by TAA-specific Tcells (mean 19.6� 3.3 pg/106 cells/mL; control T cells mean7.6 � 1.1 pg/106 cells/mL) after coculture with autologousblasts (P¼ 0.017; Fig. 4C). Furthermore, a specific inhibitionof colony formation was observed in CFU assays after 14days when leukemia blasts were cocultured with TAA-

specific T cells compared with control T cells. As shownin Fig. 4D, a mean relative inhibition of 42.3% of colonyformation was observed (P ¼ 0.012).

Representative examples for the leukemia-specific activ-ity of TAA-specific T cells are shown in Fig. 5 and supple-mentary Figs. S2–S8 respectively. In patient ID#1032 des-ignated as standard-risk, peptide reactivity against thepooled TAAs in ELISpot assay after initial (Fig. 5A) andthree (Fig. 5B) stimulations showed recognition ofWT1 and PRAME. Furthermore, cytolytic activity againstthese antigens was shown in a 51Cr-release assay usingpeptide-pulsed autologous PHA-blasts as targets (Fig. 5C).Coculture of TAA-specific T cells with autologous blastsshowed reduction of autologous leukemia cells to 13%of the initial cell count after 1 day and to 4.8% after 3 days.In contrast, when nonspecific T cells derived from thesame patient were incubated with autologous blasts,23% of leukemia blasts remained on day 1 and 10% onday 3 of coculture (Fig. 5D). Reduction of leukemia cellnumbers was also apparent when applying absolute

BA

DC

Control

CTL

P = 0.13 P = 0.04

% r

em

ain

ing leukem

ia c

ells

80

0

60

40

100

20

120

140

Day +3Day +1

P = 0.012

% r

ela

tive inhib

itio

n 80

0

60

40

100

20

CTLControl

IL-4

CTLControl

20

0

15

10

25

5

IFN-γ

pg

/10

6ce

lls/m

L

CTLControl

800

0

600

400

1,000

200

P = 0.04 P = 0.017

SF

C/1

05

ce

lls

CTLControl

500

0

400

300

600

200

100

P = 0.23

Figure 4. Reactivity of tumor antigen-specific T-cell lines against autologous leukemia cells. Summary of coculture experiments of eight patient-derivedtumor antigen-specific T-cell lines against their autologous bone marrow leukemia blast samples cryopreserved at the time of diagnosis. NonspecificT cells or unstimulated cells of the same patient were used as controls in all experiments. A, summary of the IFN-g-ELISpot coculture results of T-cell lines(n ¼ 8; CTL, black bar) versus nonspecific T cells (n ¼ 8; control, white bar; mean � SE) at an E:T ratio of 1:1. B, in a 3-day coculture at an E:T ratio of 10:1leukemia cell counts were assessed by FACS analysis and compared with the absolute leukemia cell count obtained on day 0. Leukemia blasts werequantified by anti-CD10 and CD19 costaining; T cells were quantified by CD3 positivity. Absolute cell numbers were assessed by quantification withFACScounting beads and the percentage of remaining leukemia cells was calculated comparedwith cell numbers on day 0 (nonspecific T cells; control, whitebars; specific T cell lines, CTL, black bars; mean � SD). C, IFN-g and IL-4 ELISA of the coculture supernatants after 24 hours (mean � SE; control, n ¼ 8,white bars; CTLs, n ¼ 8, black bars). D, relative inhibition of colony formation of leukemic cells in CFU assays when cocultured with specific T-celllines (n ¼ 8; CTL, black bar) versus nonspecific cells (n ¼ 8; control, white bar; E:T ratio 10:1; mean � SE).

