Sarcoma Eradication by Doxorubicin and Targeted TNF Relies ... · Milan, Italy). Cell lines were received between 2010 and 2017, expanded, and stored as cryopreserved aliquots in

Microenvironment and Immunology

Sarcoma Eradication by Doxorubicin andTargeted TNF Relies upon CD8þ T-cellRecognition of a Retroviral AntigenPhilipp Probst1, Janine Kopp1, Annette Oxenius2, Mario P. Colombo3,Danilo Ritz4, Tim Fugmann4, and Dario Neri1

Abstract

Antibody–cytokine complexes may offer new tools to treatcancer. Here, we show how TNF-linked antibodies, which recog-nize tumor-selective splice isoforms of fibronectin (F8-TNF), canbe exploited to eradicate sarcomas in immunocompetent mice.We treated mice bearing WEHI-164 fibrosarcoma with a combi-nation of F8-TNF and doxorubicin, curing the majority of treatedanimals (29/37). Notably, cured mice were resistant to rechal-lenge not only by WEHI-164 cells but also heterologous C51 orCT26 colorectal tumor cells in a CD8þ T-cell–dependent process.Mechanistic analyses revealed that each tumor cell line presented

AH1, a common endogenous retroviral peptide. Numbers ofAH1-specific CD8þ T cells exhibiting cytotoxic capacity wereincreased by F8-TNF plus doxorubicin treatment, arguing thatcognate CD8þ T cells contributed to tumor eradication. Sequenceanalysis of T-cell receptors ofCD8þ T cells revealed the presence ofH-2Ld/AH1-specific T cells and an expansionof sequence diversityin treated mice. Overall, our findings provide evidence thatretroviral genes contribute to tumoral immunosurveillance in aprocess that can be generally boosted by F8-TNF and doxorubicintreatment. Cancer Res; 77(13); 3644–54. �2017 AACR.

IntroductionSoft tissue sarcomas (STS) are a heterogeneous group of more

than 50malignancies, which account for 1% of all adult and 15%of pediatric cancers. These tumors form in nonepithelial extra-skeletal tissues, including muscle, fat, and fibrous supportingstructures, arising mainly from embryonic mesoderm (1). Theprimary tumor is typically treated by surgical excision plus radio-therapy. The overall 5-year survival rate for STS patients is 50% to60% in adults and 75% in children, depending on the tumor size,depth, site, grade and histologic subtype (2). However, if thetumor develops into a metastatic disease, the prognosis for thepatient is dismal, with an expectedmedian overall survival of only8 to 12months. Themajority of STSs are treatedwithdoxorubicin-based regimens as first-line chemotherapy. The reported responserates for this type of intervention ranges between 12% and 24%(3). Combination therapies involving doxorubicin, ifosfamide,and dacarbazine were developed to increase response rates andimprove survival of patients.However, the combinations havenot

been proven to substantially increase response rates, progression-free survival or overall survival, while increasing the toxicityburden (2, 4). Thus, the improvement of treatment modalitiesfor metastatic STSs has remained an unmet medical need.

More than 100 years ago, William Coley observed a sponta-neous regression of an established STS in a patient who hadexperienced a streptococcal infection of the skin (erysipelas infec-tion) and went on to treat patients with a mixture of heat-inactivated mycobacteria (hence named Coley toxin). A compar-ative evaluation, published inNature in 1992, revealed that morethan 50% of sarcoma patients, who had been treated with Coleytoxin, enjoyeddurable complete remissions (CR) from thedisease(5), while CRs are virtually never observed with modern chemo-therapy (2, 3). The author concluded that "in the light of thepredominantly disappointing results with chemotherapy in thetreatment of such advanced stages of cancer," an approach basedon Coley toxin or on related immunostimulatory strategies "iscertainly a reasonable place to concentrate our efforts."

The endotoxins in Coley vaccine stimulated the release of highconcentrations of TNF, among other proinflammatory cytokines.The sensitivity of tumors of mesodermal origin to TNF hasprompted numerous investigations. Carswell and colleagues (6)used a sarcoma in the initial discovery of TNF, whereas Berendtand colleagues (7) used STS to describe the essential importance oftumor immunogenicity and a corresponding T-cell immuneresponse to the curative effects of endotoxin therapy. The systemicuse of recombinant TNF was not successful in the clinic. However,the use of TNF in isolated limb perfusion procedures in combina-tion with melphalan for the treatment of inoperable STSs wasfound to be potently active even for the eradication of large tumormasses and has received marketing authorization in Europe (8).

We have previously reported that the therapeutic index ofmurine TNF can be dramatically enhanced by fusion to suitableantibody fragments capable of selective localization to the tumor

1Department of Chemistry and Applied Biosciences, Swiss Federal Institute ofTechnology (ETH Z€urich), Z€urich, Switzerland. 2Department of Biology, SwissFederal Institute of Technology (ETH Z€urich), Z€urich, Switzerland. 3MolecularImmunology Unit, Department of Experimental Oncology and Molecular Med-icine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy. 4Philochem AG,Otelfingen, Switzerland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Dario Neri, Department of Chemistry and AppliedBiosciences, Swiss Federal Institute of Technology, HCI G 392.4 Vladimir-Prelog-Weg 1-5/10, Z€urich 8093, Switzerland. Phone: 414-4633-7401; Fax:414-4633-1358; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-2946

�2017 American Association for Cancer Research.

