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Translational Cancer Mechanisms and Therapy Targeting the Leukemia Antigen PR1 with Immunotherapy for the Treatment of Multiple Myeloma Gheath Alatrash 1 , Alexander A. Perakis 1 , Celine Kerros 1 , Haley L. Peters 1 , Pariya Sukhumalchandra 1 , Mao Zhang 1 , Haroon Jakher 1 , Madhushree Zope 1 , Rebecca Patenia 1 , Anna Sergeeva 1 , Shuhua Yi 2 , Ken H. Young 2 , Anne V. Philips 3 , Amanda M. Cernosek 1 , Haven R. Garber 1 , Na Qiao 3 , Jinsheng Weng 4 , Lisa S. St John 1 , Sijie Lu 1 , Karen Clise-Dwyer 1 , Elizabeth A. Mittendorf 3 , Qing Ma 1 , and Jeffrey J. Molldrem 1 Abstract Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross- presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross- presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies. Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples. Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo. Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 338696. Ó2018 AACR. Introduction Despite therapeutic advances, multiple myeloma remains an incurable disease. Patients with high-risk disease features have a median survival of approximately 3 years (1). While immu- notherapy is currently not part of the standard regimens for the management of multiple myeloma, the role of immunother- apy and the immune system has been clearly demonstrated in multiple myeloma with allogeneic stem cell transplantation (allo-SCT), a prime example of immunotherapy and currently the only available curative option for multiple myeloma patients. However, the high treatment-related mortality asso- ciated with allo-SCT, in many cases, outweighs the graft- versus-myeloma (GvM) effect, and may limit the use of allo-SCT to selected patients who have failed standard-of-care treatments. Instead of infusing a large number of cells that contain only a small fraction of cytotoxic T lymphocytes (CTL) with multiple myeloma specicity, thereby risking nonspecic reactivity of the infused cells with normal tissue as in the case with allo-SCT, a few studies have demonstrated promising results with immunothera- pies that target distinct antigens that are expressed by multiple myeloma or with cellular immunotherapies using marrow-inl- trating lymphocytes (24). Because of the heterogeneity of anti- gen expression by multiple myeloma cells and immune evasion mechanisms (5), identifying and targeting novel antigens is critical to the success of curative immunotherapy for multiple myeloma. PR1 (VLQELNVTV) is a human leukocyte antigen (HLA)-A 0201- specic nonameric peptide derived from the myeloid-restricted primary granule proteins (PGP) neutrophil elastase (NE) and proteinase 3 (P3; ref. 6). NE and P3 are normally expressed by myeloid progenitor cells, polymorphonuclear leukocytes (PMNs) and monocytes, and are overexpressed or aberrantly 1 Department of Stem Cell Transplantation and Cellular Therapy, Section of Transplant Immunology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 2 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 3 Department of Breast Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 4 Department of Lymphoma/Myeloma, The University of Texas MD Anderson Cancer Center, Houston, Texas. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). G. Alatrash and A.A. Perakis contributed equally to this article. Corresponding Authors: Gheath Alatrash, Department of Stem Cell Transplan- tation and Cellular Therapy, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 900, Houston, TX 77030. Phone: 713-563-3334; Fax: 713-563-3364; E-mail: [email protected]; and Jeffrey J. Molldrem, [email protected] doi: 10.1158/1078-0432.CCR-17-2626 Ó2018 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 24(14) July 15, 2018 3386 Research. on September 24, 2020. © 2018 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst April 16, 2018; DOI: 10.1158/1078-0432.CCR-17-2626

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Translational Cancer Mechanisms and Therapy

Targeting the Leukemia Antigen PR1 withImmunotherapy for the Treatment of MultipleMyelomaGheath Alatrash1, Alexander A. Perakis1, Celine Kerros1, Haley L. Peters1,Pariya Sukhumalchandra1, Mao Zhang1, Haroon Jakher1, Madhushree Zope1,Rebecca Patenia1, Anna Sergeeva1, Shuhua Yi2, Ken H.Young2, Anne V. Philips3,Amanda M. Cernosek1, Haven R. Garber1, Na Qiao3, Jinsheng Weng4, Lisa S. St John1,Sijie Lu1, Karen Clise-Dwyer1, Elizabeth A. Mittendorf3, Qing Ma1, and Jeffrey J. Molldrem1

Abstract

Purpose: PR1 is a human leukocyte antigen (HLA)-A2nonameric peptide derived from neutrophil elastase (NE) andproteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presentingcells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptibleto killing by PR1-targeting immunotherapies. As multiplemyeloma is derived from B cells, we investigated whethermultiple myeloma is also capable of PR1 cross-presentationand subsequently capable of being targeted by using PR1immunotherapies.

Experimental Design: We tested whether multiple myelomais capable of cross-presenting PR1 and subsequently becomessusceptible to PR1-targeting immunotherapies, using multiple

myeloma cell lines, a xenograft mouse model, and primarymultiple myeloma patient samples.

Results: Here we show that multiple myeloma cells lackendogenous NE and P3, are able to take up exogenous NE andP3, and cross-present PR1 on HLA-A2. Cross-presentation bymultiple myeloma utilizes the conventional antigen processingmachinery, including the proteasome and Golgi, and is notaffected by immunomodulating drugs (IMiD). Following PR1cross-presentation, we are able to target multiple myeloma withPR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo.

Conclusions: Collectively, our data demonstrate that PR1 is anovel tumor-associatedantigen target inmultiplemyelomaand thatmultiple myeloma is susceptible to immunotherapies that targetcross-presented antigens. Clin Cancer Res; 24(14); 3386–96.�2018AACR.

IntroductionDespite therapeutic advances, multiple myeloma remains an

incurable disease. Patients with high-risk disease features havea median survival of approximately 3 years (1). While immu-notherapy is currently not part of the standard regimens for themanagement of multiple myeloma, the role of immunother-apy and the immune system has been clearly demonstrated in

multiple myeloma with allogeneic stem cell transplantation(allo-SCT), a prime example of immunotherapy and currentlythe only available curative option for multiple myelomapatients. However, the high treatment-related mortality asso-ciated with allo-SCT, in many cases, outweighs the graft-versus-myeloma (GvM) effect, and may limit the use ofallo-SCT to selected patients who have failed standard-of-caretreatments.

