Purpose: This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers.

Patients and Methods: Tumor-infiltrating lymphocytes (TIL) were expanded from resected ovarian cancer metastases, which were analyzed by whole-exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen-presenting cells and then cocultured with TIL fragment cultures. Secretion of IFNγ or upregulation of 41BB indicated a T-cell response.

Results: Seven women with metastatic ovarian cancer were evaluated, and 5 patients had clear, dominant T-cell responses to mutated neoantigens, which were corroborated by comparison with the wild-type sequence, identification of the minimal epitope, human leukocyte antigen (HLA) restriction element(s), and neoantigen-specific T-cell receptor(s). Mutated neoantigens were restricted by HLA-B, -C, -DP, -DQ, and/or -DR alleles and appeared to principally arise from random, somatic mutations unique to each patient. We established that TP53 “hotspot” mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S) expressed by two different patients' tumors were both immunogenic in the context of HLA-DRB3*02:02.

Conclusions: Mutation-reactive T cells infiltrated ovarian cancer metastases at sufficient frequencies to warrant their investigation as adoptive cell therapy. In addition, transfer of TP53 “hotspot” mutation-reactive T-cell receptors into peripheral blood T cells could be evaluated as a gene therapy for a diverse range of tumor histologies. Clin Cancer Res; 24(22); 5562–73. ©2018 AACR.

See related commentary by McNeish, p. 5493

Translational Relevance

This study demonstrated that T cells with specificity to mutated neoantigens were present in tumor-infiltrating lymphocytes in patients with metastatic ovarian cancer, sometimes at high frequencies and with multivalent reactivity, suggesting that these T cells could be used for adoptive cell therapy. Furthermore, T cells from two of the patients were specific for TP53 “hotspot” mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S), which are also expressed in a broad range of tumor types in unrelated individuals. Genetic transfer of the TP53 “hotspot” mutation-specific T-cell receptors into autologous lymphocytes could be used to generate cells for use in the adoptive cell transfer immunotherapy of cancer.

Ovarian cancer is expected to account for 22,440 new cases and 14,080 deaths in the United States in 2017 (1). No curative treatments exist for patients with metastatic ovarian cancer. An increased presence of tumor-infiltrating lymphocytes (TIL), composed primarily of T cells, in ovarian tumors was associated with better prognosis in patients receiving standard-of-care treatment suggesting T cells were actively interacting with ovarian cancer cells (2–5). Preliminary evidence suggested that adoptive cell transfer (ACT) of bulk TILs to patients with metastatic ovarian cancer was safe and well tolerated (6, 7). Despite these early advances, TIL therapy for the treatment of ovarian cancer has otherwise gone untested and the specificity of ovarian cancer TILs is largely unknown.

ACT using TILs expanded from metastatic tumor lesions has led to durable, complete tumor regressions in 24% of patients with metastatic melanoma in several studies at different institutions (8–12). The infused TILs of some of the patients with melanoma with complete responses recognized unique, somatic, nonsynonymous mutated neoantigens, suggesting mutation-reactive T cells were critical in mediating durable tumor regressions (12–14). More recently, long-term partial regressions of metastatic colon cancer and cholangiocarcinoma were achieved following transfer of TILs recognizing autologous mutated KRASG12D and ERBB2IPE504G neoantigens, respectively (15–17). Thus, the “final common pathway” for the immunotherapy of cancer appears to be based on the targeting of somatic mutations expressed by the tumor and recognized by T cells.

Ovarian cancer falls in the middle of the continuum of numbers of expected somatic mutations stratified by cancer histology, which ranges from dozens in brain cancers to many hundreds of mutations in melanomas and lung cancers (18–20). Mutations in the TP53 tumor suppressor gene were estimated to occur in >95% of high-grade serous ovarian cancer, but were less frequently observed (≤10%) in other ovarian cancer subtypes (refs. 21, 22; Catalogue of Somatic Mutations in Cancer (COSMIC) database; http://cancer.sanger.ac.uk/cosmic). Mutations occur across the TP53 gene and a single TP53 mutation is not ubiquitous to all ovarian cancer subtypes, but there are 6 codons (R175, Y220, G245, R248 and R273), which are most frequently mutated are thus called TP53 “hotspot” mutations (23–25). These TP53 “hotspot” mutations may be oncogenic because they have wild-type loss-of-function and can have gain-of-function activity, although these phenomena have not been directly observed in ovarian cancer (26–29). However, there are no reports to date of intratumoral T-cell responses to these TP53 “hotspot” mutations.

