Antigen Experienced T Cells from Peripheral Blood ... · 22/01/2020 · T cells could recognize processed and presented p53 neoantigens. Conclusions: PBL was a noninvasive source

CLINICAL CANCER RESEARCH | CLINICAL TRIALS: IMMUNOTHERAPY

Antigen Experienced T Cells from Peripheral BloodRecognize p53 Neoantigens A C

Parisa Malekzadeh1, Rami Yossef1, Gal Cafri1, Biman C. Paria1, Frank J. Lowery1, Mohammad Jafferji1,Meghan L. Good1, Abraham Sachs1, AmyR. Copeland1, Sanghyun P. Kim1, Scott Kivitz1, Maria R. Parkhurst1,Paul F. Robbins1, Satyajit Ray1, Liqiang Xi2, Mark Raffeld2, Zhiya Yu1, Nicholas P. Restifo1,Robert P.T. Somerville1, Steven A. Rosenberg1, and Drew C. Deniger1

ABSTRACT◥

Purpose: The purpose of this study was to evaluate antigenexperienced T cells in peripheral blood lymphocytes (PBL) forresponses to p53 neoantigens.

Experimental Design: PBLs from patients with a mutated TP53tumor were sorted for antigen-experienced T cells and in vitrostimulation (IVS) was performed with p53 neoantigens. The IVScultures were stimulated with antigen-presenting cells expressingp53 neoantigens, enriched for 41BB/OX40 and grown with rapidexpansion protocol.

Results: T-cell responses were not observed in the PBLs of 4patients who did not have tumor-infiltrating lymphocyte (TIL)responses to mutated TP53. In contrast, 5 patients with TILresponses to mutated TP53 also had similar T-cell responses intheir PBLs, indicating that the PBLs and TILs were congruent in p53

neoantigen reactivity. CD4þ and CD8þ T cells were specific forp53R175H, p53Y220C, or p53R248W neoantigens, including a 78%reactive T-cell culture against p53R175H andHLA-A�02:01. TrackingTCRB clonotypes (clonality, top ranked, and TP53 mutation-spe-cific) supported the enrichment of p53 neoantigen–reactive T cellsfromPBLs. The same T-cell receptor (TCR) from the TIL was foundin the IVS cultures in three cases and multiple unique TCRs werefound in another patient. TP53 mutation–specific T cells alsorecognized tumor cell lines bearing the appropriate human leuko-cyte antigen restriction element andTP53mutation, indicating theseT cells could recognize processed and presented p53 neoantigens.

Conclusions: PBL was a noninvasive source of T cells targetingTP53 mutations for cell therapy and can provide a window intointratumoral p53 neoantigen immune responses.

IntroductionAdoptive cell therapy (ACT) using autologous tumor-infiltrating

lymphocytes (TIL) mediated durable, complete cancer regressionsin patients with melanoma, breast, colon, cervical, and bile ductcancers (1–6). Collectively, these responses were likely based onrecognition of unique, patient-specific mutated neoantigensthrough the T-cell receptor (TCR; refs. 3–5, 7). TP53 is the mostfrequently mutated gene across all cancers and encodes the tumorsuppressor p53 protein (8). Approximately 30% of TP53 mutationsare shared “hotspots” in unrelated individuals (9). However, TP53-targeted therapies have not demonstrated efficacy beyond in vitromodels and mutant TP53 immunotherapies are not currentlyavailable (9–11). We previously evaluated the immunogenicity ofthe 12 most common hotspot TP53 mutations according tothe Catalog of Somatic Mutations in Cancer (COSMIC) databaseby measuring T-cell responses of autologous TILs. Approximately1 in 4 of our patients with metastatic epithelial cancers seen inour clinic expressed one of these twelve TP53mutations, and 40% of

patients expressing a TP53 hotspot mutation had TIL recognizingan autologous p53 neoepitope (12–14). Thus, TP53 appears to beimmunogenic when mutated.

Whether similar T-cell responses to p53 neoantigens exist in theperipheral blood T-cell repertoire remains largely unknown. Prelim-inary studies showed evidence of p53 neoantigen responses in periph-eral blood lymphocytes (PBL) using either in vivo peptide vaccinationin a small number of patients (15) or in vitro stimulation (IVS) withpredicted a p53 peptide in a patient with squamous cell carcinoma ofthe head and neck (16).Major advantages of IVS are that it can increaseantigen-specific precursor pools fromPBLs for research or therapy andis agnostic to the human leukocyte antigen (HLA) haplotype of thepatient, obviating the need for identifying candidate epitopes usingHLAprediction algorithms (17–19). This approach has been leveragedfor clinical translation using IVS of bulk PBL with Wilms tumor-1(WT-1) and NY-ESO-1 cancer germline antigens (20, 21). It has beendemonstrated that the na€�ve T cells (CD62LþCD45RO�), which arepresent within the bulk PBL, can have depressed effector function andproliferation, low avidity TCRs, and were unlikely to have previouslyexperienced naturally occurring processed and presented peptidesin vivo relative to antigen-experienced T cells (effector memory:CD62L�CD45ROþ, central memory: CD62LþCD45ROþ, and effec-tor: CD62L�CD45RO�; refs. 22, 23). The antigen-experienced T-cellpopulations were recently shown to contain all or most of the mutatedneoantigen-reactive T cells in PBLs, including private neoantigens andshared KRAS mutations, following IVS with patient-specific tandemminigenes (TMG) and peptide pools (24). Thus, we hypothesized thatantigen-experienced subsets of PBLs would contain TP53 mutation-reactive T cells in the circulation of patients with known intratumoralT-cell responses to TP53.

To test this hypothesis, antigen-experienced T cells from PBLs ofpatients with metastatic epithelial cancers expressing a TP53 hotspot

1Surgery Branch, National Cancer Institute, Bethesda, Maryland. 2Hematopathol-ogy Section, Center for Cancer Research, National Cancer Institute, Bethesda,Maryland.