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environment. In particular, a strong activity in mouse models ofsarcoma has been observed for TNF fusions to the F8 or the L19antibody, specific to the alternatively spliced EDA and EDBdomains of fibronectin, respectively (9, 10). These splice isoformsof fibronectin are virtually undetectable in normal adult tissues(exception made for placenta, endometrium, and some vessels inthe ovaries; ref. 11), but are abundantly found around the tumorblood vessels in most malignancies (11, 12). In two immuno-competentmousemodels of STS, doxorubicin did not exhibit anydetectable inhibition of tumor growth, while its combinationwith F8-TNF was curative (9). Similarly, potent therapeutic activ-ity in sarcomahasbeen reported for L19-TNF in combinationwithmelphalan (10). The fully human version of L19-TNF has beenshown tobe clinically active in isolated limbperfusionprocedures(13) and for the intralesional administration to patients withstage III melanoma (14). The systemic administration of L19-TNFhas been found to be safe for up to 1 mg/patient. A clinical trialfeaturing a combination with doxorubicin in STS patients iscurrently ongoing in Italy and Germany (Eudra-CT no. 2012-000950-75).

Here, we present a detailed analysis of how the antibody-baseddelivery of TNF to sarcoma potently synergizes with doxorubicinand confers a protective immunity against homologous andheterologous tumors. The combination of T-cell receptor andexome sequencing, as well as the analysis of MHC class I–boundpeptides, led to the identification of the retroviral AH1 peptide(SPSYVYHQF) as a contributor to the tumor rejection process.

Materials and MethodsCell lines, animals, and tumor models

All tumor cell lines were obtained from the ATCC with theexception of C51 colon carcinoma and F1F fibrosarcoma (bothkindly provided by M.P. Colombo, Istituto Nazionale Tumori,Milan, Italy). Cell lines were received between 2010 and 2017,expanded, and stored as cryopreserved aliquots in liquidnitrogen. Cells were grown according the supplier's protocoland kept in culture for no longer than 2 months. Authentica-tion of the cell lines also including check of postfreeze viabil-ity, growth properties, and morphology, test for mycoplasmacontamination, isoenzyme assay, and sterility test were per-formed by the cell bank before shipment. Eight-week-oldfemale BALB/c mice were purchased from Charles River Lab-oratories (Germany). All animal experiments were performedunder a project license granted by the Veterin€aramt des Kan-tons Z€urich, Z€urich, Switzerland (42/2012, 27/2015) in agree-ment with Swiss regulations.

Antibodies and drugs for therapy experimentsThe F8-TNF immunocytokine was produced as described pre-

viously (9). Doxorubicin was purchased in the commerciallyavailable form of 10 mg/5 mL solution for injection (SandozPharmaceuticals AG). Rat anti-CD4 (GK1.5, BioXCell), ratanti-CD8 (YTS169.4, Bio X Cell) and rabbit anti-Asialo GM1(Wako Chemicals) antibodies were used for in vivo depletion.

Therapy study and in vivo depletion of NK, CD4þ, and CD8þ

cellsExponentially growing WEHI-164 cells were harvested, repeat-

edly washed, and resuspended in saline prior to injection. Tumorcells were implanted subcutaneously in the right flank of BALB/c

mice using 3� 106 cells permouse. Tumor volumewas calculatedas follows: [length (mm) � width (mm)� width (mm)]/2. Micewere randomly divided into two groups and injected into thelateral tail vein. The treatment group (n ¼ 37) received an initialinjection of doxorubicin (5mg/kg) followed by three injections of2 mg F8-TNF every 48 hours starting on the same day. The negativecontrol group was treated with saline (n ¼ 5). For the in vivodepletion of natural killer (NK), CD4þ, and CD8þ cells,WEHI-164 tumor–bearing mice (n ¼ 5/group) were repeatedlyinjected intraperitoneally with 30 mL anti-Asialo GM1, 250 mganti-CD4, or 250 mg anti-CD8 antibodies on days 2, 5, 8, and 11after tumor implantation. An additional group (n ¼ 5) wasinjected with 250 mg anti-CD4 antibodies on days �1, 2, 4, and8. A saline group (n¼ 5) and a treatment groupwithout depletion(n¼ 5)were included as controls. Animals were euthanized whentumor volumes reached amaximum of 2,000mm3 or weight lossexceeded 15%.

Tumor rechallengeCured mice were injected subcutaneously with 3 � 106

WEHI-164, 1 � 106 C51, 1 � 106 CT26, or 1 � 106 F1F cells21 days after completion of the therapy study. In addition, tumorchallenge of curedmicewithWEHI-164 cellswas also tested inNKcell-, CD4þ T cell-, and CD8þ T-cell–depleted mice (n ¼ 5 pergroup). Depletion antibodies were injected intraperitoneallyevery third day starting on day 2 before tumor implantation. Ascontrols, na€�ve BALB/c mice of the same age were also injectedwith the tumor cells (n ¼ 3 per group) to monitor tumor growthand cell viability after injection.

Generation of H-2Ld tetramersAPC-conjugated H-2Ld tetramers were produced as described

by Toebes and colleagues (15) with minor modifications. Plas-mids of the H-2Ld heavy chain and of human b2-microglobulinwere a kind gift from A. Oxenius (ETH Z€urich, Z€urich, Switzer-land). AH1 (SPSYVYHQF) and p29 (YPNVNIHNF) peptides wereordered from Biomatik.