Instead of infusing a large number of cells that contain only asmall fraction of cytotoxic T lymphocytes (CTL) with multiplemyeloma specificity, thereby risking nonspecific reactivity of theinfused cells with normal tissue as in the case with allo-SCT, a fewstudies have demonstrated promising results with immunothera-pies that target distinct antigens that are expressed by multiplemyeloma or with cellular immunotherapies using marrow-infil-trating lymphocytes (2–4). Because of the heterogeneity of anti-gen expression by multiple myeloma cells and immune evasionmechanisms (5), identifying and targeting novel antigens iscritical to the success of curative immunotherapy for multiplemyeloma.

PR1 (VLQELNVTV) is ahuman leukocyte antigen (HLA)-A�0201-specific nonameric peptide derived from the myeloid-restrictedprimary granule proteins (PGP) neutrophil elastase (NE) andproteinase 3 (P3; ref. 6). NE and P3 are normally expressed bymyeloid progenitor cells, polymorphonuclear leukocytes(PMNs) and monocytes, and are overexpressed or aberrantly

1Department of Stem Cell Transplantation and Cellular Therapy, Section ofTransplant Immunology, The University of Texas MD Anderson Cancer Center,Houston, Texas. 2Department of Hematopathology, The University of Texas MDAnderson Cancer Center, Houston, Texas. 3Department of Breast SurgicalOncology, The University of Texas MDAnderson Cancer Center, Houston, Texas.4Department of Lymphoma/Myeloma, The University of Texas MD AndersonCancer Center, Houston, Texas.

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

G. Alatrash and A.A. Perakis contributed equally to this article.

Corresponding Authors: Gheath Alatrash, Department of Stem Cell Transplan-tation and Cellular Therapy, University of Texas MD Anderson Cancer Center,1515 Holcombe Boulevard, Unit 900, Houston, TX 77030. Phone: 713-563-3334;Fax: 713-563-3364; E-mail: [email protected]; and Jeffrey J. Molldrem,[email protected]

doi: 10.1158/1078-0432.CCR-17-2626

�2018 American Association for Cancer Research.

ClinicalCancerResearch

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expressed in acute myeloid leukemia (AML) and chronic mye-logenous leukemia (CML), making them ideal leukemia asso-ciated antigens (LAA; refs. 6–8). We discovered PR1 on thesurface of AML and showed that CTL immunity to PR1 con-tributes to cytogenetic remission in patients with CML afterallo-SCT (9, 10). We first developed a PR1-peptide vaccine andshowed that it can induce immunity and objective clinicalresponses in patients with relapsed/refractory AML, CML, andmyelodysplastic syndrome (MDS; ref. 11). As vaccines haveshown the highest antitumor efficacy in the setting of minimaldisease burden (12), we also developed a T-cell receptor (TCR)-like mAb, 8F4, which targets PR1/HLA-A2 for use in the hightumor burden setting. 8F4 has demonstrated anti-leukemiaactivity in vitro and in vivo against myeloid leukemia cell linesand primary samples from AML patients (13), and is currentlybeing developed for clinical trials.

In addition to myeloid leukemia, published reports haveshown that nonmyeloid tumors lacking endogenous P3 and NEexpression (i.e., lack PR1) are able to take up NE and P3 from theextracellular environment (14–18). Furthermore, we showed thatbreast cancer, melanoma, lung cancer, and malignant lymphoidcells cross-present NE and P3 (i.e., present PR1 on surfaceHLA-A2), and are rendered susceptible to PR1-CTL and 8F4(14, 15, 19). Cross-presentation is a mechanism predominantlyfound in antigen-presenting cells (APC), whereby exogenousantigens are taken up, processed, and presented on HLA class Ito prime an immune response (20).

As a natural extension of our prior work and because (i)multiplemyeloma originates from B cells, which are known APCs(15, 21, 22); and (ii) myeloid progenitor cells and PMNs (thesources for NE and P3) are abundantly found in the bonemarrowand may play a role in the multiple myeloma microenvironment(6, 23), we hypothesized that multiple myeloma cross-presentsNE and P3 and subsequently becomes susceptible to PR1-target-ing immunotherapies.

Here we report that multiple myeloma lacks endogenousexpression of NE and P3. We show that multiple myeloma takesupNE and P3 and cross-presents PR1 in the context of HLA-A2 on

the cell surface.We demonstrate that PR1 cross-presentation leadsto specific killing of multiple myeloma by PR1-CTL and 8F4in vitro and in vivo. Finally, we show that PR1 is expressed on thesurface of multiple myeloma cells from primary HLA-A2þ patientsamples. Our data point to a potential role for PR1-targetingimmunotherapies for patients with multiple myeloma and high-light the importance of cross-presented antigens as potentialclinical targets for immunotherapy.

Materials and MethodsCells and cell culture

The Arkansas (ARK), LP-1, ARP-1, IM-9, OPM-2, RPMI 8226,and U266 multiple myeloma cell lines, U937 histiocytic leuke-mia, T2 T/B cell hybridoma, H2023 lung cancer, and T-47D andMDA-MB-453 breast cancer cell lines were obtained from ATCC.Cell lines were grown in RPMI1640 media with 25 mmol/LHEPES þ L-glutamine (Hyclone) supplemented with 10% FBS(Gemini Bio-Products), 100 U/mL penicillin, and 100 mg/mLstreptomycin (Cellgro). All cell lines were cultured and main-tained in 5% CO2 at 37�C. Cell lines were validated using shorttandem repeat DNA fingerprinting by our institutional sequenc-ing facility.