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

Corresponding Author: Steven A. Rosenberg, National Cancer Institute, 10Center Dr, MSC 1201, CRC Room 3-3940, Bethesda, MD 20892. Phone: 240-858-3080; Fax: 301-402-1738; E-mail: [email protected]

Clin Cancer Res 2020;XX:XX–XX

doi: 10.1158/1078-0432.CCR-19-1874

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mutation and TIL screening results were subjected to a modified IVSprotocol. Antigen-experiencedCD4þ andCD8þTcells were given IVSwith either a single p53 neoantigen long peptide (LP) corresponding totheTP53mutation expressed in the autologous tumor or a TP53-TMGthat was used for TIL screening which contain multiple hotspot TP53mutations. After 12 days of growth, a coculture with autologousantigen-presenting cells either pulsed with mutant p53-LP or electro-porated with mutated TP53-TMG was performed, and the followingday activated T cells were sorted by 41BB and/orOX40 expression. Thesorted T cells were given a rapid expansion protocol (REP) and werescreened for p53 neoantigen responses. Collectively, this strategyidentified T cells reactive against TP53 hotspot mutations, suggestingthat PBLs may be a viable source of generating p53 neoantigen–targeted cancer immunotherapies.

Materials and MethodsSubjects and samples

Written, informed consent was granted from all study participants.This study was approved by the Investigational Review Board inaccordance with an assurance filed with and approved by the U.S.Department ofHealth andHuman Services at theNCI (Bethesda,MD)and was registered at https://clinicaltrials.gov under NCT01174121.The study was conducted in accordance with the U.S. Common Rule.Patients were chosen on basis of availability of pretreatment leuka-pheresis and TIL screening results and had received prior therapies(surgery, chemotherapy, radiotherapy) per standard of care (12, 14).Ficoll–Hypaque was used to isolate PBLs from leukapheresis and cellswere cryopreserved for further use. All patients studied had a con-firmedTP53mutation bywhole-exome sequencing in linewith clinicalprotocols as described previously (12).

Tumor cell linesCEM/C1,HCC1395, SKMEL5, Saos2, andKLE tumor cell lineswere

purchased from ATCC. TYKNU tumor cell line was acquired fromJapanese Collection of Research Bioresources Cell Bank. Mycoplasmatesting and cell authenticity were not independently performed by ourgroup as we relied on the commercial vendor's testing. Saos2-R175Hand TC#4266 cell lines were generated and authenticated as describedpreviously (12). All cell lines were acquired within the past 3 years andused within 2 to 10 passages from receipt from the commercial vendorand within 1 to 5 passages from the time of thaw. Tumor cell lines weregrown for at least a week from cryopreserved stocks before coculture.

Antibodies and FACSFluorescently labeled antibodies used in flow cytometry are detailed

in Supplementary Table S1. Analytic flow cytometrywas performed onFACSCanto II (BD Biosciences) with analysis by FlowJo software(TreeStar). All cells were gated by lymphocytes and live cells byexclusion of cells stained with propidium iodide (PI). Cells were sortedfor IVS and 41BB/OX40 on a FACS Aria II (BD Biosciences). 41BBþ

and/or OX40þ cells were sorted separately through CD3þCD4þCD8�

(CD4) and CD3þCD4�CD8þ (CD8) gates. Cocultures were sorted bySH800S sorted (Sony Biotechnology) for single-cell PCR to identifyTCR genes.

TP53 hotspot mutation screening reagentsThe wild-type (TMG-WT-TP53) and mutated TP53 (TMG-MUT-

TP53) tandem minigene constructs were generated for TIL screeningas described previously (12). In brief, each TP53 hotspot mutation(R175H, Y220C, G245S, G245D, R248L, R248Q, R248W, R249S,R273C, R273H, R273L, and R282W) was composed into a minigenewith mutated codon in the middle and 12 normal codons upstreamand downstream and the minigenes were concatenated into a TMG. Asimilar sequence corresponding to wild-type sequences was alsoderived. TMGs were synthesized as DNA and cloned in frame to aLAMP signal sequence and a DC-LAMP localization sequence thenin vitro transcribed to mRNA using mMESSAGE mMACHINE T7Ultra Kit according to themanufacturer's instructions (Thermo FisherScientific). In addition, wild-type and mutated peptides were synthe-sized for p53R175H, p53Y220C p53R248W, p53R248Q, and p53R273H andpurified to >95% by high-performance liquid chromatography (Gen-script). All peptides were reconstituted in DMSO.

Antigen-presenting cellsMonocyte-derived immature dendritic cells (DC)were generated by

adherence method (25). Briefly, PBLs were plated in AIM-V media(Life Technologies) containingDNase (Genentech Inc.) and incubatedfor 1.5 to 2 hours at 37�C. Nonadherent cells were removed and usedfresh or cryopreserved. Adherent cells were washed in AIM-V, incu-bated for 1 hour at 37�C, and media were exchanged to DC media[RPMI1640, 2mmol/L L-glutamine, with 5% human serum, 100U/mLpenicillin, 100 mg/mL streptomycin, amphotericin B, 800 IU/mLgranulocyte-macrophage colony-stimulating factor (GM-CSF; Leu-kine) and 200 U/mL IL4 (Peprotech)]. Cells were fed every 2 to 3 dayswith cytokines and harvested on days 5 to 6.