IHC and immunofluorescence analysisTumors were excised and immediately embedded in frozen

section medium (Thermo Fisher Scienfic) 48 hours after thefirst injection. Staining was performed on 10-mm cryosectionsfixed in ice-cold acetone. For immunofluorescence analysis,antibodies against the following antigens were used: CD11c(N418, BioLegend), F4/80 (BM8, eBiosciences), Asialo GM1(Wako Chemicals), CD4 (GK1.5, BioLegend), CD8 (53-6.7,BioLegend), Foxp3 (FJK-16s, eBiosciences), EDA (F8), andCD31/PECAM-1 (390, Invitrogen; M-185, Santa Cruz Biotech-nology). Primary antibodies were detected either with AlexaFluor 488- or Alexa Fluor 594–coupled secondary antibodies(Invitrogen). For immunofluorescence staining with H2-Ld

tetramers, freshly frozen tissue sections were generated fromF8-TNF/doxorubicin–treated and saline-treated mouse spleensfrom day 18 of the therapy, from tumors 48 hours after the firstinjection and from spleens of healthy BALB/c mice. Immuno-fluorescence experiments with H2-Ld tetramers were performedaccording to established protocols (16). Stained slideswere analyzed with an Axioskop2 mot plus microscope (with10�/0.30 and 20�/0.50 objective lenses, Zeiss) and documen-ted with an AxioCam color or an AxioCam MRm (Zeiss)camera, using the AxioVision software (4.7.2 Release, Zeiss).

Sarcoma Eradication by Targeted TNF

www.aacrjournals.org Cancer Res; 77(13) July 1, 2017 3645




Images were processed in Photoshop CS6 for Macintosh(Adobe), and the number of cells was counted manually usingImageJ.

T-cell receptor beta library preparation and sequencingF8-TNF/doxorubicin–treated and saline control mouse

spleens were excised at day 18 of the therapy. CD8þ T cellswere isolated from single-cell suspensions through depletionof CD8� leukocytes using the Dynabeads Untouched MouseCD8 Cells Kit (Thermo Fisher Scientific). RNA was isolatedusing TRIzol reagent (Thermo Fisher Scientific) accordingto the manufacturer's protocol. Reverse transcription wasperformed using an OneStep RT-PCR Kit (Qiagen) withiRepertoire mouse T-cell receptor beta primers (MTBIvc, iRe-pertoire Inc.) under the conditions specified by iRepertoire.PCR products were purified using SPRIselect magnetic beads(Beckman Coulter) and subjected to a second PCR using aMultiplex PCR Kit (Qiagen) with iRepertoire primers contain-ing communal Illumina sequencing adaptors. Quality andquantity of the libraries were determined using Qubit quan-titation (Thermo Fisher Scientific) and the D1000 ScreenTapeAssay (Agilent). Final libraries were pooled according to themanufacturer's instructions and sequenced on an IluminaMiSeq platform.

T-cell receptor beta sequencing data analysisSequencing analysis, including filtering and sequence align-

ment, was performed by iRepertoire Inc. The Lorenz curve and theGini coefficient are commonly used to measure the inequalityamong values in economic distributions, such as the levels ofincome.However, in recent studies, theyhave alsobeen applied tocharacterize the frequency distribution of T-cell clones (17). TheLorenz curve was constructed by plotting the cumulative percent-age of the whole CD8þ T-cell repertoire sequences on the y-axisrelative to the cumulative proportion of unique T-cell receptor b(TCRb) clonotype sequences on the x-axis. The Gini coefficientcorresponds to the ratio of the area under the Lorenz curve and theuniform distribution line to the total area under the uniformdistribution line. It ranges between 0 and 1, a value of 1 indicatinga monoclonal sample and 0 meaning equal distribution of allclones. The Gini coefficient can be approximated with the trap-ezoidal rule as follows:

G ¼ 1�Xn

k¼1

Xk � Xk�1ð Þ Yk � Yk�1ð Þ

Unique clonotype sequences were ranked from least frequent(k¼ 1) to most frequent (k¼ n). Xk is the cumulative proportionof unique clonotype sequences 1 to k and Yk is the cumulativepercentage of clonotypes 1 to k.

Whole-exome sequencing of the WEHI-164 cell lineDNA from the tumor cell line and from liver cells of a healthy

8-week-old female BALB/c mouse was extracted using theQIAamp DNA Mini Kit (Qiagen). Exome capture was performedwith the use of the SureSelectXT Mouse All-Exon Kit (AgilentTechnologies). Exome libraries were enriched using the SureSe-lectXT Target Enrichment System for Illumina Paired-End Multi-plexed Sequencing Library protocol for 3 mg DNA (Version B4,August 2015, Agilent Technologies) and sequenced on the HiSeq2500 v4 platform (Illumina). Sequence reads were aligned to the

mm10/GRCm38 genome using Bowtie 2 version 2.2.6 (options:no-mixed, no-discordant, very-sensitive; ref. 18). The average on-target coverage was 159� with approximately 93% of the targetsequence being covered by at least 30 reads. GATK v3.5 (19) wasused for base quality recalibration (known sites from dbSNPv137), variant calling/genotyping (HaploTypeCaller, GATK), andvariant filtering. We used a hard-filtering strategy with the fol-lowing criteria: DP > 20, FS < 30, QD > 2. The resulting variantswere annotated with snpEff version 4.11 (20), and only variantswith impact levels "moderate" and "high" were kept. Finally,variants thatwere also detected inwild-type BALB/cwere removedfrom the candidate list.

Affinity purification of mouse MHC I molecules (H-2 class Imolecules)

Affinity purification of H-2 class I complexes was performed aspreviously described for human HLA class I molecules (21).Lysis was performed at a cell density of approximately 2.5 �107 cells/mL, andH-2 class I complexes were purified from lysatesby incubation with M1/42 (Bio X Cell) antibody-coupled resin.