RT-PCRmRNA was purified using RNA Stat 60 kit (TelTest). cDNA was

synthesized using the Gene AMP RNA kit (Perkin Elmer). Thefollowing primers were used: NE forward primer 50-CACG-GAGGGGCAGAGACC-30 and reverse primer 50-TATTGTGCCA-GATGCTGGAG-30; P3 forward primer 50-GACCCCACCATGGCT-CAC-30, and reverse primer 50-ATGGGAAGGACAGACAGGAG-30;actin forward primer 50-CCAGAGCAAGAGAGCTATCC-30 andreverse primer 50-CTGTGGTGGTGAAGCTGTAG-30 (14, 18).cDNA amplification was performed using an iCycler (Bio-Rad).Samples were separated on a 1.5% agarose gel. Bands werevisualized using GelDoc2000 (Bio-Rad) and analyzed usingQuantity One software (Bio-Rad).

Western blottingCell lysates were generated by resuspending cell pellets at 4�C

for 30 minutes in lysis buffer [10 mmol/L HEPES (pH 7.9), 10mmol/L KCl, 0.1 mmol/L EGTA 0.1 mmol/L EDTA, 1 mmol/LDTT] containing protease inhibitors (Thermo Fisher Scientific).Protein was loaded on 10% SDS gels (Bio-Rad), separated byelectrophoresis under reducing conditions, transferred onto poly-vinylidene fluoride membranes, and blocked with 5% milk.Membranes were probed using anti-NE (Santa Cruz Biotechnol-ogy), anti-P3 (NeoMarkers), anti-actin (Millipore) antibodies,andperoxidase-conjugated secondary antibodies (Jackson Immu-noResearch). Chemiluminescence was captured on Kodak film(Kodak).

Antigen cross-presentation and flow cytometryCells were cultured with 10 mg NE, P3 (Athens Research &

Technology), EndoGrade ovalbumin (Ova; Hyglos) or withirradiated (7500 cGy) PMNs or peripheral blood mononuclearcells (PBMCs; 1:1 ratio) at varying durations. Cells were per-meabilized by washing in BD Biosciences Perm/Wash bufferand stained with fluorochrome-conjugated anti-P3 (CloneMCPR3-2; Thermo Scientific) or anti-NE (Santa Cruz Biotech-nology) antibodies using Alexa Fluor-488 or -647 conjugation

Translational Relevance

Multiple myeloma is considered an incurable hematologicmalignancy despite advances in its management. Althoughallogeneic stem cell transplantation (allo-SCT) can providecures for some patients, it is highly toxic and oftentimes isreserved for patients with a good performance status in whomall lines of therapy have failed. Nevertheless, even in theaforementioned clinical setting, the efficacy of allo-SCT isovershadowed by its toxicity. Our results identify PR1 as anovel target for immunotherapy in multiple myeloma. Thesefindings are highly valuable from a clinical perspective,because there are several therapies that target PR1, includingPR1 peptide vaccine, anti-PR1/HLA-A2 antibody, and PR1-specific T-cell therapies. Furthermore, in addition to its use inpatients with refractory multiple myeloma, PR1-targetingimmunotherapy may be offered in the setting of autologous(auto)-SCT either as consolidation after auto-SCT or as apurging strategy during stem cell collection.

Multiple Myeloma Cross-Presents PR1

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kits from Invitrogen. Cross-presentation of peptides wasdetected by staining cell surface with fluorescently conjugated,anti-PR1/HLA-A2 antibody (clone 8F4) as previously described(14, 15, 19). Costimulatory molecule surface expression wasanalyzed by staining myeloma patient bone marrow for CD19,CD33, CD34, CD38, CD138, CD40, CD80, CD86, and HLA-DR (all from Biolegend). Data were analyzed using FlowJosoftware (Tree Star). Inhibition of cross-presentation wasaccomplished by treating cell cultures with the endoplasmicreticulum (ER) to Golgi antegrade inhibitor brefeldin A (Sigma-Aldrich), or proteasome inhibitors lactacystin (Sigma-Aldrich)or bortezomib (Millennium Pharmaceuticals). To determinethe effects of IMiDs on cross-presentation, cell cultures weretreated with lenalidomide (Celgene).

Peptide-specific CTL generationPR1-specific CTL were generated from HLA-A2–positive

healthy donor (HD) PBMC by stimulating with PR1 peptide(Bio-synthesis Inc.) in vitro, as previously described (14, 24).Briefly, PBMC from healthy donor leukapheresis were isolatedusing Histopaque 1077 gradient centrifugation (Sigma-Aldrich)and were cocultured with PR1 (20 mg/mL)-pulsed T2 cells at 1:1ratio in RPMI1640 media supplemented with 10 % human ABserum (Gemini Bio-Products). Cell cultures were restimulatedwith PR1-pulsed T2 cells on days 7, 14, and 21, and 20 IU/mL ofrecombinant human IL2 (rhIL2; Biosource International).

Cell-mediated cytotoxicity assayCytotoxicity assays were performed as previously described

(14, 18). In brief, 1 � 103 target cells/mL were fluorescentlylabeled with calcein-AM (Invitrogen) for 15 minutes at 37�C andthoroughly washed with RPMI1640 to remove free calcein-AM.Target cells were cocultured with peptide-specific CTL at theindicated effector-to-target (E:T) ratios for 4 hours at 37�C in60-well Terasaki plates. Trypan blue was added to each well tostop the reaction and fluorescence was detected on a CytoFluor IIplate reader (Applied Biosystems). The percent specific cytotox-icity was calculated as follows:

1� fluoresencetargetþeffector �fluoresencemedia =���

½fluoresencetarget alone�fluoresencemedia�Þ � 100

Complement-mediated cytotoxicity assayComplement-mediated cytotoxicity (CDC) assays were per-

formed as previously described (13, 14). Briefly, U266 cells werecultured with NE or P3 for 24 hours and stained with calcein-AM.Labeled cells (1 � 106) were resuspended in serum-freeRPMI1640 media and treated with anti-PR1/HLA-A2 (clone8F4) antibody or isotype control antibody for 10 minutes at37�C. Standard rabbit complement (C') (Cedarlane Labs) wasadded and cells were incubated for 60 minutes at 37�C. Fluores-cence was measured as described in the previous section.