Intratumoral T-cell responses to human ovarian cancer–mutated neoantigens have not been comprehensively evaluated. In a pilot study, one mutation T-cell response was detected among tumor-associated lymphocytes from ascites of 3 women with ovarian cancer (30), and a murine model of ovarian cancer neoantigen vaccination generated some murine mutation T-cell responses, but was deemed an ineffective strategy due to the low mutational load of ovarian cancer (31). Recently, low frequencies (<2%) of mutated neoantigen-reactive CD8+ T cells were detected from TILs in 4 of 14 patients with ovarian cancer after screening with predicted Class-I human leukocyte antigen (HLA) neoepitopes (32), and mutation-reactive T cells were generated from peripheral blood lymphocytes (PBL) of patients with ovarian cancer vaccinated with tumor cell lysate (33). The aim of this study was to evaluate whether mutation-reactive TILs were present in resected ovarian cancer metastases independent of neoepitope prediction. All mutations identified in the cancer were introduced into both intracellular and extracellular pathways of autologous antigen-presenting cells (APC), containing all of the patient's HLA Class-I and Class-II alleles, followed by coculture with TIL microcultures. Mutation-reactive T cells were characterized in terms of the specificity to the mutated versus wild-type variants, the minimal determinants of mutated sequences, the HLA restriction elements of the neoantigens, and the identification of T-cell receptors (TCR) recognizing neoantigen:HLA complexes. Thus, a comprehensive characterization of T-cell interactions with ovarian cancer–specific mutated neoantigens was performed.

Neoantigen screening with TILs

All patients were enrolled after written, informed consent was granted on NIH protocol 10-C-0166 (NCT01174121) in accordance with ethical guidelines and with approval by the NIH institutional review board. Referring physicians provided initial diagnoses and pathology, and the eligibility requirements for enrollment on the clinical trial included progressive disease at the time of surgery at the National Cancer Institute (NCI) with at least two metastases (>1 cm), one of which was amenable for harvest for TIL growth. The diagnosis date and classification (Federation of Gynecology and Obstetrics 2013), time to initial progression, histologic subtype (World Health Organization 2014 Classification), therapies prior to enrollment, date of resection, metastatic sites, resection site at the NCI, and key mutations (ARID1A, CCNE1, NF1, PIK3CA, PTEN, KRAS, TP53) detected in the resected metastases can be found in Supplementary Table S1. Growth of TILs, exome and transcriptome sequencing, and bioinformatic methods can be found in the Supplementary Material. After mutation calling was complete, quality control was manually performed for each called mutation with the Interactive Genomics Viewer (IGV) software (Broad Institute, Cambridge, MA; http://software.broadinstitute.org/software/igv/). Called mutations were excluded from the analysis if similar reads were observed in the normal exome or if there was an obvious mapping issue, that is, multiple random mutations along multiple reads. A list of screened neoepitopes with patient ID, gene name, amino acid change, TMG, and peptide pool is given in Supplementary Table S2. Each mutated neoepitope was synthesized as a peptide or gene and cloned into a pcRNA2-SL expression vector (GenScript). The TMG plasmids were linearized with NotI and in vitro transcribed mRNA was made with mMESSAGE mMACHINE T7 ULTRA Transcription Kit according to manufacturer's instructions (Thermo Fisher). Peptides were reconstituted in dimethyl sulfoxide (DMSO) then peptide pools were generated congruent to the TMG neoepitopes. Autologous immature dendritic cells (DCs) were generated by adherence method with IL4 and GM-CSF as described previously (15). After 4–6 days, immature DCs were harvested and either transfected with TMG mRNA (80 μg/mL) using ECM 830 square electroporator (BTX) or pulsed with peptides either individually or as pools (10 μg/mL unless otherwise stated). Mutation-loaded immature DCs (0.8–1 × 105) were cocultured with individual TIL microcultures (2 × 104) overnight in an IFNγ ELISPOT plate (Mabtech). The following day, the ELISPOT plate was developed according to manufacturer's instructions after cocultured cells were transferred to a new 96-well plate and stained for OX40, 41BB, CD3, CD4, CD8 (BD Biosciences), and/or mouse TCRβ (mTCRβ; Thermo Fisher Scientific). Stained cells were analyzed on BD FACSCanto I or II or sorted by FACS on BD FACSAria II (BD Biosciences). FlowJo software (v.10.3) was used to analyze flow cytometry data. Spots on ELISPOT plates were enumerated with the Immunospot machine (Cellular Technology Ltd.).