Analysis of H-2 class I peptides by LC/MSAnalysis of H-2 class I peptides by LC/MS was carried out as

previously described for human HLA class I peptides (22).MS/MS data were searched against all murine proteins(84,716 entries) of the UniProt database, downloaded onJune 5, 2016. To this protein database, additional sequenceswere added corresponding to nonsynonymous mutationsidentified by exome sequencing. Both wild-type and mutatedsequences were added as 39mers, with the mutation presentat position 20. A total of 1,735 mutated and 1,727 corre-sponding wild type sequences were added. The followinganalysis settings were used with SEQUEST: (i) no-enzyme(unspecific); (ii) precursor mass tolerance 4 ppm; (iii) frag-ment mass tolerance 0.02Da; and (iv) two variable modifica-tions (oxidation of methionine, and phosphorylation ofserine, threonine, or tyrosine). FDRs were calculated with thePercolator plug-in. MaxQuant parameters were set asdescribed previously (23).

Bioinformatics processingBioinformatics processing using the DeepQuanTR (24) soft-

ware suite was performed as described previously (22). The sixreplicate analyses of the WEHI-164 MHC class I peptidome werealigned, resulting in one dataset. Sequence identifications wasremoved byDeepQuanTR if ambiguity remained after alignment,a process that improves data quality and is necessary whencombining results from two or more search engines (22). Peptideto protein annotation was also performed with the DeepQuanTRsoftware (24).

MHC binding prediction of identified peptidesUnique peptide identifications with a length between 8 and 11

amino acids were subjected to MHC class I binding predictionanalysis using NetMHCpan 3.0 net (25). Each peptide wasassigned the minimal rank for the three BALB/c H-2 alleles Dd,Kd, and Ld. Peptides were annotated as being predicted to bind ifthe rank calculated by NetMHCpan was below 2% for at least oneof the three alleles.

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Cancer Res; 77(13) July 1, 2017 Cancer Research3646




Gibbs clustering of MHC class I peptides and annotation ofclusters to MHC

All 9mers of the WEHI-164 MHC class I peptidome weresubjected to GibbsCluster-1.1 Server (26) analysis using thedefault settings without alignment (1–5 clusters, and "use trashcluster to remove outliers" enabled). On the basis of the resultingclusters, 9mers were annotated to Dd, Kd, and Ld alleles accordingto the motifs presented by the SYFPEITHI database (27).

Flow cytometry and intracellular cytokine stainingSpleens were excised at day 18 of the therapy or at day 3 after

rechallenge, mashed in Red Blood Cell Lysis Buffer (Roche),and passed through a 40-mm cell strainer (EASYstrainer, Greiner

Bio-One). Single-cell suspensions were either used directly forFACS staining or cultured for 2 days in RPMI1640 (ThermoFisher Scientific, supplemented with 10% FBS, 2 mmol/L glu-tamine, 50 mmol/L 2-mercaptoethanol, and antibiotic-antimy-cotic solution) with 10 mg/mL AH1 or p29 peptide. Afterwards,100 U/mL IL2 was added, and cells were cultured for another3 days. For flow cytometry analysis, 1 � 106 splenocytes wereincubated for 1 hour at 4�C with H-2Ld tetramer, LIVE/DEADFixable Near-IR Dead Cell Stain (Thermo Fisher Scientific), andfluorochrome-conjugated antibodies against CD8 (FITC, KT15,Thermo Fisher Scientific), CD4 (PerCP, GK1.5, BioLegend), andB220 (PerCP, RA3-6B2, BioLegend) in PBS containing 2% FBSand 2 mmol/L EDTA.

Figure 1.

Therapeutic activity of the F8-TNF/doxorubicin combination againstsubcutaneous murine WEHI-164fibrosarcoma and in vivo depletion ofNK cells, CD4þ T cells, and CD8þ T cells.A, Mice were challenged with 3 � 106

WEHI-164 tumor cells, and treatmentwas started when tumors reached a sizeof approximately 75 mm3. Mice wererandomly grouped and received a singleinjection of 5 mg/kg doxorubicin (grayarrow) and three injections of 2 mg F8-TNF (black arrow) intravenously into thelateral tail vein (n¼ 37). Saline was usedas negative control (n ¼ 5). �� , P <0.0001 (regular two-way ANOVA testwith the Bonferroni posttest). Data,mean tumor volumes (�SEM). B,Representative images of a treatedmouse and tumor regression duringtherapy at days 5, 7, 12, and 30 aftertumor implantation. C, WEHI-164tumor–bearing mice were treated withthe F8-TNF/doxorubicin combination.Depletion antibodieswere administeredon days 2, 5, 8, and 11 (gray arrows,square) after tumor implantation. Onegroup was included where CD4þ T cellswere depleted on days �1, 2, 5, and 8(circle, gray arrows). A saline-treatednegative control group and anundepleted, F8-TNF/doxorubicin–treated positive control group wereincluded. Data, mean tumor volumes(�SEM), n ¼ 5 mice per group. � , P <0.05; �� , P < 0.0001 (regular two-wayANOVA test with the Bonferroniposttest).






For intracellular cytokine staining, splenocyte cultures werestimulated with the AH1 peptide and GolgiStop for 4 hoursaccording to the manufacturer's instructions (BD Cytofix/Cyto-perm Plus Fixation/Permeabilization Kit, BD Biosciences). Cellswere surface stained as described above. Following fixation andpermeabilization, cells were stained against mouse IFNg (PE,XMG1.2, BioLegend) for 1 hour at 4 �C. Cells were analyzed ona CytoFLEX cytometer (Beckman Coulter), and data were pro-cessed using FlowJo (v.10, Tree Star).