IVS, mutant TP53 coculture, 41BB/OX40 enrichment, and REPTo perform the antigen experienced sort, pretreatment PBLs were

processed as described previously (24). Cryopreserved apheresis sam-ples were thawed, washed, set to 5–10 � 106 cells/mL with AIM-Vmedia containing DNase, and 1.75–2� 108 viable cells were plated perT175 flasks (Corning Inc.) and incubated at 37�C, 5% CO2 for 90minutes. After 90 minutes, the nonadherent monocyte-depleted PBLswere harvested, centrifuged, and incubated overnight at 37�C and 5%CO2 in 50/50 media [AIM-V media, RPMI1640 media (Lonza), 5%human AB serum, 100 U/mL penicillin and 100 mg/mL streptomycin(Life Technologies), 2mmol/L L-glutamine (Life Technologies), 10 mg/mL gentamicin (Quality Biological Inc.), 12.5 mmol/L HEPES (LifeTechnologies)]. Adherent monocytes were differentiated into imma-ture DCs as described above. After resting the nonadherent cellsovernight, the cells were harvested, and 1–2 � 108 cells were resus-pended in 50 mL of staining buffer (PBS, 0.5% BSA, 2 mmol/L EDTA)with CD3, CD8, CD4, CD62L, CD45RO antibodies. Cells were incu-bated for 30 minutes at 4�C and washed twice before acquisition. To

Translational Relevance

TP53 is the most commonly mutated gene in cancer but has notbeen an effective target to date. This study demonstrated thatantigen experienced T cells from peripheral blood lymphocytesrepresent a source of T cells with specificity to TP53-mutatedneoantigens. In vitro stimulation was effective in increasing lowfrequency precursors (roughly less than 1 in 105) to frequencies ashigh as 70% p53 neoantigen-reactive and to 106–108 cells. Theperipheral blood and intratumoral T-cell responses were congruentsuggesting that the peripheral blood could be a viable noninvasiveoption for any patient with a tumor expressing a TP53 mutation,including those with inoperable cancers. This strategy could beused for direct cell therapy or to isolate T-cell receptor sequencesand generate genetically engineered T-cell therapy.

Malekzadeh et al.

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determine the sorting population, gating was performed on livecells (propidium iodide negative), single cells, CD3þ T cells thenantigen experienced cells (CD62L�CD45ROþ, CD62LþCD45ROþ,CD62L�CD45RO�), which were further subdivided into CD4þ orCD8þ. TheCD8þ andCD4þ antigen experiencedmemory T cells weresorted separately, collected, counted, and resuspended in 50/50 mediacontaining concentration of 60 ng/mL IL21. Autologous DCs wereelectroporated 18 to 24 hours in advance with TMG-MUT-TP53 orpulsed for 2 to 4 hours the day of the FACS sort with patient-specificmutant p53-LP. Target cells (DCs) were washed in 50/50 media twiceand resuspended in 50/50mediawith no cytokines. IVSwas performedwith a 1:3 to 1:6 ratio (DC:T cell) coculture in a final concentration of30 ng/mL IL21 after the addition of the DCs. Following 14 days ofgrowth and 3 feedings with IL21 and IL2 (aldesleukin) as describedpreviously (24), autologous DCs were again electroporated or pulsedwith TMG-MUT-TP53 or mutated LP, respectively, and coculturedwith IVS cultures for 18 to 24 hours at 37�C and 5% CO2. In parallel,IVS cultures were cocultured with DCs electroporated with irrelevantTMG or pulsed with DMSO for negative controls during the 41BB/OX40 enrichment sort. Following coculture, the cells were harvestedand resuspended in 50 mL of staining buffer containing CD3, CD4,CD8, 41BB, and OX40 antibodies, incubated for 30 minutes at 4�C,and washed twice before acquisition. The sorted 41BBþ/OX40þ

enriched T cells were expanded by REP using irradiated PBL feeders,30 ng/mL OKT3 antibody (Miltenyi Biotec) and 3,000 IU/mL IL2 in50/50 media. The REP cultures were fed 3 times and were tested forreactivity or cryopreserved on day 14.

CocultureScreening of IVS and enriched T cells was accomplished through

same strategy used to screen TIL fragments (12). Briefly, autologousDCs were electroporated with TMG (105 cells/well) and rested over-night or pulsed with peptide or DMSO (8 � 104 cells/well) for 2 to4 hours. Target cells were washed twice and resuspended in 50/50media and cocultured with 2 � 104 T cells in IFNg ELISPOT plates(EMD Millipore). Phorbol 12-myristate 13-acetate (PMA) and iono-mycin (Thermo Fisher Scientific) were used as a positive control andmedia only was a negative control. The cocultured cells were removed,stained, and analyzed by flow cytometry as above, while the ELISPOTplate was processed according to the manufacturer's instructions.Tumor cells were cocultured at 1:1 ratio with T cells (2 � 105 totalcells) overnight in round-bottom 96 well plates. Following harvestingof coculture supernatant to assess IFNg secretion by ELISA (ThermoFisher Scientific), the cocultured cells were and analyzed by flowcytometry.

Minimal peptide assay and HLA restriction mappingSimilar method previously described was used to identify minimal

peptides and determine HLA restrictions (12). In short, NetMHCpeptide binding affinity algorithm (v3.4) was used to predict neoepi-topes for HLA class-I alleles (26). Candidate 9–11 amino acids werecocultured as described above. To investigate the CD4þ minimalneoantigens, 15 amino acid peptides overlapping 14 amino acids werecocultured as described above. To determine the HLA restrictions,COS7 tumor cells were plated at 2.5 � 104 cells/well in RPMI1640, 2mmol/L L-glutamine and 10% FBS in flat-bottom 96-well plates andincubated overnight at 37�C. Patient-specific individual HLA class-Ialleles (300 ng/well) or both HLA class-II a and b chains (150 ng/welleach) in DNA plasmids (pcDNA3.1) were transfected with Lipofecta-mine2000 according to the manufacturer's instructions (ThermoFisher Scientific). When TMGs (100 ng/well) were cotransfected with

HLA, the concentration of HLAs reduced to 150 ng/well for class-I andto 100 ng/well for class-II. The wild-type TMGs used for theseexperiments were the TMG-MUT-TP53 reverted to wild type onlyat the position of interest, for example, R175H. Following 24-hourincubation, transfectionmedia were removed, peptides orDMSOwerepulsed for 2 to 4 hours in 50/50 media when applicable, wells werewashed twice with 50/50 media, and 105 T cells were incubatedovernight. Coculture supernatants were analyzed for IFNg secretionby ELISA and cells were stained for upregulation of 41BB and analyzedby flow cytometry.