TCR identification and expression in PBL

Highly oligoclonal neoantigen-reactive TIL cultures were sequenced with TCRAD and TCRB surveys by Adaptive Technologies to pair TCRα with TCRβ by frequency. The TCRs from bulk TIL cultures were also paired using statistical modeling on the PairSeq platform (Adaptive Biotechnologies; ref. 34). Single-cell reverse transcriptase PCR specific for TCRα and TCRβ was performed to pair TCRs after coculture with neoantigen (peptide or TMG) and sorting of 41BB+ T cells into single wells of 96-well plates, as was done in other studies (35, 36). All TCRs were chimeras of human variable regions and mouse constant regions to enable detection with mTCRβ antibody and limit mispairing with the endogenous TCR. TCRs for patients 1, 4, and 6 were transduced into PBL using standard protocols with transiently produced γ-retroviral supernatants (35). TCRs for patients 5 and 7 were transposed into PBL with the Sleeping Beauty transposon/transposase system similarly to previous studies (37).

HLA restriction mapping

The HLA restriction elements were determined by transfecting COS7 tumor cell line (Green monkey; Cercopithecus aethiops) with individual Class-I alleles or both Class-II α and β chains, pulsing the mutated peptides and coculture with T cells. Secretion of IFNγ into supernatants was evaluated by ELISA. Expression of 41BB was assayed by flow cytometry following cocultures.

Patient demographics, mutation identification, and neoepitope screening

A total of 7 women with metastatic ovarian cancer were enrolled on the clinical trial evaluating autologous TILs for the possible treatment of metastatic disease (NCT01174121). Each patient received standard of care (surgery and chemotherapy) and had progressive disease prior to enrollment to the trial at the NCI Surgery Branch (Supplementary Table S1). At least one tumor was resected from a metastatic deposit from each patient for generation of TILs and mutation sequencing. Following whole-exome and transcriptome sequencing, a total of 1,714 putative mutated neoantigens were screened and a median of 228 mutations were identified per patient (range: 64–333; Table 1). Each mutation was assembled into a minigene encoding a 25 amino acid sequence by flanking the mutation with 12 amino acids of the wild-type sequence as was done in previous studies (12, 15). Minigenes were concatenated in tandem (median 18, range 13–21) to generate tandem minigene (TMG) constructs, which were synthesized as DNA and in vitro transcribed to mRNA. Twenty-five amino acid long peptides (LP) were also synthesized corresponding to each mutated minigene and peptide pools (PP) were assembled to increase throughput of screening. Peptides were pulsed and TMG mRNA was electroporated into autologous APCs. Thus, each mutation was screened using intracellular (TMG) and extracellular (LP) antigen presentation pathways on any possible HLA Class-I and -II molecules of the APC.

Table 1.