Weber et al.





quantification with fluorescence-activated cell sorting(FACS) counting beads. After 3 days, elimination of leu-kemia cells was observed when blasts were cocultured with

autologous TAA-specific T cells but not control T cells(Fig. 5E). Activation of TAA-specific T cells by the auto-logous blasts was further shown in IFN-g-ELISpot assay,

BA

C

SF

C/1

05

ce

lls

TAAControl

250

0

150

100

50

200


SF

C/1

05

ce

lls

100

250

200

150

0

50

350

300

1.2:12.5:15:110:120:140:1

80

0

60

40

100

20

% lysis

PHAB

PHAB + TAA

PHAB + WT1

PHAB + Survivin

PHAB + MAGE

PHAB + PRAME

E:T ratio

MAGE-A3

PRAMESurvivin

WT1

D

G

E

F

SF

C/1

05

ce

lls

CTLControl

300

0

200

150

100

250

50

Control

CTL

% r

em

ain

ing leukem

ia c

ells

Day +3Day +1

20

0

15

10

25

5

80

0

60

40

100

20

Rem

ain

ing leukem

ia

ce

lls*1

03/m

L

Day +3Day +1Day 0

CTL

Control

Figure 5. Antigen recognitionand reactivity against autologousleukemia cells in a patient withstandard-risk ALL. Antigen-specific T cells of patient ID #1032showed recognition of TAA afterthe initial stimulation (A), as well asrecognition of PRAME and WT1after the third stimulation (B) in IFN-g-ELISpot (mean�SD).C, cytolyticactivity in a standard 51Cr-releaseassay against antigen-pulsedautologous target cells (PHA-blasts, PHAB) at E:T ratios from40:1 to 1.2:1. D, percentage ofremaining leukemia cells incocultures of specific T-cell lines(CTL, black bars) with autologousleukemia blasts on day 1 and 3 inpresenceof IL-2 (50U/mL) at anE:Tratio of 10:1. Leukemic cells weredetected by costaining with anti-CD10 and CD19 antibodies, T cellsby stainingwith anti-CD3 antibody;nonspecific T cells (control, whitebars) were used as control forunspecific lysis. E, assessment ofabsolute leukemia cell counts incoculture with specific T-cell lines(CTL, black line) and nonspecific Tcells (control, dashed line) at an E:Tratio of 10:1 by FACS analysisusing counting beads. F, cocultureat a 1:1 ratio of autologousleukemia cells to specific T cells(CTL, black bar) or nonspecificT cells (control, white bar) inIFN-g-ELISpot assay (mean � SD).G, immunohistochemistry ofleukemia blasts.






where the T cells showed increased IFN-g production(mean, 252 SFC/105 cells) compared with the control(mean, 128 SFC/105 cells; Fig. 5F). To evaluate the expres-sion of the targeted antigens on the ALL blasts, cytospinswere stained by immunohistochemistry and showed weakpositivity for MAGE-A3 and higher for Survivin (Fig. 5G).Similarly, in Supplementary Figs. S2–S8 the results ofthe coculture experiments with autologous blasts of theremaining patients are shown, including the 3-day cocul-ture and ELISpot assays as well as the immunohistochem-istry of the leukemia blasts. There was no correlationbetween the generation of effective T-cell lines and leuke-mia antigen expression in the blasts in the 8 patients whereleukemia blasts and peripheral blood samples wereavailable.

DiscussionPatients with ALL who have high-risk disease or who

relapse have a high rate of mortality and the best chance ofcure is the allogeneic HSCT. There is a need for moreeffective treatment options for patients not eligible forallogeneic transplant and immunotherapy may be mosteffective for preventing relapse in high-risk patients afterchemotherapy. Therefore, we sought to develop a strategy togenerate autologous T cells that target multiple TAAs andshow specific antileukemic activity for use as adoptive T-cellimmunotherapy to prevent relapse in pediatric ALL. Ourrationale for obtaining samples during maintenance ther-apy was to show feasibility of collection at this time pointfor potential use as a first-line strategy, to augment therapyin high-risk patients in first remission. We show here thatTAA-specific T cells can be generated frompatients with ALLduring maintenance therapy regardless of NCI risk groupand despite low lymphocyte counts at the time of sampleacquisition. Patients with high-risk ALL might have morecompromised immune systems, correlating with their pooroutcome. Nevertheless, we observed no difference in thequality of TAA-specific T cells that could be generated fromhigh- and standard-risk patients. None of the commonlyknown risk factors for relapse, including age and gender,affected our ability to ex vivo expand TAA-specific T cellsfrom these patients and extensive in vitro testing showedantileukemic activity of the expanded products againstautologous ALL blasts in long- and short-term cocultureexperiments.