TCRB sequencingTCRB survey sequencing was performed from genomic DNA by

Adaptive Biotechnologies. A minimum 5 � 104 cells were sent forsequencing. Analysis of productive TCR rearrangements was per-formed using ImmunoSEQ Analyzer 3.0 (Adaptive Biotechnologies).

TCR identification and reconstructionTCRs were identified by sorting cocultures of T cells and DCs

expressing p53 neoantigens into single wells followed by single cellreverse transcriptase polymerase chain reaction (RT-PCR) of TCRgenes similar to previous studies (27, 28). The PCR products were keptseparate for TCRa and TCRb and were analyzed by Sanger sequenc-ing. These partial TCR sequences were analyzed with IMGT/V-Quest(http://www.imgt.org/IMGT) and IGBLAST (https://www.ncbi.nlm.nih.gov/igblast) websites, which identified the CDR3 and J or D/Jregions and inferred the TRAV and TRBV family. The human full-length variable sequences were fused to murine constant chains as wasdone in other studies (27, 29). The murinized TCRa and TCRb geneswere linked with a RAKR-SGSG sequence and P2A ribosomal slipsequence to result in stoichiometric expression of the TCR chains in asingle cistron. This sequence was synthesized and cloned intoMSGV1vector for generation of transient retroviral supernatants.

TCR transductionPBL donors were adjusted to 3 � 106 cells/ml in 50/50 media

supplemented 50 ng/mL soluble OKT3 and 300 IU/ml IL-2 and wereactivated on low adherence plates for two days prior to retroviraltransduction. The pMSGV1 plasmid encoding mutation-specific TCR(1.5 mg/well) and the envelope-encoding plasmid RD114 (0.75 mg/well) were co-transfected into 106 HEK293GP cells/well of a 6-wellpoly-D-lysine-coated plate using Lipofectamine2000 (Life Technolo-gies). Retroviral supernatants were collected two days after transfec-tion, diluted 1:1 with DMEM media, and centrifuged onto non-tissueculture-treated 6-well plate coated with Retronectin (10 mg/well,Takara) at 2,000� g for 2 hours at 32�C. Supernatants were aspiratedand 2� 106 stimulated T cells at 5� 105 cells/mL were added to eachwell in 50/50 media with 300 IU/mL IL2. The T cells were centrifugedonto the retronectin coated plates for 10minutes at 300� g. Themediawere exchanged 3 to 4 days later with 300 IU/mL IL2 and transducedcells were assayed 10 to 14 days posttransduction.

ResultsIVS of antigen experienced peripheral blood T cells with TP53mutations

Antigen experienced T cells from PBLs were evaluated in 7 colon, 1rectal, and 1 ovarian cancer patients with a TP53-mutated tumor(Table 1). Patients 4217 (p53R175H), 4213 (p53R248Q), 4257(p53R248W), and 4254 (p53R273H) did not have TIL responses to p53neoantigens. In contrast, TILs were reactive to the autologous TP53

TP53-Specific T Cells from PBL

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mutation in patients 4141 (p53R175H), 4285 (p53R175H), 4149(p53Y220C), 4266 (p53R248W), and 4273 (p53R248W; refs. 12, 14). Cryo-preserved aphereses (prior to any ACT) were used to sort antigenexperienced CD4þ or CD8þ T cells from PBLs (Fig. 1A, left). SortedCD4þ or CD8þ T cells were in vitro stimulated with DCs expressingmutatedTP53-TMGmRNA (TP53-TMG-IVS) or pulsedwith patient-specific mutated p53-LP (p53-LP-IVS). After 12 days of culture in thepresence of IL21 and IL2, in vitro stimulated CD4þ andCD8þmemorycells were cocultured with DCs electroporated with mutated TP53-TMG mRNA, in the case of TP53-TMG-IVS, or cocultured withpatient-specificmutated p53-LP, in the case of p53-LP-IVS, and sortedthe following day based on expression of T-cell activation markers41BB and/or OX40 (Fig. 1A, middle). The cell yields afterTP53-TMG-IVS and p53-LP-IVS were comparable for both CD4þ and CD8þ Tcells ranging from 5� 105 to 3.6� 107 cells from 3� 105 to 1.6� 107

starting T cells (Table 2). A portion of CD4þ41BBþ/OX40þ andCD8þ41BBþ/OX40þ T cells were sorted from all populations forcompleteness and symmetry of the experiment, which ranged from2 � 102 to 1.7 � 105 cells and 0.1% to 6.9% from the parent CD4 orCD8 gate (Table 2). The sorted T cells underwent a REP and wereanalyzed after of 14 days rapid expansion. The final cell yields rangedfrom 7 � 106 to 4.2 � 108 T cells, which were likely influenced by theinput cell numbers into the REP (Table 2).