Somatic mutated neoantigens recognized by ovarian cancer TIL

Peptide:HLA predicted
PtFrTu## mutMutated gene (location)Protein (AA sub.)Neoepitope (mutation)HLARankAffinity [nmol/L]
4046 280 USP9X (X:41075846 c.6026A>G) USP9X (Y2009C) RMQYSMECB*15:01:01 0.2 31.1 
     YSMECFQFM C*03:03:01 0.04 7.6 
     RMQYSMECFQFMKKL DPA1*01:03:01 DPB1*04:02:01 0.9 16.4 
4067 122 – –  – – – 
4068 284 – – – – – – 
4097 317 HIST1H1B (6:27835096 c.212C>A) Histone H1.5 (A71D) LADGGYDVEKNNSRI DQA1*03:01:01 DQB1*04:02:01 65 8,884.3 
     SLAALKKALADGGYD DRB4*01:01:01 19 387.5 
   INPP5K (17:1401379 c.526C>G) INPP5K (L272V) RIVWRLKRQPCAGPD Class–II – – 
4098 333 RPTOR (17:78858926 c.1961A>G) RAPTOR (D654G) HNVAMMLAQLVSGGS DPA1*01:03:01 DPB1*10:01:01 20 115.6 
 4173 138    DPA1*02:01:01 DPB1*10:01:01 17 113.1 
4127 176 TP53 (17:7577548 c.733G>A) p53 (G245S) HYNYMCNSSCMGSMN DRB3*02:02:01 21 533.4 
4149 64 CTAGE5 (14:39796122 c.1613A>T) CTAGE5 (E576V) NERGVSSCDRLTDPH DQA1*01:03:01 DQB1*06:03:01 95 4,640.6 
   HUWE1 (X:53560337 c.13058T>C) HUWE1 (F4353S) SEKLRHMLL B*40:02:01 0.8 199.5 
   TP53 (17:7578190 c.659A>G) p53 (Y220C) NTFRHSVVVPCEPPE DRB3*02:02:01 17 433.8 
Peptide:HLA predicted
PtFrTu## mutMutated gene (location)Protein (AA sub.)Neoepitope (mutation)HLARankAffinity [nmol/L]
4046 280 USP9X (X:41075846 c.6026A>G) USP9X (Y2009C) RMQYSMECB*15:01:01 0.2 31.1 
     YSMECFQFM C*03:03:01 0.04 7.6 
     RMQYSMECFQFMKKL DPA1*01:03:01 DPB1*04:02:01 0.9 16.4 
4067 122 – –  – – – 
4068 284 – – – – – – 
4097 317 HIST1H1B (6:27835096 c.212C>A) Histone H1.5 (A71D) LADGGYDVEKNNSRI DQA1*03:01:01 DQB1*04:02:01 65 8,884.3 
     SLAALKKALADGGYD DRB4*01:01:01 19 387.5 
   INPP5K (17:1401379 c.526C>G) INPP5K (L272V) RIVWRLKRQPCAGPD Class–II – – 
4098 333 RPTOR (17:78858926 c.1961A>G) RAPTOR (D654G) HNVAMMLAQLVSGGS DPA1*01:03:01 DPB1*10:01:01 20 115.6 
 4173 138    DPA1*02:01:01 DPB1*10:01:01 17 113.1 
4127 176 TP53 (17:7577548 c.733G>A) p53 (G245S) HYNYMCNSSCMGSMN DRB3*02:02:01 21 533.4 
4149 64 CTAGE5 (14:39796122 c.1613A>T) CTAGE5 (E576V) NERGVSSCDRLTDPH DQA1*01:03:01 DQB1*06:03:01 95 4,640.6 
   HUWE1 (X:53560337 c.13058T>C) HUWE1 (F4353S) SEKLRHMLL B*40:02:01 0.8 199.5 
   TP53 (17:7578190 c.659A>G) p53 (Y220C) NTFRHSVVVPCEPPE DRB3*02:02:01 17 433.8 

NOTE: Patient (Pt) TILs from fresh tumor (FrTu) resections were screened against putative mutated neoantigens (mut) in 25 amino acid peptides or tandem minigenes (TMGs). The gene name, genetic mutation, protein name, and amino acid substitution (AA sub.) are shown for each mutated neoantigen recognized by TILs. A minimal epitope is shown with the mutated amino acid in bold underline followed by the matching HLA restriction element. The predicted peptide:HLA complex rank and affinity (nmol/L) were determined by NetMHCpan 3.0 for Class–I (http://www.cbs.dtu.dk/services/NetMHCpan/) and NetMHCIIpan 3.1 for Class–II (http://www.cbs.dtu.dk/services/NetMHCIIpan/).