Antigen-specific responses were detectable within 7 to 10days after just a single stimulation in more than half of thetested patients, indicating that the immunosuppression dueto the chemotherapeutic drugs administered during main-tenance therapy did not preclude the expansion of TAA-specific T cells in vitro. Moreover, upon subsequent restim-ulation, TAA-specific T cells were expanded frommore than90% of patients, suggesting that the in vivo immunosup-pressive environment could be overcome in vitro. At least aminimal 1.2-fold expansion of the autologous T-cell lineswas possible in all cases, using methods approved for goodmanufacturing practices (GMP). Nevertheless, for a clinical

scale product, the conditions need to be adjusted to achievecell numbers necessary for infusion, for example, throughuse of gas-permeable cell culture flasks (30).

After 3 weeks in culture, the T-cell lines expanded frompatients with ALL displayed predominantly an effector-memory phenotype, although small populations of T cellswith central memory and na€�ve phenotypes were alsodetected. Effector-memory T cells have been shown to haveeffective antitumor and antiviral efficacy, but are short-livedin vivo, leading to tumor outgrowth after loss of the adop-tively transferred cells (31). In contrast, it has been shownthat na€�ve T cells or na€�ve-derived effector T cells exert themost effective antitumor effects as well as having betterin vivo expansion and persistence than effector cells derivedfrom memory subsets (31, 32) We and others previouslyshowed that virus- and TAA-specific T cells can be expandedfrom na€�ve T cells derived from cord blood as well as fromhealthy donors (15, 23, 28, 33, 34). In contrast to virus-specific T cells where memory T cells are the main source ofexpanded T cells, na€�ve T cells seem to be an importantsource of TAA-specific T cells (15, 34). The lymphopeniafollowing high-dose chemotherapy may favor the in vitrogeneration of T cells from na€�ve cell populations. However,further experiments will be needed to define the origin ofthe TAA-specific T-cell lines. In addition to the ability togenerate T-cell lines, the adoptive transfer of in vitro–primedT cells into a lymphopenic milieu may favor the persistenceand expansion of the transferred T cells, as has been shownby the infusion of predominantly effector-memory T cells inthe transplantation setting (35–37).

The antigens we selected for T-cell generation are exp-ressed in a wide variety of hematologic disorders as well assolid tumors. Immunohistochemistry of the availablepatient samples showed only low levels of antigen expres-sion in the leukemia cells, which might limit the antileu-kemic effects of the T cells in vivo, despite their high spec-ificity. However, antigen expression in leukemic cells can beenhanced by epigenetically modifying drugs (38) and clin-ical trials in adults and pediatric patients with acute leuke-mia are currently under investigation. Furthermore, epige-netic modulation has been shown to rendermalignant cellsmore susceptible to T-cell recognition without impairingexpansion or function of in vitro–generated T cells (38–40).These observations suggest the feasibility of combiningepigenetic modification and adoptive T-cell transfer.

Immunotherapeutic approaches for patients with acuteleukemia have included vaccines, which generally target asingle-peptide epitope and clinical responses have beenobserved that seem to correlate with expansion of epi-tope-specific T cells in vivo. More recently, CD19-specificantibodies (41, 42) and CD19CAR gene-modified T cellstargeting the CD19-receptor on B cell malignancies haveshown remarkable success for CD19þmalignancies includ-ing pediatric ALL (10–12). Our strategy offers a synergisticapproach to these strategies. First, our technique coversmultiple immunogenic epitopes. This offers the potentialfor a combined immunotherapeutic approach with vac-cines to rechallenge the patients with the antigens used

Weber et al.