TP53mutation-reactive T cells were present in PBLs of patientswith intratumoral TIL responses to p53 neoantigens

An analytic screen was performed on the cultures after IVS, 41BB/OX40 enrichment and REP where reactivities were evaluated by cellsurface marker 41BB upregulation via flow cytometry and IFNgsecretion using an ELISPOT assay (Fig. 1A, right). Peripheral bloodT cells were not reactive to p53 neoantigens in patients 4213, 4217,4254, and 4257, which corroborated the TIL screening results(Table 1). These patients may not have had an immunogenic com-bination of HLA and p53 neoepitope. In contrast, TP53 mutation–specific T cells were identified in antigen experienced T cells from PBLof 5 patients (4141 and 4285: p53R175H, 4149: p53Y220C, 4266 and 4273:p53R248W) who had intratumoral T-cell responses to mutated TP53 byTILs (Fig. 1B; Table 1; ref. 12). TP53-TMG-IVS resulted in p53neoantigen-specific T cells in 4141-CD8, 4285-CD4, 4149-CD4, and4266-CD8 cultures (Fig. 1B, left), and p53-LP-IVS resulted in p53neoantigen-specific T cells in 4285-CD4 and 4273-CD4 cultures(Fig. 1B, right). The specificity of the responses to mutated TP53(Fig. 1B, closed shapes) was exemplified by the lack of response to thewild-type counterpart (Fig. 1B, open shapes). The highest frequency ofTP53 mutation-reactive cells was 78% in the 4141-CD8 TP53-TMG-

IVS culture against p53R175H (Fig. 1C). The 4285-CD4 TP53-TMG-IVS culture exemplified a positive IFNg secretion screening result as Tcells were reactive to the mutant TMG-MUT-TP53 and p53R175H LPbut not against wild-type TMG-WT-TP53, irrelevant TMG, DMSO(peptide vehicle), or wild-type p53R175 LP (Fig. 1D). Selected cultureswere deemed reactive based on 41BB upregulation and/or IFNgsecretion and were studied further (Supplementary Table S2). The4285-CD4 TP53-TMG-IVS culture showed specific recognition of thecognate p53R175H LP with peptide concentrations down to 10 ng/mL(Fig. 1E). Thus, through IVS and 41BB/OX40 enrichment, highlyspecificCD8þ andCD4þTcells targeting publicTP53mutations couldbe identified.

TCRB tracking demonstrated enrichment of p53 neoantigen-reactive T cells from PBLs

We wanted to further characterize the TCR diversity for eachpatient's TP53 mutation reactive T-cell population and determinewhether the IVS and 41BB/OX40 enrichment altered the T-cellrepertoire. To accomplish this, we performed TCRB deep sequenc-ing (27) and measured productive T-cell clonotype frequencies basedon unique CDR3B sequences and overall sample clonality, which is anormalized measurement of the population diversity where moreoligoclonal samples approach 1 (30, 31). TCRB clonality significantlyincreased inTP53-TMG-IVS and p53-LP-IVS cultures generated fromall patient PBL samples relative to the pre-IVSPBL (Fig. 2A). Similarly,the most frequent unique TCRB clonotype in each TP53-TMG-IVSand p53-LP-IVS sample was of higher frequency than in the pre-IVSPBL (Fig. 2B). The ranking of p53 neoantigen-reactive TCRBsequences ranged from 1 to 167 in the final p53-LP-IVS or TP53-TMG-IVS cultures but were not detected or ranked 5,020 in theoriginal PBL (Supplementary Table S3). The increased clonality ormaximum TCRB clonotype frequencies were not restricted to cultureswith T-cell responses to p53 neoantigens (Fig. 2A and B, asterisksdenoted positive cultures). This suggested that there was T-cellrepertoire skewing through the IVS and 41BB/OX40 enrichment butassessments of top frequency and clonality were not sufficient topredict a culture's response to mutated TP53.

Isolation of p53 neoantigen-reactive TCRs and tracking by TCRBsequencing

We then identified TCRs from the p53 neoantigen-reactive IVSpopulations for potential therapeutic and research use and to track theTCRB clonotypes during the culture period to evaluate the extent ofTP53 mutation-reactive T-cell enrichment. The TCRs were identifiedfollowing a co-culture of reactive IVS cultures with the cognate p53

Table 1. Summary of p53 neoepitope responses by TILs and PBLs.

Patient # Age/Sex Cancer typep53 Amino acidsubstitution

p53 NeoepitopeHLA restriction

TILResponse

PBLResponse

4141 52M Colon R175H A�02:01 CD8 CD84217 51M Colon R175H n/a — —4285 46M Colon R175H DRB1�13:01 CD4 CD44149 36F Ovarian Y220C DRB3�02:02 CD4 CD44213 65M Colon R248Q n/a — —

4257 65M Colon R248W n/a — —

4266 41F Colon R248W A�68:01 CD8 CD84273 49M Rectal R248W DPB1�02:01 CD4 CD44254 55F Colon R273H n/a — —

Note: Patient number, age, gender, cancer diagnoses, p53 amino acid substitutions, HLA restriction for the p53 neoepitope, and the type of T-cell response are given.

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neoantigen (TMG or LP), sorting 41BBþ T cells and single-cell RT-PCRofTCRalpha and beta genes, similar to previous studies (27). TCRpairs were reconstructed, cloned into retroviral vectors and transducedinto donor PBL. A total of 11 TCRs targeting p53R175H (patients 4141and 4285) and p53R248W (patients 4266 and 4273) neoantigen wereidentified (Supplementary Table S3).Wewere unable to determine theTCR for patient 4149 due to limited availability of T cells. The samep53neoantigen-reactive TCRs from TILs (12) were identified in PBLs afterIVS and 41BB/OX40 enrichment in patients 4141, 4266 and 4273targeting p53R175H/HLA-A�02:01, p53R248W/HLA-A�68:01 andp53R248W/HLA-DPB1�02:01, respectively. No additional TP53 muta-tion-reactive T cells were identified in these patients. In contrast, 7unique p53R175H neoantigen-specific TCRs from patient 4285 wereidentified from PBL, which were not present in the TIL study, and theTCR derived from TILs was not found in the PBL populations. Thefunctional avidities of the PBL-derived TCRs (4285-PBL-TCR) were

comparable to the TIL-derivedTCR (4285-TIL-TCR)with recognitionof p53R175H LP to 10 ng/mL and no response to the wild type p53R175