Coculture of APCs with TIL was performed with the TMG or PP to identify the recognized mutations by assaying IFNγ secretion by ELISPOT and upregulation of 41BB expression by flow cytometry. Twenty-four tumor fragments from different parts of a resected tumor were cultured in high-dose IL2 and the resulting expanded TILs were screened against autologous APCs expressing all autologous mutations. Each TIL fragment was kept as an individual mini-culture to evaluate multiple, diverse, oligoclonal populations. A total of 8 neoantigens were identified from 5 of the 7 patients (Table 1). Neoantigens were not identified for patients 2 and 3. The minimal determinants of the recognized mutations (Table 1, column 6) demonstrated that the amino acid substitution could occur within the neoepitope near the N-terminus (Histone H1.5A71D, INPP5KL272V, CTAGE5E576V and HUWE1F4353S), the middle (USP9XY2009C), or the C-terminus (Histone H1.5A71D, RAPTORD654G, p53G245S, p53Y220C and USP9XY2009C). Both USP9XY2009C and Histone H1.5A71D developed more than one neoantigen minimal neoepitope. All of the mutations recognized by CD8+ T cells were predicted by an HLA-binding algorithm (NetMHCpan 3.0; http://www.cbs.dtu.dk/services/NetMHCpan/) to have a rank <1 and affinity <200 nm. Five of seven HLA Class-II epitopes recognized by CD4+ T cells were predicted to have an affinity <500 nmol/L, and all 7 had a rank >17, except for USP9XY2009C, which had a rank of 0.9 (Table 1, far right column). The overall mutation burden did not predict for T-cell responsiveness as patient 3 had the third most mutations (n = 284) but no corresponding mutation reactivity, whereas patient 7 had three independent immunogenic neoantigens and the fewest mutated neoepitopes (n = 64).

Multivalent T-cell responses to USP9XY2009C neoantigen

The TIL from patient 1 demonstrated high frequency and multivalent T-cell response to USP9XY2009C, a putative driver mutation (38). She presented to the NCI Surgery Branch at 38 years old and a metastasis was resected from an axillary lymph node for TIL growth and sequencing. The 280 identified mutations were concatenated into fifteen TMGs (4046-TMG) and the top 79 mutations were assembled into 6 peptide pools (4046-PP) after being prioritized for having high variant allele frequency in the exome and RNAseq. Fragments 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 20, and 21 displayed IFNγ secretion in response to 4046-TMG15, and fragments 7, 11, 13, 14, 20, and 21 secreted IFNγ when cocultured with 4046-PP6 (Fig. 1A). Coculture of selected TIL fragments with individual LP congruent between 4046-TMG15 and 4046-PP6 showed USP9XY2009C as the immunogenic neoantigen (Fig. 1B). To increase the number of T cells for further experiments, fragments 7, 11, 13, 14, 20, and 21 were grown using a rapid expansion protocol (REP) consisting of irradiated PBL feeder cells, OKT3 (agonistic pan-CD3 antibody) and IL2. The six expanded fragments were pooled to generate the 4046-REP-TIL culture, which was specific for 4046-TMG15 and USP9XY2009C LP as evidenced by 41BB upregulation in cocultures compared with minimal upregulation caused by irrelevant TMG (TMG-IRR) or 4046-TMG15 reverted back to wild-type only at USP9X (4046-TMG15wtUSP9X), peptide vehicle (DMSO), and wild-type USP9XY2009 LP (Fig. 1C). Both CD4+ and CD8+ T cells in the 4046-REP-TIL expressed 41BB in response to USP9XY2009C neoantigen and suggested that a multivalent attack on USP9XY2009C occurred in patient 1.

Figure 1.

Multivalent T-cell responses to USP9XY2009C neoantigen. A, TIL fragments from FrTu#4046 were cocultured with autologous APCs (i) electroporated with irrelevant TMG (TMG-IRR) or 4046-TMG15 or (ii) pulsed with DMSO (peptide vehicle) or 4046-PP6. Media (T cells only) and OKT3 were negative and positive control, respectively. Secretion of IFNγ was evaluated by ELISPOT. B, Selected TIL fragments were cocultured with autologous APCs pulsed with peptides, and IFNγ secretion was evaluated by ELISPOT. C, Expression of 41BB in T cells following coculture of 4046-REP-TIL with autologous APCs electroporated with TMGs or pulsed with peptides. D, Coculture of TIL or TCR-transduced T cells with autologous APCs pulsed with minimal neoepitope peptides and measurement of 41BB expression in gated CD8+ (top graph) or CD4+ T cells (mean ± SEM; n = 3). E, Transfection of HLA genes into COS7 cell line, pulsing of identified minimal mutated neoantigen peptides, and coculture with TCR-transduced T cells. Expression of 41BB is displayed for CD8+mTCR+ (4046-TCR1a1/2/3, 4046-TCR3, 4046-TCR4 and 4046-TCR5), and CD4+mTCR+ (4046-TCR32a1/2) gates. mut, mutated; wt, wild-type.