In vitro cytotoxicity assayIn vitro cytotoxicity assays were performed using the CytoTox96

Non-Radioactive Cytotoxicity Assay Kit (Promega) according tothe manufacturer's protocol. Briefly, splenocytes of cured micewere cultured as described above and used as effector cells. Tumorcells (5 � 103) were cocultured with the effector cells at a ratio of1:0.1, 1:1, 1:5, 1:25, and 1:50 in 96-well plates for 4 hours. Eachratiowasmeasured inquadruplicates. Absorbance (A) valuesweremeasured at 492 nm. The percentage of specific lysis for each ratiowas calculated as follows:½AðexperimentalÞ � Aðeffector spontaneousÞ � Aðtarget spontaneousÞ�

½Aðtarget maximumÞ � Aðtarget spontaneousÞ�� 100

Quantitative PCRRNA of cell lines and tissues of an 8-week-old female BALB/c

mouse was prepared using TRIzol (Thermo Fisher Scientific). Twomicrogram RNA was reverse transcribed with GoScript reversetranscriptase (Promega) using Oligo(dT)23 primer (SigmaAldrich). The gene expression levels were analyzed in a SYBRGreen real-time RT-PCR reaction with the AB7900 HT FastRT-PCR system (Life Technologies) in three different cell isolatesin triplicates. Rplp0 was used as reference gene for normalization.Primers were as follows:murine Rplp0 (forward) 50-AGATTCGGGATATGCTGTTGG-30,murine Rplp0 (reverse), 50-TCGGGTCCTAGACCAGTGTTC-30;human Rplp0 (forward) 50-CAGATTGGCTACCCAACTGTT-30,human Rplp0 (reverse), 50-GGGAAGGTGTAATCCGTCTCC-30;gp70 (forward), 50-CACCAATTTGAAAGACGAGCC-30,gp70 (reverse) 50-CAATTCCGCCCATAGTGAGTC-30.

Statistical analysesData were analyzed using Prism 6.0 (GraphPad Software, Inc.).

Statistical significance of in vivo experiments, the in vitro cytotox-icity assay, the usage of T-cell receptor beta joining (TRBJ) and

T-cell receptor beta variable (TRBV) gene segments, the number oftrimmed TRBJ and TRBV nucleotides, and the CDR3 lengthdistribution were determined with a regular two-way ANOVAtestwith the Bonferroni posttest. A Student t testwas used to assessdifferences of the Gini coefficients between the F8-TNF/doxoru-bicin and the saline treatment group. Data represent means �SEM. P < 0.05was considered statistically significant (� ¼ P < 0.05,�� ¼ P < 0.01, �� P ¼ < 0.001, �� ¼ P < 0.0001).

ResultsTherapy experiments, tumor rechallenge, and depletionstudies

BALB/c mice, bearing subcutaneous WEHI-164 fibrosarcoma,were treated with doxorubicin (5 mg/kg) and F8-TNF (threeinjections of 2 mg) as described previously (9). Figure 1A showsthat 29 of 37 mice were cured in the treatment group, whiletumors grew in the saline control group. The cancer cure pro-ceeded with the conversion of the tumor mass into a black scab,which eventually fell off without regrowing (Fig. 1B).

Execution of the therapy procedure after selective antibody-based leukocyte depletion revealed a requirement forCD8þT cellsand NK cells. CD4þ T cells were dispensable if depleted aftertumor implantation. However, depletion of CD4þ T lymphocytesprior to tumor growth had an impact on the therapeutic outcome(Fig. 1C).

Cured mice were rechallenged with WEHI-164 or with heter-ologous C51 or CT26 tumor cells. In all cases, the tumors did notgrow in the cured mice, whereas they grew in na€�ve control mice.Depletion experiments indicated that the tumor protection wasmediated by CD8þ T lymphocytes (Table 1).

A microscopic analysis of tumor sections, performed 48 hoursafter the first intravenous injection of mice with the F8-TNF/doxorubicin combination or with saline, confirmed that themajority of the tumor had been converted into necrotic tissue,most probably by adirect cytotoxic activity of doxorubicin and theTNF moiety (Fig. 2A). Analysis of the leukocyte infiltrate, usingCD11c, F4/80, asialo GM1, CD4, CD8, and Foxp3 antibodies,revealed an increased infiltration of macrophages, dendritic cells,NK cells, and CD8þ T cells into the tumor mass, while CD4þ Tcells and regulatory T cells were essentially not affected (Fig. 2Band G). Fibronectin molecules, containing the alternativelyspliced EDA domain that is targeted by the F8 antibody, couldstill be found in the dying tissue (Fig. 2H).

Table 1. Rejection of tumor challenges by WEHI-164–cured mice

Mouse type Depletion TumorTumor growth (%)at day 7

Tumor growth (%)at day 21

Na€�vea None WEHI-164 3/3 (100) 3/3 (100)Cured None WEHI-164 0/5 (0) 0/5 (0)Na€�vea None C51 3/3 (100) 3/3 (100)Cured None C51 0/3 (0) 0/3 (0)Na€�vea None CT26 3/3 (100) 3/3 (100)Cured None CT26 0/3 (0) 0/3 (0)Na€�vea None F1F 3/3 (100) 3/3 (100)Cured None F1F 3/3 (100) 3/3 (100)Cured CD4þ T cells WEHI-164 0/5 (0) 0/5 (0)Cured CD8þ T cells WEHI-164 5/5 (100) 1/5 (20)b

Cured NK cells WEHI-164 0/5 (0) 0/5 (0)aTumor cell injection in healthy BALB/c mice was used as control for tumor growth.bInitial tumor growth observed in all CD8þT-cell–depletedmice. Tumors reached a size of 40 to 50mm3. Nodules started to shrink again after day 8 to 10 as injectionsof depleting antibodies were stopped. One tumor reached termination criteria after 21 days.