LP (Fig. 2C). Tracking of p53R175H neoantigen-specific TCRB clono-types from patients 4141 and 4285 demonstrated exponential expan-sion after IVS and 41BB/OX40 enrichment compared to the startingPBL (Fig. 2D, left).Moreover, theCDR3B sequences frompatient 4285were below the limit of detection (<0.001% from 2� 105 reads) in bulkPBL but were of sufficient frequencies following IVS and 41BB/OX40enrichment, including four TCRs in the top 10 total CDR3B clonotypes(Supplementary Table S3). The p53R248W neoantigen-specific TCRsfrompatient 4266were also below the limit of detection in the PBLs butwere 2.6% (4266-PBL-TCR3) and 7.5% (4266-PBL-TCR2) in the 4266-CD8 TP53-TMG-IVS culture (Fig. 2D, right). The 4273-PBL-TCRwith specificity to p53R248W neoantigen enriched from 0.002% to0.017% in the 4273-CD4 p53-LP-IVS culture (Fig. 2D, right). Collec-tively, the data demonstrated that PBL can be source of public TP53

Figure 1.

TP53mutation-specific T cells can be isolated and expanded from PBL.A, Schematic for experimental designwhere the parent gate was lymphocytes! single cells! live (PI negative) ! CD3þ (T cells). B, Results from screening the cultures after IVS and 41BB/OX40 enrichment. Cultures deemed positive are bolded. C,Representative flow cytometry plots from the 4141-CD8 TP53-TMG-IVS culture following coculture with autologous antigen-presenting cells electroporated withwild-type (WT) ormutated (MUT) TP53 tandemminigenes (TMG).D,Representative IFNg ELISPOT frompatient 4285-CD4 TP53-TMG-IVS cultures demonstrating allscreening target populations. DMSO was peptide vehicle and PMA/ionomycin was a positive control for nonspecific T-cell activation. E, IFNg secretion as measuredby ELISA into 4285-CD4 TP53-TMG-IVS and peptide-pulsed autologous antigen-presenting cell coculture supernatants. Data are mean � SEM (n ¼ 3 technicalreplicates).






mutation-reactive TCRs identical or comparable to intratumoralTCRs.

Common HLA restriction elements and p53 neoepitopes wereimmunogenic

The recognized minimal p53 neoepitopes and correspondingHLA restrictions were then assessed. HLA mapping was accom-plished by transfecting DNA plasmids corresponding to each ofthe patient's individual HLA alleles into COS7 monkey cell line(lacking HLA) and pulsing peptides or cotransfecting with TMG,similar to previous reports (12, 14). The 4141-CD8 TP53-TMG-IVS culture was specific for the p53R175H HMTEVVRHC neoe-pitope in the context of HLA-A�02:01, a highly frequent HLA inthe U.S. population (32), as measured by 41BB expression(Fig. 3A). HLA-A�68:01 restricted the p53R248W neoepitopeSSCMGGMNWR recognized by the 4266-CD8 TP53-TMG-IVSculture as measured by IFNg secretion (Fig. 3B). This wasexpected as the TCRs from TIL in patients 4141 and 4266 werefound in these IVS cultures and had already established minimalneoepitopes and HLA restriction elements (12). Similarly, the4273-PBL-TCR was found in the 4273-CD4 p53-LP-IVS cultureand had a known p53R248W and HLA-DPB1�02:01 combinationfrom the TIL studies. Even though the TCRs were differentbetween PBL and TIL for patient 4285, the 4285-CD4 TP53-TMG-IVS culture was specific for the same p53R175H and HLA-DBR1�13:01 combination found in the TIL (Fig. 3C). Further-more, all 4285-PBL-TCRs were specific for p53R175H and HLA-DBR1�13:01 (data not shown) and were all present in the 4285-CD4 TP53-TMG-IVS culture by TCRB sequencing (Supplemen-tary Table S3). Fifteen amino acid p53R175H peptides overlapping14 amino acids were pulsed on DCs from patient 4285 andcocultured with TCR transduced T cells, and the core peptide

EVVRHCPHHER was determined to be the common sequencerecognized by 4285-PBL-TCRs in the context of HLA-DRB1�13:01(Fig. 3D). In sum, we found the same TCRs from TILs in PBLsthat recognized the same HLA and minimal p53 neoepitopes in 3cases and in one case found additional TCRs with the same p53neoantigen specificity as the intratumoral T cells.

Tumor cells process and present p53 neoepitopes on HLA,which are recognized by PBL-derived T cells following IVSthrough their TCR

TP53 mutation–reactive T cells were evaluated for the capacity torecognize naturally processed and presented antigen expressed on thetumor cell surface. Saos2-R175H osteosarcoma tumor cell line (HLA-A�02:01 and overexpressing full length p53R175H) and TC#4266human xenograft tumor cell line (p53R248W and HLA-A�68:01:02colon cancer) were co-cultured overnight with CD8þ T cells fromthe 4141-CD8 TP53-TMG-IVS and 4266-CD8 TP53-TMG-IVS cul-tures, which upregulated 41BB in response to Saos2-R175H andTC#4266 cell lines, respectively, with minimal activation against thecross-matched cell line (Fig. 3E). To test whether p53 neoantigen-reactive TCRs identified from peripheral blood would be of value asgene-modified cell therapy, T cells transduced with the p53R175H/HLA-A�02:01-specific 4141-PBL-TCR were cocultured with alloge-neic cell lines expressing p53R175H, HLA-A�02:01 or both. TYKNU,KLE and Saos2-R175H (p53R175H, HLA-A�02:01) were recognized by4141-PBL-TCR asmeasured by specific upregulation of 41BB (Fig. 3F)and secretion of IFNg into coculture supernatants (Fig. 3G). Incontrast, 4141-PBL-TCR did not recognize CEM/C1 (p53R175H,HLA-A�02:01negative), HCC1395 (p53R175H, HLA-A�02:01negative),SKMEL5 (p53WT, HLA-A�02:01) and Saos2 cell lines (13), suggestingthat the TCR was specific to cell lines expressing p53R175H and HLA-A�02:01 and that sufficient p53 neoepitope was on the tumor cell

Table 2. T-cell yields after IVS, 41BB/OX40 enrichment, and rapid expansion in patients with a p53 neoantigen-reactive culture.