Figure 1.

Multivalent T-cell responses to USP9XY2009C neoantigen. A, TIL fragments from FrTu#4046 were cocultured with autologous APCs (i) electroporated with irrelevant TMG (TMG-IRR) or 4046-TMG15 or (ii) pulsed with DMSO (peptide vehicle) or 4046-PP6. Media (T cells only) and OKT3 were negative and positive control, respectively. Secretion of IFNγ was evaluated by ELISPOT. B, Selected TIL fragments were cocultured with autologous APCs pulsed with peptides, and IFNγ secretion was evaluated by ELISPOT. C, Expression of 41BB in T cells following coculture of 4046-REP-TIL with autologous APCs electroporated with TMGs or pulsed with peptides. D, Coculture of TIL or TCR-transduced T cells with autologous APCs pulsed with minimal neoepitope peptides and measurement of 41BB expression in gated CD8+ (top graph) or CD4+ T cells (mean ± SEM; n = 3). E, Transfection of HLA genes into COS7 cell line, pulsing of identified minimal mutated neoantigen peptides, and coculture with TCR-transduced T cells. Expression of 41BB is displayed for CD8+mTCR+ (4046-TCR1a1/2/3, 4046-TCR3, 4046-TCR4 and 4046-TCR5), and CD4+mTCR+ (4046-TCR32a1/2) gates. mut, mutated; wt, wild-type.

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Putative USP9XY2009C-reactive TCRs were identified by coculture with USP9XY2009C neoantigen, isolation of 41BB+ T cells by FACS and performing single-cell RT-PCR with primers spanning the TCRα and TCRβ chains similar to previous studies (35). T-cell receptors were constructed with murine constant chains thus enabling the detection of the introduced TCR with anti-mTCRβ antibody. The most abundant clonotypes from the 4046-REP-TIL (TCRs named based on their rank) were evaluated following γ-retroviral transduction of autologous PBL. Minimal epitopes were used to evaluate TCR specificity. Peptides RMQYSMECF and YSMECFQFM (mutation in bold underline) embedded in the 25 amino acid neoantigen were predicted to bind HLA-B*15:01:01 and HLA-C*03:03:01 alleles, respectively, from patient 1 (NetMHCpan 3.0). The minimal epitope for CD4+ T-cell responses to USP9XY2009C was empirically determined to be RMQYSMECFQFMKKL by coculturing HLA Class-II–restricted USP9XY2009C TIL with 15 amino acid peptides overlapping 14 amino acids (Supplementary Fig. S1). The top ranking TCR in the 4046-REP-TIL (23.6% by TCRB sequencing) was reactive to YSMECFQFM peptide when paired with three different TCRA chains (4046-TCR1a1, 4046-TCR1a2, and 4046-TCR1a3; Fig. 1D, blue bars). The second ranked TCR (4046-TCR2; 10.3%) was not reactive to USP9XY2009C but the third (4046-TCR3; 7.0%), fourth (4046-TCR4; 6.4%), and fifth (4046-TCR5; 4.8%) ranked TCRs were each reactive to RMQYSMECF peptide (Fig. 1D, black bars). T cells transduced with 4046-TCR32a1 and 4046-TCR32a2 (rank 32 and 0.2% by TCRB of 4046-REP-TIL; unique TCRα chains) reacted to RMQYSMECFQFMKKL and the majority of the response was seen by CD4+ T cells suggesting that this was a HLA Class-II–restricted response (Fig. 1D, red bars, bottom graph). The 4046-REP-TIL culture was reactive to all three minimal peptides, whereas mock-transduced T cells (open repertoire from PBL) did not respond to any of the peptides. The specificity of the responses was supported by an absence of reactivity to wild-type variants of the USP9XY2009 peptides (Fig. 1D, hatched bars).