Probst et al.





Exome sequencing, MHC class I peptidome, and tetrameranalysis

Exome sequencing of WEHI-164 was performed to identifymutations in protein-coding regions of the DNA of the tumor cellline. The analysis revealed the presence of 1,648 missense muta-tions, 85 nonsense variants, and 94 short insertions and deletionsin the WEHI-164 exome compared with wild-type BALB/c (Sup-plementary Table S1). The high mutation rate observed inWEHI-164 cells is in line with similar recent analyses of mouse tumorexomes (28, 29).

Using a combination of immunocapture and mass spectrom-etry, we have recently reported the confident identification ofthousands of peptide sequences bound to HLA class I onhuman tumor cells (22). For this work, the methodology wasadapted to allow the study of the murine MHC class I pepti-dome, using the M1/42 antibody for immunocapture. WEHI-164 cell lysates were analyzed in six independent experiments,which led to the identification of 4,639 unique peptides withhigh confidence (i.e., less than 1% FDR). The majority of theidentified peptides (4,151, 90%) were 8-11 amino acid long,which represents the typical length of MHC class I–boundpeptides (Fig. 3; Supplementary Table S2). Binding predictionanalysis revealed that 84% of the 8-11mers were predicted tobind to the cognate MHC class I molecule. The identified 9merswere clustered into the three alleles H-2Kd, H-2Dd, and H-2Ld

leading to MHC-specific motifs in agreement with the literature(Fig. 3; ref. 27). Even though mass spectrometric data wereinterrogated against a database containing not only wild-typesequences, but also the catalog of somatic mutations identifiedby whole-exome sequencing, no neoepitope was found withhigh confidence. Seventeen phosphorylated 8-11mers wereobserved (Supplementary Table S2), which is in line withprevious reports on the incidence of this class of epitopes incancer cell peptidomes (30, 31).

AsCT26andC51 tumorswere rejected after the initial cure fromWEHI-164 sarcomas, we hypothesized that the three malignan-cies would share at least one common tumor rejection antigen.The AH1 (SPSYVYHQF) peptide, which is derived from the gp70envelope protein of the murine leukemia virus, had been previ-ously reported as the immunodominant antigen of CT26 tumorcells (32). Interestingly,we could also identify theAH1peptide onWEHI-164 cells by MHC I peptidome analysis (SupplementaryTable S2). Presentation of the retroviral peptide onC51 and CT26cells was verified by peptidome analysis (data not shown), and ahigh expression of the gp70 genewas detected in all three cell lines(Fig. 4).

Tetramer analysis was performed with recombinant H-2Ld

molecules loaded with AH1 (SPSYVYHQF) or with p29(YPNVNIHNF), an irrelevant peptide (33) serving as negativecontrol. Spleen sections from F8-TNF/doxorubicin treated,saline treated, and from healthy BALB/c mice were comparedin immunofluorescence analysis. A specific staining of AH1-binding T cells was observed in sections of mice, which hadbeen cured with F8-TNF and doxorubicin. In parts of theanalyzed sections, more than 10% of the CD8þ T cells werefound to recognize the retroviral AH1 peptide (Fig. 5A). Flowcytometry analysis revealed an expansion of AH1-specificCD8þ T cells in F8-TNF/doxorubicin–treated mice comparedwith saline-treated mice. Highest levels of AH1-specific CD8þ

T cells were found in cured mice that were rechallenged in vivowith WEHI-164 cells (Fig. 5B). Samples from healthy mice or

Figure 2.

Ex vivo IHC and immunofluorescence analysis on WEHI-164 tumor sections48 hours following treatment with saline or the F8-TNF/doxorubicincombination. A, H&E staining. Magnification, �10. B–G, Immunofluorescenceanalysis of tumor-infiltrating cells. Cellular antigens were detected withAlexa Fluor 488 (green fluorescence); blood vessels were visualized bystaining for CD31 (Alexa Fluor 594, red fluorescence). Magnification, �20.H, Analysis of EDA antigen expression by immunofluorescence with F8antibody (Alexa Fluor 488, green fluorescence) and anti-CD31 (Alexa Fluor594, red fluorescence). Magnification, �20. Scale bar, 100 mm.






from tumor-bearing mice, which had received saline injec-tions, were largely negative in these analyses (Fig. 5; Supple-mentary Figs. S1 and S2).

We performed a rechallenge experiment with syngeneic F1Ffibrosarcoma cells to demonstrate the importance of the anti-AH1response in cross-protection of WEHI-164–cured mice againstother tumors. F1F had been previously reported to be gp70negative (34). Analysis of gp70 expression by qPCR revealed anextremely low expression level of the retroviral protein comparedwith other BALB/c cell lines (Fig. 4). Interestingly, cured mice didnot reject F1F tumor cells (Table 1). In addition, an in vitrocytotoxicity assay revealed a specific lysis of CT26 and WEHI-164 cells by AH1-stimulated splenocytes, whereas no specific lysisof F1F cells could be detected (Fig. 5C).