Patient #p53 a.a.sub

T-celltype IVS

# Cellsto start IVS(�106)

# Cellsafter IVS(�106)

% 41BB/OX40Sorted

# Cellsto start REP(�106)

# Cellsafter REP(�106)

4141 R175H CD4 p53-LP 13.2 12.6 0.9 0.0146 15.9TP53-TMG 15.6 18.3 2.3 0.0179 42.2

CD8 p53-LP 1 9.1 0.1 0.0002 12.9TP53-TMG 1.5 7.9 4.9 0.1185 44.2

4285 R175H CD4 p53-LP 5 31 2.0 0.0360 360TP53-TMG 5 36 2.2 0.0428 420

CD8 p53-LP 1.5 10 2.4 0.0192 34TP53-TMG 1.5 19 0.2 0.0788 10

4149 Y220C CD4 p53-LP 1 14 0.7 0.0127 19.3TP53-TMG 1 5.7 0.1 0.0002 7.1

CD8 p53-LP 1 6.6 0.2 0.0040 45.4TP53-TMG 1 7.2 0.1 0.0011 48.4

4266 R248W CD4 p53-LP 5 0.9 0.1 0.0041 305TP53-TMG 7.8 11.5 0.1 0.0351 294

CD8 p53-LP 0.3 0.5 0.2 0.0010 136.8TP53-TMG 0.4 13.3 0.1 0.0123 131.2

4273 R248W CD4 p53-LP 8.4 1.6 1.1 0.0642 15.1TP53-TMG 8.4 3.3 2.2 0.1719 68.6

CD8 p53-LP 0.4 3.9 6.9 0.0084 66.6TP53-TMG 0.8 5.6 2.9 0.0419 44.8

Note: The numbers of cells to start the IVS depended upon the frequencies of CD4 and CD8 antigen-experienced T cells in the PBL. The percentage of 41BB/OX40 Tcells sorted was based on control gating to eliminate asmany nonspecific T cells as possible and is likely an underestimate of the percentage of reactive T cells in thepopulation. All enriched cells were placed in a REP.

Malekzadeh et al.





surface to trigger TCR-engineered T-cell recognition. Mock-transduced T cells were not reactive to any of the cell lines, furthersupporting the specificity of the 4141-PBL-TCR. Thus, TP53 muta-tion-specific T cells and TCRs from PBL could recognize autologousand allogeneic tumor cells with naturally processed and presented p53neoepitopes.

DiscussionThe intratumoral (TIL) and antigen experienced T responses

from PBLs were congruent in 9 patients, including identical TCRs in3 cases. This suggested that TP53mutation-reactive T cells circulate

in peripheral blood, which can provide insight into the intratumoralT-cell response and can be a source of p53 neoantigen-reactive Tcells and TCRs for ACT. The initial amount of blood necessary tofind the reactive cells will likely depend upon the relative precursorfrequencies, which in this study were less than one in 105 PBLs inmost cases. Thus, leukapheresis or a large volume venipuncture maybe needed to start with 106 antigen experienced CD4þ or CD8þ Tcells, but a thorough study may be needed to determine the limits ofdetection for this technique. The ability to translate the findingsfrom this study to clinical application may depend on the frequencyand quality of intratumoral T-cell responses to TP53mutations thatmake their way to the peripheral circulation.

Figure 2.

TCRs with specificity to mutated TP53 have exponential expansion from PBL following IVS and 41BB enrichment. For (A, B, and D) TCRB sequencing was performedon PBL prior to or after expansion with IVS and 41BB/OX40 enrichment with either LP or TMG. In A and B, the cultures with verified p53 neoantigen responses arehighlightedwith an asterisk.A,Clonality of the total populations, which is a normalized estimate of sample diversitywhere numbers closer to 1 are less diverse.B, Themaximum productive unique CDR3B frequency from each population. C, IFNg secretion as measured by ELISA into supernatants from TCR-transduced T cells andcocultured with autologous antigen-presenting cells pulsed with wild-type (left) or mutated (right) p53R175H peptides. Mock-transduced T cells were a negativecontrol for TCRexpression. Data aremean�SEM (n¼ 3 technical replicates).D,Trackingof CDR3Bwith known specificity tomutated TP53before (PBL) andafter theIVS and 41BB enrichment protocol.






Figure 3.

TP53mutation–specific TCRs fromPBL recognize p53 neoepitopes in the context of commonHLA and endogenously processed and presented neoantigen on tumorcells. ForA–C, COS7monkey cell line was transfected with HLA and either pulsed with minimal p53 peptides or cotransfected with TMGs. Results from the 4141-CD8TP53-TMG-IVS (A), 4266-CD8 TP53-TMG-IVS (B), and 4285-CD4 TP53-TMG-IVS (C) cultures. The TMG-wtR175 had mutated TP53 at all positions except for R175H.D, IFNg secretion as measured by ELISpot following coculture of TCR or mock (-) transduced T cells with autologous antigen-presenting cells pulsed with 15 aminoacid p53R175H peptides overlapping 14 amino acids. DMSO was peptide vehicle. E, Upregulation of 41BB on CD8þ T cells from TP53-TMG-IVS cultures (4266-CD8on left and 4141-CD8 on right) following coculture with TC#4266 (autologous xenograft from patient 4266; A�68:01; p53R248W) and Saos2 cells (A�02:01)overexpressing full-length p53R175H gene. F and G, Cocultures of mock or 4141-PBL-TCR–transduced T cells with tumor cell lines that expressed either HLA-A�02:01,p53-R175H or both. F, Expression of 41BB on cocultured T cells by flow cytometry. G, Secretion of IFNg into coculture supernatants by ELISA. Data for A–C, F, andG were mean � SEM (n ¼ 3 technical replicates) and representative or two independent experiments or donors.