Staining for PR1-CTL in multiple myeloma patient samplesPatient and HD peripheral blood (PB) samples and bone

marrow aspirates were collected after informed consent to par-ticipate in anMDAnderson Cancer Center (MDACC) institution-al review board-approved study. Peripheral blood mononuclearcells (PBMC) were isolated using Histopaque 1077 gradientcentrifugation (Sigma-Aldrich). PBMC were stained using the

following fluorescent antibodies: CD8 APC-H7 (BD Biosciences),CD3 FITC (BD Biosciences), PE-conjugated PR1/HLA-A2-dextra-mer (Immudex) or tetramer (Baylor College of Medicine MHCTetramer Core, Houston, TX) and the following Pacific blue–conjugated lineage antibodies: CD4 (BD Biosciences), CD14 (BDBiosciences), CD16 (BD Biosciences), and CD19 (Biolegend).Samples were fixed with 4% paraformaldehyde. Data wereacquired on a FACSCanto flow cytometer (BD Biosciences)and analyzed using FlowJo software (Tree Star). The frequencyof PR1-CTL was determined as the percentage of live cells thatwere lineage�, CD3þ, CD8þ, and PR1-dextramerþ or PR1-tetramerþ. Phenotype of PR1-CTL (PR1/HLA-A2-dextramerþ)was investigated using CCR7 PE-Cy7 (Biolegend) and CD45RAPerCP-Cy5.5 (Biolegend) staining, and was classified as centralmemory (CCR7þ/CD45RA�), effector memory (CCR7�/CD45RA�), na€�ve (CCR7þ/CD45RAþ), or terminally differen-tiated (CCR7�/CD45RAþ).

Confocal staining and imagingBone marrow smears and U266 cells were fixed with cold

acetone and blocked with 5% normal mouse serum (JacksonImmunoResearch). Fixed slides were washed with PBS and thendouble stained with Alexa-647–conjugated 8F4 antibody andAlexa-488–conjugated mouse anti-human HLA-A2 antibody(Serotec) or Alexa-488–conjugated rabbit anti-CD138 antibody(Bioss). Slides were stained with antibodies for 90 minutes atroom temperature. After washing, ProLong Gold antifade reagentwith DAPI (Invitrogen) was added. Confocal imaging was per-formed using Leica Microsystems SP2 SE confocal microscopewith 10�/25 air, 63�/1.4 oil objectives. Leica LCS software(version 2.61) was used for image analysis.

U266 xenograft mouse modelNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) female mice were

purchased from Jackson Laboratory and housed at MDACCfollowing International Animal Care and Use Committee–approved protocol. Mice received sublethal irradiation 24 hoursprior to intravenous injection with 2� 106 U266 cells (25). U266engraftment and disease burden was measured by quantifyingblood human IgE level by ELISA (Bethyl Laboratories). Mice weretreated intravenously with 0.5 � 106 PR1-CTL, irrelevant peptide(CG1, E75)-CTLor left untreated (PBS-treated) 14days afterU266engraftment. Mice were treated intravenously with 8F4 antibody(10 mg/kg) or IgG2A isotype control (10 mg/kg; Jackson Immu-noResearch) three times per week beginning on day 28 for a totalof 10 injections (26). Mice were sacrificed 35 days after CTLinfusion or 3–4 days after the last antibody treatment, and bonemarrow was harvested, stained with mouse CD45, human (h)CD45, hCD138, and HLA-A2 fluorescently conjugated antibo-dies, and then analyzed by flow cytometry.

Statistical analysisStatistical analyses were performed using GraphPad Prism 6.0

software. P values less than 0.05 were considered significant.

ResultsMultiple myeloma cells lacking endogenous NE and P3internalize exogenous proteins

To determine whether multiple myeloma cells express NE andP3, a panel of multiple myeloma cell lines was analyzed for

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endogenous expression of these PGP. Our data indicate that NEand P3 are absent in multiple myeloma cells at the protein andtranscript levels (Fig. 1A and B). These findings are in agreementwith reports from the Cancer Cell Line Encyclopedia (27), whichconfirm the lack of NE and P3 in multiple myeloma (Supple-mentary Fig. S1). The absence of NE and P3 in multiple myelomais expected as multiple myeloma is of lymphoid origin, which isknown to be deficient in myeloid PGP (6). The myelomonocyticU937 leukemia cell line that endogenously expresses NE and P3was used as a positive control in these experiments.

Becauseweandothers previously showed that solid tumors andB cells take up NE and P3 (14–19), we next tested whethermultiple myeloma cells take up NE and P3, the first step inantigen cross-presentation. We cocultured a number of multiplemyeloma cell lines with soluble NE or P3 over increasing dura-tions. Flow cytometry analysis of intracellular NE and P3 stainingdemonstrates that NE and P3 are taken up by multiple myeloma(Fig. 1C and D). Time-dependent internalization of NE and P3 isobserved,with amarkeddifference in the kinetics betweenNEandP3. The uptake of both proteins is sustained throughout theduration of the cultures. The kinetics and degree of NE and P3uptake by the multiple myeloma cell lines closely resembles thepattern of NE and P3 uptake observed in breast cancer andmelanoma (14).