TCR sequencingTo learn about the dynamics of the TCR sequences as a result of

the therapy intervention, CD8þ T cells were purified from spleensof mice treated with F8-TNF/doxorubicin or saline, 18 days afterthe start of therapy. TCRb sequencing libraries of the isolated Tcells were constructed and analyzed on the Illumina MiSeqplatform. In total, 41,200 � 2,571 and 46,007 � 25,379 uniqueTCRb sequences were determined in the two study groups. Theaverage number of productive TCRb sequence reads was262,318 � 36,756 for the treatment group and 1,012,046 �198,544 for the saline-treated group samples. A significantincrease in sequence diversity was observed for lymphocytes fromthe F8-TNF/doxorubicin group, as evidenced by using the Ginicoefficient (Fig. 6A and B). Analysis of the usage of V and J

Figure 3.

MHC class I peptidome analysis of theWEHI-164 cell line.A, Number of MHC class I–bound peptides identified ineach peptidome sample. B, Length distribution ofpeptides identified from the WEHI-164 cell line.C,H-2–specificmotifs from theMHC class I peptidome ofWEHI-164. All unique 9mers were subjected to Gibbsclustering with the GibbsCluster-1.1 Server (26), andidentified motifs were annotated by comparing theexperimental data with H-2Kd, -Dd, and -Ld allele motifspresented by the SYFPEITHI database (27).

Figure 4.

Quantitative PCR analysis of gp70 expression. Relativeexpression of the retroviral gp70 gene, assessed byquantitative PCR, in cancer cell lines of BALB/c,C57BL/6, or other origin (e.g., human origin for Ramosand SKRC52, Sv129 mice for F9, C3H/HeN mice forK1735M2). Lowexpressionwasobservedonly in F1F cells,among tumors of BALB/c origin (arrow). In addition, lowor undetectable expression levels (�) were found innormal BALB/C tissues. Rplp0 was used as referencegene for the normalization of the gp70 expression levels.Data represent mean relative expression values (�SEM)of three independent experiments.

Probst et al.





segments in the TCR b-chain revealed small but significantchanges in the usage of the TRBV 5 and TRBV 19 gene segments,the frequency of trimmed TRBJ and TRBV nucleotides, and theCDR3 sequence length distribution (Fig. 6C–E).

Ten sequences of the beta chain of T cells specific to the AH1peptide have recently been reported on the basis of anticancerpeptide vaccination experiments (35). Nine of these sequenceswere found in our analysis (Supplementary Table S3), thusproviding additional evidence for the specific recognition of AH1in mice with WEHI-164 sarcomas.

DiscussionIn this article, we showed that immunocompetent mice, bear-

ing syngeneic WEHI-164 fibrosarcoma, could be cured using acombinationof the F8-TNF immunocytokine anddoxorubicin. Incontrast, the use of doxorubicin as a single agent showed nodetectable tumor growth inhibition in sarcoma-bearing mice,confirming the essential contribution of targeted TNF (9). Anthra-cyclines are commonly used for the first-line treatment of meta-static STSs in humans but are rarely associated with objective

responses and lead tomedian progression-free survival of approx-imately 4 months (36, 37).

Cancer cures in mice crucially relied on the action of CD8þ Tcells and of NK cells, as evidenced by depletion experiments.Cured mice could reject subsequent challenges with WEHI-164,C51, or CT26 cells, in a process that was due to the action of CD8þ

T cells. The bulk of the neoplastic mass is rapidly killed by thecombined biocidal activity of F8-TNF and doxorubicin. However,CD8þ T cells are crucially important for the selective eliminationof the last residual tumor cells and for the development ofprotective immunity.

It is often assumed that immune rejection of solid tumors relieson the recognition of mutated peptides presented onMHC class Iby CD8þ T cells (28, 38). However, in this work and in otherrecent reports, mass spectrometric analysis of MHC class I com-plexes has shown that mutated peptides are rarely found (21, 39,40). For example, only one mutated peptide could be observedamong over 10,000 peptide sequences presented onHLA class I inhuman melanoma cells (21). In our work, over 1,700 nonsynon-ymous mutations were observed in exome sequencing ofWEHI-164 cells comparedwithwild-type BALB/c, but nomutated

Figure 5.

Analysis of AH1-specific CD8þ T cells.A, Representative images of CD8 andH-2Ld tetramer costaining. Frozentissue sections were generated fromF8-TNF/doxorubicin–treated andhealthy BALB/c mouse spleens.Staining was performed with eitherH-2Ld/AH1 tetramers (red) or H-2Ld/p29 tetramers (red) and anti-CD8(green). Numbers correspond to thepercentage of double positive cells inthe CD8þ cell population.Magnification,�20. Scale bar, 100 mm.B, Frequency of AH1-specific T cellswas measured 7 days after in vitrostimulation. Cells were stained withH-2Ld/AH1 tetramers and against CD8,CD4, and B220. H-2Ld tetramersloaded with the p29 peptide wereused as negative control. Numbersindicate the frequency of antigen-specific CD8þ T cells. C, Lytic activityof AH1-specific T cells. Splenocytes ofrepresentative cured mice werecultured for 7 days in the presence ofAH1 (black) or p29 (gray) peptide andused as effector cells. Lytic activity ofthe effector cells against WEHI-164,CT26, andF1F tumor cellswas tested ina 4-hour nonradioactive cytotoxicityassay. The average specific lysis of arepresentative experiment is shown.






epitope could be found to be associated with MHC class I.However, the analysis of theMHC class I peptidome ofWEHI-164allowed the identification of potential tumor-associated antigensand in particular of the AH1 peptide (SPSYVYHQF, derivedfrom the gp70 envelope protein of the murine leukemia virus),which was also presented on C51 and CT26 tumor cells. TheSPSYVYHQF sequence was recognized by cognate T cells, whichhad been expanded as a result of the F8-TNF/doxorubicin

pharmacologic intervention. Flow cytometry analysis andthe staining of spleen sections revealed a specific detection ofSPSYVYHQF by CD8þ T cells, which had increased in numberafter cancer cure, while being largely undetectable before phar-macological intervention and in suitable negative controls. Anin vitro cytotoxicity assay confirmed the cytotoxic potential ofAH1-specific T cells against gp70þ tumor cells. F1F, a syngeneicBALB/c fibrosarcoma cell line that showed extremely low levels of

Figure 6.