Malekzadeh et al.





Both p53-LP-IVS and TP53-TMG-IVS were able to expand p53neoantigen-reactive T cells. The TMG approach may be moreadvantageous as it covers 12 mutations at once, shows the T cellsan intracellularly processed and presented neoepitope and resultedin more positive cultures than the p53-LP-IVS (Fig. 1B). This TP53-focused strategy could be expanded to other TP53 mutations, someof which are shared in unrelated people, to interrogate T cells fromboth TIL and PBL in their capacity to recognize p53 neoantigens ina more comprehensive manner. Indeed, patients with gastrointes-tinal tumors have demonstrated T-cell responses by TILs to non-hotspot TP53mutations (33), suggesting that mutated TP53 is likelyimmunogenic outside of the most frequent genetic changes. In ourprevious studies, almost all neoantigens with a verified T-cellresponse were unique to the individual patient with the exceptionof KRAS and TP53, which have immunogenic mutations recognizedby unrelated people with the appropriate HLA restrictionelement (3, 5–7, 12, 14, 25, 29, 33–37). This indicates that KRASand TP53 could be high value targets for ACT.

Targeting TP53 mutations with T cells may be an efficaciousstrategy because of the importance of the mutated TP53 to the tumor.The loss of HLA is a limiting aspect to any ACT strategy, the extentof which is largely unknown and likely dependent upon the targetor HLA/p53 neoepitope combination. In contrast toHLA loss, antigenloss is less likely in the case of mutated TP53. Tumors expressingTP53 missense mutations typically have loss-of-heterozygosity of thewild type allele thereby limiting the chances of antigen loss afterACT (12, 38). Furthermore, some TP53 mutations can have gain-of-function activity, further emphasizing their importance to tumorsurvival and fitness (39).TP53 is expressed at high levels in cells but thep53 protein is degraded by MDM2 in normal cells (40). Mutations inTP53 can interrupt the p53 degradation process and lead to accumu-lation in the cytosol (41) indicating that there will likely be high levelsof neoepitope available for T-cell recognition.

This study lays the foundation for the generation of cell therapydirectly using PBL after IVS and 41BB/OX40 enrichment or bygenetic modification with TCRs. Furthermore, TCRs identifiedfrom IVS and 41BB/OX40 enrichment could be used for anyunrelated donors with matching TP53 mutation and HLA expres-sion in an off-the-shelf setting. Animal models of mutated TP53xenografts can potentially be established to evaluate the pre-clinicalefficacy of p53 neoantigen-reactive TCR gene–engineered T cellsin vivo though these experiments of human cells in the mouse areoften difficult to interpret. An all-in-one noninvasive strategy could

be achieved by combining IVS and 41BB/OX40 enrichment withcirculating tumor DNA detection of TP53 mutations in patientplasma or serum, which has been shown to strongly correlate withgenetic features of the tumor (42–45) and is a promising diagnosticand prognostic tool (44, 46–48). Identification of TP53 mutationsfrom circulating tumor DNA using a liquid biopsy could make PBLthe sole source for neoantigen and T cells. This strategy can likelybenefit patients ineligible for surgery and deepen the scope oftargeting the most commonly mutated gene in cancer.

Disclosure of Potential Conflicts of InterestN.P. Restifo is an employee/paid consultant for Lyell Biopharma and holds

ownership interest (including patents) in Lyell Immunopharma. No potential con-flicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: P. Malekzadeh, R. Yossef, M. Jafferji, P.F. Robbins,N.P. Restifo, S.A. Rosenberg, D.C. DenigerDevelopment of methodology: P. Malekzadeh, R. Yossef, G. Cafri, P.F. Robbins,S. Ray, N.P. Restifo, D.C. DenigerAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.):P.Malekzadeh, G.Cafri, B.C. Paria, F.J. Lowery,M. Jafferji,M.L.Good,A. Sachs, A.R. Copeland, S.P. Kim, S. Kivitz, M.R. Parkhurst, S. Ray, L. Xi, Z. Yu,N.P. Restifo, R.P.T. Somerville, S.A. Rosenberg, D.C. DenigerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Malekzadeh, F.J. Lowery, M. Jafferji, A.R. Copeland,S.P. Kim, S. Kivitz, P.F. Robbins, S.A. Rosenberg, D.C. DenigerWriting, review, and/or revision of the manuscript: P. Malekzadeh, R. Yossef,F.J. Lowery, M. Jafferji, M.L. Good, A.R. Copeland, S.P. Kim, S. Kivitz, P.F. Robbins,M. Raffeld, N.P. Restifo, S.A. Rosenberg, D.C. DenigerAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): P. Malekzadeh, A. Sachs, L. Xi, R.P.T. Somerville,S.A. Rosenberg, D.C. DenigerStudy supervision: N.P. Restifo, S.A. Rosenberg, D.C. Deniger

AcknowledgmentsWe thank the TIL lab (NCI Surgery Branch) for their efforts processing the

apheresis, and Arnold Mixon and Shawn Farid for their assistance with FACS. Thisworkwas supported by an award to S. Rosenberg by the Intramural Research Programof the NIH at the Center for Cancer Research, NCI.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 11, 2019; revised September 30, 2019; accepted November 12, 2019;published first January 29, 2020.

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Published OnlineFirst January 29, 2020.Clin Cancer Res Parisa Malekzadeh, Rami Yossef, Gal Cafri, et al. Recognize p53 NeoantigensAntigen Experienced T Cells from Peripheral Blood

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Antigen Experienced T Cells from Peripheral Blood ... · 22/01/2020 · T cells could recognize processed and presented p53 neoantigens. Conclusions: PBL was a noninvasive source