HLA-A2þ multiple myeloma cells take up and cross-present NEand P3 from soluble and cell-associated sources

As PR1 is an HLA-A2–restricted epitope, we focused ourcross-presentation studies on the HLA-A2þ (i.e., HLA-A�0201)U266 multiple myeloma cell line. In agreement with the panelof multiple myeloma cell lines we examined (Fig. 1C and D),we confirmed the internalization of soluble and PMN-associ-ated NE and P3 by U266 cells (Fig. 2A and B; SupplementaryFig. S2). Previously, we demonstrated that solid tumor cells

can cross-present PR1 from both soluble and cell-associatedsources of NE and P3 (14, 19). These findings were recapitu-lated in the U266 multiple myeloma cells that were coculturedwith soluble NE, P3, or irradiated HLA-A2� PMNs, the latterserving as the cellular source of NE and P3 that lack endog-enous PR1 (Fig. 2C and D). As shown previously, cross-pre-sentation was demonstrated by staining cells with anti-PR1/HLA-A2 Alexa-647–conjugated antibody (14). Because of theunique and inherent HLA-binding properties of TCR-likeantibodies, we have observed low-affinity binding of 8F4 toHLA-A2 in other cell lines (13, 14), and also observed lowbackground 8F4 staining in nonpulsed U266 cells. However,to highlight the specificity of 8F4 for PR1/HLA-A2, the HLA-A2� multiple myeloma cell lines OPM-2 and RPMI-8226 werealso cocultured with HLA-A2� PMN and examined for cross-presentation (Supplementary Fig. S3).

Thus, these data confirm NE and P3 internalization by U266multiplemyeloma cells. Notably, there were kinetic differences ininternalized NE or P3 based on the protein source: PMN-derivedNE achieved higher intracellular levels than soluble NE and wascomparable with the uptake of PMN-derived P3.

Cross-presentation byU266 involves proteasome andGolgi/ERCross-presentation involves distinct, well-defined pathways

that utilize the proteasome and ER/Golgi (28–30). The protea-some plays an important role in antigen cross-presentation bycleaving intracellular proteins into small, 8–11 amino acid pep-tides, which are transported into theERby the TAP1/2 complex. Inthe ER, peptides are loaded onto MHC-I molecules using com-ponents of the antigen processing machinery and are thenexported to the cell surface via the Golgi. We hypothesized thatNE and P3 cross-presentation involves proteasome and ER/Golgishuttling, as previously shown for NE and P3 cross-presentationby solid tumors and APCs (14, 15). Our data confirm that

Figure 1.

Multiple myeloma (MM) cells lackendogenous NE and P3 and internalizeexogenous NE and P3. Protein (A) andmRNA (B) were extracted frommultiple myeloma cell lines. A,Immunoblots demonstrate lack of NEand P3 proteins in whole-cell lysatesfrom seven different multiple myelomacell lines. Gels were loaded with 20 mgof protein. B, RT-PCR was performedusing NE and P3 primers, which showslack of NE and P3 mRNA expression inmultiple myeloma cell lines. U937myelomonocyticAML cell linewasusedas a positive control. b-Actin was usedas a loading control. The first lane ineach figure represents the molecularweight (MW) marker. C and D, Flowcytometry detected intracellular NEand P3 following culture with NE (C) orP3 (10mg/mL;D). After incubation, cellswere permeabilized and stained witheither anti-NE or anti-P3 antibodies.Graphs display the mean � SEM foldincrease in median fluorescenceintensity (MFI) over untreated cellsfrom triplicate wells from sixindependent experiments.

Multiple Myeloma Cross-Presents PR1

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multiple myeloma utilizes the ER/Golgi and proteasome for NEandP3 cross-presentation, as incubationof cellswith lactacystin, aproteasome inhibitor, and brefeldin A, which inhibits ER toGolgiantegrade transport, both decreased PR1/HLA-A2 expression(Fig. 3A and B).

Next, we translated our in vitro studies into a clinically relevantsystem by examining PMN as the source for NE and P3 and usingbortezomib, a proteasome inhibitor approved for treatment ofmultiple myeloma. In agreement with our findings with lacta-cystin, bortezomib reduced PR1/HLA-A2 on the U266 myelomacell surface (Fig. 3C). In addition, the IMiD lenalidomide did notalter surface levels of PR1/HLA-A2 on U266 after coculture withPMNs (Fig. 3D). We also observed a consistent decrease in thesurface expression of overall HLA-A2 due to inhibition of theproteasome (Supplementary Fig. S4). As conventional antigencross-presentationmechanisms employ proteasome (20), it is notsurprising that overall expression of HLA-A2 decreases with theuse of lactacystin and bortezomib, and highlights the role of theproteasome in PR1 cross-presentation.

PR1 cross-presentation increases the susceptibility of multiplemyeloma to PR1-CTL and anti-PR1/HLA-A2 antibody

Immunotherapy targeting PR1 has shownpromising efficacy inthe treatment of myeloid malignancies (11, 13, 31, 32). Thus, weinvestigated whether PR1 cross-presentation by multiple

myeloma cells could lead to their lysis by PR1-CTL and thecomplement-fixing, anti-PR1/HLA-A2 8F4 antibody. Calcein-AMcytotoxicity assays demonstrate that cross-presentation of NE andP3 by U266 cells renders them susceptible to killing by PR1-CTLand 8F4 antibody in a dose-dependent manner (Fig. 4). Specif-ically, NE enhanced the killing of U266 cells by PR1-CTL at 10:1and5:1 effector:target (E:T) ratios,when comparedwithuntreatedor ova-supplemented U266 cells (Fig. 4A). The efficacy of PR1-CTL in eliminating PR1-cross-presenting multiple myeloma cellswas further validated by U266 cells that were supplemented withP3, where killing was demonstrated at the 10:1, 5:1, and 2.5:1 E:Tratios (Fig. 4A). As we have previously shown that the anti-PR1/HLA-A2antibody (8F4) lysesmalignant cells viaCDC(13, 14),wetested whether multiple myeloma could be killed by 8F4. In astandard CDC assay, we demonstrate significantly higher 8F4-mediated killing of U266 target cells that were cultured with NEand P3 (Fig. 4B). Combined, these studies show that multiplemyeloma is rendered susceptible to killing by PR1-targetingimmunotherapies and further confirm PR1 cross-presentation bymultiple myeloma.