for T-cell generation and boosts the adoptively transferredT cells in vivo. This may prolong their persistence andincrease their potency. Second, our approach permits T-cellgeneration from all patients without limitation to certainHLA-types, unlike single-peptide vaccine studies (14, 27,43). Finally, the polyfunctionality and simultaneous target-ing of multiple antigens may decrease the risk of tumorimmune escape, such as the downregulation of antigenexpression, elimination of clones expressing the targetedepitope, or outgrowth of antigen-negative populations,already observed with CD19-directed therapy (12). There-fore, to increase the breadth of the tumor-specific activityby T cells, TAA-specific T cells could be administered incombination with CD19 antibodies or could be geneticallymodified to express a CD19CAR.Previous studies have shown the importance of CD4þ T-

helper cells for the strength and persistence of in vivoimmune responses against viral and tumor antigens (44,45). In this study, tumor-specific responses were seen inboth the CD4þ and CD8þ T-cell populations and immu-nogenic peptides restricted by MHC class I and II weredetected by epitope mapping for WT1. The combination ofCD4þ-restricted epitopes with the commonly used CD8þ

epitopes for T-cell stimulation can improve the survival ofthe generated T-cell lines and vaccination studies using amix of MHC class I- and II–restricted peptides show asustained activation and better survival of specific T cells(44–46). Furthermore, CD4þ T cells can also have a directcytolytic effect on tumor cells (47, 48). Thus, the infusion ofTAA-specific T cells containing antigen-specific CD4þ andCD8þ T cells may facilitate a better antileukemic effectin vivo.Although it is encouraging that our TAA-specific T cells

reduced autologous leukemia blasts in coculture experi-ments, it remains to be determined whether TAA-specificT cells have therapeutic efficacy in patients with ALL. Itwould be possible to generate a severe combined immu-nodeficient (SCID)/hu mouse model of ALL to further testthe antileukemic effect of TAA-specific T cells. However,while such a model, using autologous T cells to prevent ortreat ALL, might yield positive results, it would not guar-antee success in man. Conversely, were the TAA-specific Tcells ineffective in the murine model, we would not wish toabandon evaluating TAA-specific T cells in a carefullydesigned clinical trial, where we can track the fate andfunction of in vitro–generated TAA-specific T cells and

evaluate antileukemic efficacy by measuring residual dis-ease. Given the safety of infused TAA-specific T cells gener-ated in healthy donors (13) and the lack of relevance ofanimal models for establishing safety, our next step will beto generate GMP grade TAA-specific T cells for a phase Iclinical trial.

This study shows that the generationof TAA-specific T-celllines from the peripheral blood of patients with ALL onmaintenance therapy irrespective of NCI risk group is fea-sible, and that these T cells show antileukemic activityagainst autologous blasts in vitro. Therefore, adoptiveimmunotherapy with autologous TAA-specific T cells toboost antileukemic immune responses and prevent relapseseems to be a promising approach and may improve out-comes in high-risk ALL both in the first-line setting, and as anew approach to treat relapsed patients not eligible forallogeneic stem cell transplant in combination with othertherapeutic options.

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

Authors' ContributionsConception and design:G.Weber, I. Caruana, A.J. Barrett, K.R. Rabin, C.M.BollardDevelopment of methodology: G. Weber, A.J. Barrett, A.M. Leen, U.Gerdemann, K.R. Rabin, C.M. BollardAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): G. Weber, R.H. Rouce, K.R. Rabin, C.M. BollardAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): G. Weber, I. Caruana, K.R. Rabin, C.M.BollardWriting, review, and/or revisionof themanuscript:G.Weber, I. Caruana,R.H. Rouce, A.J. Barrett, A.M. Leen, K.R. Rabin, C.M. BollardAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.H. Rouce, C.M. BollardStudy supervision: C.M. Bollard

Grant SupportThis work was supported in parts by NIH grant 1P01CA148600-01 and

Cancer Prevention and Research Institute of Texas (CPRIT) grant RP100484.C.M. Bollard was also supported by the career development award from theLeukemia Lymphoma Society and an award from the Gillson LongenbaughFoundation. K.R. Rabin was supported by the Kurt Groten Family ResearchScholars’ Program, the Gillson Longenbaugh Foundation, and the St. Bal-drick’s Pediatric Cancer Foundation.

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 April 4, 2013; revised May 30, 2013; accepted June 16, 2013;published OnlineFirst July 9, 2013.

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2013;19:5079-5091. Published OnlineFirst July 9, 2013.Clin Cancer Res Gerrit Weber, Ignazio Caruana, Rayne H. Rouce, et al. Immunotherapy

Implications for−−Patients with Acute Lymphoblastic Leukemia Generation of Tumor Antigen-Specific T Cell Lines from Pediatric

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