The HLA restriction elements for USP9XY2009C were determined by transfecting a nonhuman COS7 tumor cell line with individual HLA Class-I or Class-II alleles, followed by pulsing the minimal peptides and coculture with USP9XY2009C-specific T cells. The HLA Class-I responses to RMQYSMECF and YSMECFQFM were restricted by HLA-B*15:01:01 (Fig. 1E, gray histograms) and HLA-C*03:03:01 (Fig. 1E, blue histograms), respectively, and HLA Class-II response to RMQYSMECFQFMKKL was restricted by DPA1*01:03:01 with DPB1*04:02:01 (Fig. 1E, red histograms). In sum, a single mutation could generate three epitopes capable of binding one HLA each and forming functional interactions with one or more TCRs.

Distinct neoepitopes within one Histone H1.5A71D mutation were recognized by CD4 T cells

Multiple epitopes were also identified and characterized from a single Histone H1.5A71D mutation. Patient four, 59 years old, had an iliac lymph node resected for TIL growth and mutation sequencing. All 317 mutations detected from her cancer were assembled into 17 TMGs (4097-TMG) and 18 PPs (4097-PP). Because the quantity of mutations was large, two 4097-PPs were pulsed separately then pooled into one coculture with each TIL fragment microculture. Fragments 1, 2, 3, 4, 6, 16, 19, 22, 23, and pooled fragments P1 and P2 showed specific responses to 4097-PP15/PP16, which contained mutated Histone H1.5A71D LP (Fig. 2A, red bars). The screening also revealed a CD4+ T-cell response in fragment 23 to 4097-PP5/PP6 (Fig. 2A, closed bars). Testing of the individual LP in 4097-PP6 revealed that mutated INPP5KL272V was the neoantigen, but we were unable to identify the HLA restriction element (Supplementary Fig. S2). To interrogate the Histone H1.5A71D response, fragments 3, 6, and 22 were expanded in REP and pooled into one 4097-REP-TIL culture, which displayed specific reactivity to mutated Histone H1.5A71D LP compared with wild-type LP by IFNγ secretion (Fig. 2B). Expression of 41BB by T cells cocultured with Histone H1.5A71D LP established that this response was mediated by CD4+ T cells (Fig. 2C).

Figure 2.

Distinct neoepitopes within one HIST1H1BA71D mutation were recognized by CD4+ T cells. A, Screening of TIL fragments from FrTu#4097 against autologous APCs pulsed with DMSO, a pool of 4097-PP5 and 4097-PP6 (PP5/PP6), or a pool of 4097-PP15 and 4097-PP16 (PP15/PP16). Secretion of IFNγ was evaluated by ELISPOT. B, 4097-REP-TIL was cocultured with decreasing concentrations of wild-type (wt) or mutated (mut) Histone H1.5A71D LP. Secretion of IFNγ was evaluated by ELISPOT. C, Expression of 41BB in CD4+ T cells in cocultures of 4097-REP-TIL with autologous APCs pulsed with DMSO, wt Histone H1.5A71, or mut Histone H1.5A71D LPs. D, Coculture of TIL or TCR-transduced T cells with autologous APCs pulsed with Histone H1.5A71D LPs (wt or mut) and 15 amino acid mut Histone H1.5A71D peptides overlapping 14 amino acids. No target (media) and PMA and ionomycin were negative and positive controls, respectively. Secretion of IFNγ was evaluated by ELISPOT. E, Transfection of HLA genes into COS7 cell line, pulsing of SLAALKKALADGGYD or LADGGYDVEKNNSRI peptides, and coculture with 4097-REP-TIL or TCR-transduced T cells. Secretion of IFNγ into coculture supernatants was evaluated by ELISA (mean ± SEM; n = 3).

Figure 2.