PR-1 CTL and PR1/HLA-A2 antibody reduce multiple myelomaburden in xenograft mice

As PR1 cross-presentation by U266 increases susceptibility toPR1-targeting immunotherapy in vitro, we next investigated

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HLA-A2þ U266 multiple myeloma (MM) cell line takes up and cross-presents NE and P3 from soluble and cell-associated sources. HLA-A2þ U266 MM cell linewas cultured with soluble NE (10 mg/mL) or P3 (10 mg/mL; A), or irradiated HLA-A2� PMNs (B) at the indicated time points. Cells were permeabilized, stainedwith anti-NE or anti-P3 antibody, and analyzed by flow cytometry. For cell-associated uptake, light scatter and HLA-A2 staining seen on flow cytometry provided aclear distinction between PMNs and U266 cells. PMNs alone were used as a positive control for NE and P3 (white bars). ANOVA was performed using Prism6.0 software (� , P < 0.05). Data aremeans� SEM from triplicate experiments. C andD,U266MM cells were culturedwith soluble NE or P3 (10 mg/mL;C) or irradiatedPMNs (D) then analyzed for expression of surface PR1/HLA-A2 using 8F4-Alexa 647. Mean � SEM fold increase of the median fluorescence intensity (MFI) ofPR1/HLA-A2 is shown from duplicate experiments. Time "0" indicates untreated cells. ANOVA test was performed using Prism 6.0 software (� , P < 0.05).

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whether PR1 can be targeted in vivo using multiple myelomaU266 xenograft mouse models (25). To test this hypothesis,we utilized NSG mice, which contain bone marrow–residentPMNs, providing an available source of NE and P3 for cross-presentation. In addition, the murine PR1 sequence is homolo-gous to the human sequence (VLQELNVTV), and murine CTLcan recognize PR1/HLA-A2 and are able to be expanded aftervaccination (33).

After confirming U266 engraftment in the bone marrow andfollowing treatment with 8F4, we demonstrate a significantlydecreased U266 multiple myeloma burden, as shown by adecrease in the concentration of human IgE in mouse serum incomparison with isotype and untreated groups (Fig. 5A). Further-more, 8F4 treatment also noticeably reduced the percent ofmultiple myeloma cells in mouse bone marrow in comparisonwith mice treated with isotype and untreated mice (Fig. 5B).Similar results were also seen using PR1-CTL (Fig. 5C). U266multiple myeloma cells were identified as human CD45þ andmouse CD45� cells (Fig. 5D and E). These data suggest that 8F4and PR1-CTL are a feasible and effective therapy for HLA-A2þ

multiple myeloma.

PR1/HLA-A2 and PR1-CTL are detected in patients withmultiple myeloma

Wenext investigatedwhether PR1 could bedetected in thebonemarrow from patients with multiple myeloma, and if immunityto PR1 (i.e., PR1-CTL) could be detected in peripheral blood (PB)from patients with multiple myeloma following allo-SCT. Wewere able to detect PR1/HLA-A2 on the surface of 4 of 8 HLA-A2þ

patients withmultiple myeloma (Supplementary Table S1; Fig. 6;control staining is shown in Supplementary Fig. S5).

To determine whether PR1-CTL could be detected in PB fromHLA-A2þ patients with multiple myeloma, we used PR1/HLA-A2dextramer staining and showed PR1-CTL in the PB from 10 of 14multiple myeloma patients who received allo-SCT (Supplemen-tary Fig. S6A and S6B; Supplementary Table S2). The medianfrequency of PR1-CTL in these multiple myeloma patients was0.053%of CD8þ T cells (range, 0%–1.67%), which is higher thanthe frequency of PR1-CTL reported in HLA-A2þ healthy donors(24). Furthermore, using CD45RA and CCR7 staining, PR1-CTLphenotype was analyzed in 4 of the patients and demonstratedprimarily an effector memory phenotype of the PR1-CTL (Sup-plementary Fig. S7). In addition, we investigated whether PR1-

Soluble NE or P3Lactacystin: 0.2 µmol/L

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Proteasome and ER/Golgi are involved in the cross-presentation of NE and P3 by U266 multiple myeloma (MM) cells. U266 MM cells were cultured for 24 hours inmedia containing NE or P3 (10 mg/mL) and the antigen-processing machinery inhibitors lactacystin (proteasome inhibitor) or brefeldin A (ER/Golgi transportinhibitor;A andB) or were cultured for 24 hourswith PMNs at a 1:1 U266:PMN ratio inmedia containing bortezomib (Bor) or lenalidomide (Len;C andD). PBMCswerecultured with U266 to serve as a negative control, as PBMCs lack NE and P3. Cells were then analyzed using flow cytometry for expression of PR1/HLA-A2 aftersurface staining with 8F4-Alexa-647. Mean � SEM of the median fluorescence intensity (MFI) of PR1/HLA-A2 is shown from duplicate wells from threeseparate experiments. ANOVA test was performed using Prism 6.0 software (� , P < 0.05).

Multiple Myeloma Cross-Presents PR1

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CTL are present in the PB of HLA-A2þmultiple myeloma patientsafter receiving autologous stem cell transplant (auto-SCT; Sup-plementary Fig. S8; Supplementary Table S3). PR1-CTL frequen-cies were detected by PR1/HLA-A2 tetramer staining in all 18multiple myeloma patients following auto-SCT. The medianfrequency of PR1-CTL in these patients was 0.205% (range,0.076%–1.31%). These patient data suggest that bone mar-row–derived NE and P3 is taken up and cross-presented bymultiple myeloma, and that immunity to PR1 is elicited inmultiple myeloma patients following SCT.

Finally, we used flow cytometry to study the expression of PR1on CD138þ multiple myeloma cells from patient bone marrow[to corroborate confocal microscopy data (Supplementary TableS1; Fig. 6)] and to also investigate the expression of costimulatorymolecules on PR1/CD138þ multiple myeloma cells. Our datashow surface expression of PR1 and costimulatory moleculesHLA-DR, CD40, CD80, andCD86onCD138þmultiplemyelomacells in 12 (Supplementary Table S4; Supplementary S9) patients,further supporting the observation that patientmultiplemyelomacells are capable of PR1 cross-presentation and possibly cross-priming.