Analysis of the CD8þ T-cell repertoire of salineand F8-TNF/doxorubicin–treated mice by next-generation sequencing of the TCRb CDR3region. A, Lorenz curves of TCRb sequencedistribution in samples of saline (black) andF8-TNF/doxorubicin–treated mice (gray).B, Gini coefficient values for the two differenttreatment groups. Lines, means � SEM;� , P ¼ 0.014 (unpaired, two-tailed t test). C, Barplot indicating the usage of the different TRBJgene segments (left) and comparison of thenumber of trimmed TRBJ nucleotides betweensamples of saline and F8-TNF/doxorubicin–treated mice (right). D, Usage of TRBV genesegments (left) and number of trimmed TRBVnucleotides (right). E, CDR3 sequence lengthcomparison. Data on all bar plots representmeans� SEM, n¼ 3 mice per group. � , P < 0.05;�� , P < 0.01; �� , P � 0.001; �� , P < 0.0001(regular two-way ANOVA test with theBonferroni posttest).

Probst et al.





gp70 expression, was not rejected by WEHI-164–cured mice andF1F cellswere resistant to lysis byAH1-specific T cells in vitro. Thesefindings provide additional evidence in support of the contribu-tion of an anti-AH1 response in the treatment of WEHI-164tumors and in providing cross-protective immunity.

Immune responses to endogenous retroelements have been amatter of intense investigations. Retroviral sequences have beenfound to be abundant in the genome of all vertebrate species thathave been studied. Many of the endogenous retroviral elementsthat have been integrated in the host germline have retained thecapacity to replicate (41). As for self-peptides, thymic presentationof these retroviral products leads to immunologic tolerance due todeletion of the respective TCR or BCR specificities (41). However,adaptive immunity to endogenous retroviral antigens can betriggered if the retroelements are transcriptionally induced afterthe establishment of the adaptive immune cell repertoire. Induc-tion of retroviral protein expression has been associated withchronic infection and autoimmune diseases both in mice andhumans (42–44). For example, human endogenous retrovirus(HERV) antigens were proposed as putative autoantigens for thedevelopment of systemic lupus erythematosus (45). In addition,adaptive immunity to retroelements is particularly relevant incancer, as transformed cells typically exhibit major epigeneticalterations compared with healthy cells. Consequently, recogni-tion of endogenous retroviral antigens, which are no longertranscriptionally repressed, has been frequently detected in cancer(46). Like other cancer testis antigens, the expression of theseretroviral proteins is often restricted to germ cells and testis. Theexpression of the gp70 envelope protein of the endogenousmurine leukemia virus had previously been shown to be restrictedto mouse cancer cell lines (47), and products of the HERV-Kfamily have shown abundant expression in various human can-cers (48). In addition, retroviral antigens havebeen shown to elicita potent T-cell activity against murine tumors, as well as humancancers (49).

It is thus conceivable that peptides similar to SPSYVYHQFmaybe upregulated in human malignancies and displayed on tumorcells, thereby contributing to the tumor surveillance process by T

cells. This issue may be particularly relevant for STSs, as thesetumors tend to be highly sensitive to the action of TNF (5).Encouraged by promising results in exploratory clinical trials insarcoma patients, L19-TNF (a fully human fusion protein specificto the EDB domain of fibronectin) in combination with doxo-rubicin is about to enter phase III clinical trials as a first-linetreatment for different subtypes of STSs.

Disclosure of Potential Conflicts of InterestD. Neri is a co-founder and shareholder at Philogen SpA. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: P. Probst, D. NeriDevelopment of methodology: P. Probst, D. NeriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Probst, J. Kopp, D. Ritz, T. Fugmann, D. NeriAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Probst, D. Ritz, T. FugmannWriting, review, and/or revision of the manuscript: P. Probst, D. NeriAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Oxenius, M.P. Colombo, D. NeriStudy supervision: D. Neri

AcknowledgmentsWe would like to thank Dr. J. K€uhn-Georgijevic (Functional Genomics

Center Zurich) for the help in preparing the exome sequencing libraries andDr. L. Opitz (Functional Genomics Center Zurich) for the analysis of the NGSdata.

Grant SupportD. Neri received financial support from the ETH Z€urich, the Swiss National

Science Foundation (grant nr. 310030B_163479/1), the ERC Advanced Grant"ZAUBERKUGEL," and the Federal Commission for Technology and Innova-tion (KTI, grant nr. 12803.1 VOUCH-LS).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedNovember 4, 2016; revisedMarch 14, 2017; accepted April 25, 2017;published OnlineFirst May 8, 2017.

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Probst et al.




2017;77:3644-3654. Published OnlineFirst May 8, 2017.Cancer Res Philipp Probst, Janine Kopp, Annette Oxenius, et al.

T-cell Recognition of a Retroviral Antigen+CD8Sarcoma Eradication by Doxorubicin and Targeted TNF Relies upon

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