DiscussionThis work demonstrates that multiple myeloma cells lacking

endogenousNE andP3have the ability to take upNE andP3 fromthe extracellular microenvironment and cross-present PR1 in thecontext of HLA-A2. Furthermore, PR1 cross-presentation rendersmultiple myeloma susceptible to killing by PR1-CTL and anti-PR1/HLA-A2 antibody. Our data illustrate a novel mechanism bywhich multiple myeloma is able to present antigens to theimmune system. These findings suggest that cross-presentationmay broaden the multiple myeloma tumor antigen repertoire,thus expanding the immunotherapeutic targets that could beexploited to treat multiple myeloma. Also, this study highlightsthe therapeutic potential of PR1-based therapies in multiplemyeloma.

Cross-presentation is an importantmechanism in the initiationof the CD8þ T-cell immune response. A number of studies have

shown that B cells can cross-present antigens and prime animmune response, while other reports have suggested that B cellslack the ability to cross-present (14, 34–36). This study is the firstto report antigen cross-presentation by multiple myeloma cells, afinding supported by a previous study from outside our labora-tory, demonstrating that multiple myeloma cells can act as APCs(37). In that study, CD38þ plasma cells from the bonemarrow ofpatients with multiple myeloma were shown to stimulate a T-cellresponse to tetanus toxoid and purified protein derivative. Fur-thermore, that study demonstrated that multiple myeloma plas-ma cells had low expression of the costimulatory moleculesCD40, CD80, CD86, and HLA-DR, which are known to be criticalfor priming the immune system. Our data show that a smallsubpopulation of multiple myeloma cells can express thesecostimulatory molecules (Supplementary Table S4). Although inour report we focused on the capacity of multiple myeloma cellsto cross-present antigens in the context of becoming a target for aCD8þ T-cell immune response, together with published datashowing thatmultiplemyeloma cells express costimulatorymole-cules under specific conditions, it is possible that multiple mye-loma could function as APCs and prime a CD8þ T-cell immuneresponse.

The immune system has clear implications in multiple mye-loma. For example, multiple myeloma cases have been reportedin the setting of immunodeficiency states (38, 39). Furthermore,deficiencies in the immune systemhave been reported inmultiplemyeloma patients, while higher frequencies of distinct popula-tions of immune cells have been correlated with favorable out-comes inmultiplemyelomapatients (40, 41). Recent studies havebegun to characterize the surface antigen repertoire of multiplemyeloma to discover effective antigen targets for immunothera-peutic development (42). Although some tumors downregulatesurface HLA class I molecules to evade the immune system (43),this does not appear to be the case in multiple myeloma, wherethere is an abundance of surface peptide/HLA class I molecules.A number of peptides have been shown to elicit effective CD8þ T-cell immune responses in multiple myeloma and are promisingimmunotherapeutic targets (42, 44, 45). However, in manypeptide discovery approaches, antigens that are known to be

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PR1 cross-presentation increases the susceptibility of multiple myeloma to PR1-CTL and anti-PR1/HLA-A2 antibody. A, When comparing PR1-CTL killing oftarget U266 cells supplemented with NE or P3 with ovalbumin (ova)-supplemented U266 and U266 cells alone, significantly higher killing of target cells was seen atthe 10:1 and 5:1 E:T ratios for the U266 þ NE group and 10:1, 5:1, and 2.5:1 E:T ratios for the U266 þ P3 group (�P < 0.05). B, Similarly, when comparing 8F4killing of NE-supplemented and P3-supplemented U266 target cells with ova-supplemented U266 and U266 cells alone, significantly higher killing of targetcells was seen at the 10 mg/mL and 5 mg/mL 8F4 concentrations for the NE-supplemented group and at the 10 mg/mL, 5 mg/mL, and 2.5 mg/mL 8F4concentrations for the P3-supplemented group (�, P < 0.05). Significantly higher killing was also demonstrated when comparing P3-supplemented U266 withova-supplemented U266 at the 1.25 mg/mL 8F4 concentration. Statistical analysis was performed using ANOVA test.

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absent from the tissues that are being analyzed are oftentimesexcluded from further development as immunotherapeutic tar-gets. Our studies highlight the potential importance of cross-presented antigens and critically evaluate the antigen repertoirefrom a clinically relevant perspective, as they could provide anentire class of antigens that could be targeted with immunother-apeutic approaches.

The proteasome plays an integral role in the process of cross-presentation (46). The involvement of the proteasome in NE andP3 cross-presentation by multiple myeloma is especially relevantin the context of bortezomib, which is part of the first-line therapyfor multiple myeloma patients. However, the effects of bortezo-mib on the peptide repertoire and antigen presentation has yet tobe fully determined. There is a preponderance of data thatdemonstrate negative effects of proteasomal inhibition onHLA-class I antigen presentation (46). In the setting of multiple

myeloma, one study reported a decrease in the presentation ofendogenous antigens by multiple myeloma after cells wereexposed to bortezomib (47). Other studies, however, have dem-onstrated an increase in the presentation of some antigens fol-lowing treatment with bortezomib (48–50). The variable effectsthat are seen following cell treatment with bortezomib may bespecific for bortezomib and not generalizable to all proteasomeinhibitors. Although our data presented here and our previouslypublished data support an inhibitory effect on cross-presentationfollowing proteasomal inhibition (14, 15), further studies usingantigens other than NE and P3 need to be conducted to moreconclusively determine the effects of proteasome inhibition onantigen cross-presentation.

The timing of immunotherapies has been shown to be criticalto clinical outcomes. PR1-targeting immunotherapy may be bestintegrated into standard-of-care myeloma regimens. Our in vitro

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8F4 antibody and PR1-CTL reduce multiple myeloma (MM) in U266 xenograft mice. U266 MM xenograft mice were established and treated with 8F4 antibody (A),IgG isotype control (B), or PR1-CTL (C). A, Mouse serum was collected by tail bleeding and separated by centrifugation. Human IgE was measured usingELISA. 8F4-treated mice show reduced circulating human IgE levels comparedwith isotype and untreatedmice (���� , P < 0.00001). B,At the end of treatment, micewere sacrificed and bone marrow was harvested; a single-cell suspension of bone marrow was generated and analyzed by flow cytometry for residual U266MM cells. 8F4-treated mice show a reduced percentage of U266 cells in the bone marrow compared with isotype and untreated mice (���� , P < 0.00001).The percent of myeloma cells was normalized to untreated mouse bone marrow for each experiment. C, Mouse bone marrow was analyzed by flow cytometryafter treatment with PR1-CTL. PR1-CTL–treated mice show a reduced percentage of U266 cells in the bone marrow compared with mice treated with irrelevantpeptide-CTL (�� , P < 0.001) or untreated mice (��� , P < 0.0001). D and E, Gating strategy used to identify U266 cells in mouse bone marrow. U266 cells weredefined as human CD45þ mouse CD45�.

Multiple Myeloma Cross-Presents PR1

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data suggest that lenalidomide does not interfere with the abilityof myeloma to cross-present PR1, implicating a possible benefitusing these two therapies in combination.However, our datawithbortezomib suggest that the activity of PR1-targeting immuno-therapy may be attenuated in the setting of proteasome inhibi-tion. Furthermore, 8F4 may be useful as a purging strategy in thesetting of stem cell collection in preparation for auto-SCT. Aspatients withmultiple myeloma often receive granulocyte colonystimulating factor (G-CSF) as part of their mobilization, whichincreases the expression of NE/P3 in the bonemarrow (7, 51) andthereby increases the source for PR1 cross-presentation, 8F4 maybe applicable in this setting to reduce the multiple myelomaburden in multiple myeloma cells that cross-present PR1. Ourdata using preclinical xenograft animal models, where 8F4 treat-ment alone markedly reduced multiple myeloma in the bonemarrow, supports this hypothesis.

We recognize that normal hematopoietic cells express thesource proteins from which PR1 is derived, raising a concernregarding the potential toxicity of PR1-targeting immunothera-pies. Furthermore, we note that fine epitope mapping demon-strated that 8F4 has contact residues with the HLA-A2 molecule(13). As HLA-A2 is part of the conformational epitope of PR1/HLA-A2, naturally we expect some binding of 8F4 to HLA-A2, asshown previously (14, 15, 19). However, to date, preclinicalstudies (13, 26) and clinical trials (11, 32) have demonstratedPR1-targeting immunotherapies to be safe and efficacious despitethe shared expression of NE and P3 between malignant cells andnormal counterpart, and the binding of 8F4 to HLA-A2.

Although we have shown PR1 expression by primary multiplemyeloma, we recognize that we have not directly shown killing ofprimary multiple myeloma cells by PR1-CTL or 8F4. However,taken together, our data support the continued investigation ofPR1-targeting immunotherapies, including 8F4 antibody, PR1-peptide vaccine, and PR1-CTL in the treatment of multiple mye-lomapatients. Our results also emphasize the need to evaluate therole of cross-presentation as a mechanism for the generation ofnovel tumor antigens in multiple myeloma.

Disclosure of Potential Conflicts of InterestE.A. Mittendorf is a consultant/advisory board member for Merck and

AstraZeneca. J.J. Molldrem holds ownership interest (including patents) inAstellas Pharma, for patent royalty income related to the 8F4 monoclonalantibody. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design:G. Alatrash, A.A. Perakis, A.M. Cernosek, J.J. MolldremDevelopment of methodology: G. Alatrash, A.A. Perakis, H.L. Peters,R. Patenia, A.V. Philips, A.M. Cernosek, N. Qiao, J. Weng, S. Lu, K. Clise-Dwyer,J.J. MolldremAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): G. Alatrash, A.A. Perakis, C. Kerros, P. Sukhumalchandra,H. Jakher, M. Zope, A. Sergeeva, S. Yi, K.H. Young, Q. Ma, J.J. MolldremAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Alatrash, A.A. Perakis, C. Kerros, H.L. Peters,P. Sukhumalchandra,H. Jakher,M. Zope, K.H. Young, A.V. Philips, H.R. Garber,E.A. Mittendorf, J.J. MolldremWriting, review, and/or revision of the manuscript: G. Alatrash, A.A. Perakis,C. Kerros, H.L. Peters, M. Zope, K.H. Young, H.R. Garber, S. Lu, E.A. Mittendorf,J.J. MolldremAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Alatrash, A.A. Perakis, M. Zhang,A.M. Cernosek,, L.S. St John, S. Lu, Q. MaStudy supervision: G. Alatrash, J.J. Molldrem

AcknowledgmentsThis research was funded by NIH, National Cancer Institute (NCI) grant

P50CA142509 (to G. Alatrash), NIH T32 Immunobiology Training grant5T32CA009598-24 (to H.L. Peters and C. Kerros), and NIH/NCI grantP30CA16672 (Flow Cytometry, Cell Sorting and Cell Imaging Core Facilities),and was supported by the generous philanthropic contributions to The Uni-versity of Texas MD Anderson Moon Shots Program.

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 September 11, 2017; revised February 19, 2018; accepted April 10,2018; published first April 16, 2018.

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Figure 6.

PR1/HLA-A2 is presented on the surface of patientmultiple myeloma cells. Bonemarrow smears from 2 patients with multiple myeloma were stained with(A) anti-PR1/HLA-A2 (8F4)-647 (red) and anti-HLA-A2-Alex-488 (green) or (B)anti-PR1/HLA-A2 (8F4)-647 (red) and anti-CD138-Alex-488 (green), and thenimaged using confocal laser microscopy. PR1/HLA-A2 is expressed by multiplemyeloma cells as shown by the costaining of 8F4 with HLA-A2 or CD138.

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2018;24:3386-3396. Published OnlineFirst April 16, 2018.Clin Cancer Res   Gheath Alatrash, Alexander A. Perakis, Celine Kerros, et al.   Treatment of Multiple MyelomaTargeting the Leukemia Antigen PR1 with Immunotherapy for the

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