Distinct neoepitopes within one HIST1H1BA71D mutation were recognized by CD4+ T cells. A, Screening of TIL fragments from FrTu#4097 against autologous APCs pulsed with DMSO, a pool of 4097-PP5 and 4097-PP6 (PP5/PP6), or a pool of 4097-PP15 and 4097-PP16 (PP15/PP16). Secretion of IFNγ was evaluated by ELISPOT. B, 4097-REP-TIL was cocultured with decreasing concentrations of wild-type (wt) or mutated (mut) Histone H1.5A71D LP. Secretion of IFNγ was evaluated by ELISPOT. C, Expression of 41BB in CD4+ T cells in cocultures of 4097-REP-TIL with autologous APCs pulsed with DMSO, wt Histone H1.5A71, or mut Histone H1.5A71D LPs. D, Coculture of TIL or TCR-transduced T cells with autologous APCs pulsed with Histone H1.5A71D LPs (wt or mut) and 15 amino acid mut Histone H1.5A71D peptides overlapping 14 amino acids. No target (media) and PMA and ionomycin were negative and positive controls, respectively. Secretion of IFNγ was evaluated by ELISPOT. E, Transfection of HLA genes into COS7 cell line, pulsing of SLAALKKALADGGYD or LADGGYDVEKNNSRI peptides, and coculture with 4097-REP-TIL or TCR-transduced T cells. Secretion of IFNγ into coculture supernatants was evaluated by ELISA (mean ± SEM; n = 3).

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Four TCRs in the 4097-REP-TIL culture targeting Histone H1.5A71D neoantigen were identified. A combination of TCR deep sequencing, Adaptive Biotechnologies' PairSeq (computational TCR pairing service) and single-cell RT-PCR of TCR genes was used to reconstruct TCRs ranking 1, 2, 3, 6, 8, 9, 15, 23, and 79 in the 4097-REP-TIL. Following transduction into peripheral blood T cells it was determined that 4097-TCR1a1 (TCRβ clonotype rank and frequency in 4097-REP-TIL: #1; 36.2%), 4097-TCR2 (#2; 26.2%), 4097-TCR3 (#3, 13.2%), and 4097-TCR9a1 (#9; 0.2%) were specific for Histone H1.5A71D neoantigen (Supplementary Fig. S3).

The specific neoepitopes that were targeted by Histone H1.5A71D reactive TCRs were empirically characterized. Coculture of 4097-REP-TIL and TCR-transduced T cells with 15 amino acid peptides overlapping 14 amino acids demonstrated that there were two neoepitopes recognized (Fig. 2D). More specifically, 4097-TCR1a1, 4097-TCR3, and 4097-TCR9a1 displayed strong responses to 5 peptides spanning LSLAALKKALADGGYDVEK, whereas 4097-TCR2 responded to 4 peptides covering KKALADGGYDVEKNNSRI. All tested T cells, except for the open repertoire mock-transduced culture, showed specific response to the mutated 25 amino acid Histone H1.5A71D peptide and no recognition of wild-type peptide.

Given that there were two distinct Histone H1.5A71D neoepitopes, there was likely more than one HLA restriction element. Experiments transfecting HLA genes into COS7 cells showed that the responses by 4097-TCR1a1, 4097-TCR3, and 4097-TCR9a1 to SLAALKKALADGGYD were restricted by HLA-DRB4*01:01:01 (Fig. 2E, blue bars). In contrast, the 4097-TCR2 response to LADGGYDVEKNNSRI was restricted by DQA1*03:01:01 and DQB1*04:02:01 (Fig. 2E, gray bars). Therefore, a single Histone H1.5A71D neoantigen resulted in two distinct neoepitopes restricted by either HLA-DQ or HLA-DR.

Recognition of RAPTORD654G neoantigen by TILs from a metastasis

In patient 5, we had the opportunity to evaluate the reactivity from TIL fragments from three spatially and temporally distinct tumors toward a common set of neoepitopes. An axillary lymph node was resected for TIL harvest (FrTu#4098), and an initial screen of TILs against 333 putative neoepitopes called from FrTu#4098 and assembled into 18 TMGs and 19 PPs resulted in no clear and/or dominant reactivities. Five months later, patient 5 underwent another surgery to resect progressive abdominal tumor lesions (FrTu#4173-A and FrTu#4173-B). Given that there were TILs grown from fragments from 3 lesions, the TIL from fragments remaining after the initial screen (FrTu#4098) were rescreened in parallel with 7 fragments from FrTu#4173-A/B against 138 mutations present in FrTu#4173-A and/or FrTu#4173-B, which were assembled in eight TMGs (4173-TMG) and eight PPs (4173-PP).

Fragment 9 from FrTu#4098 (4098-F9) displayed IFNγ secretion in response to 4173-PP4 (Fig. 3A, red squares). Parsing of the peptides present in 4173-PP4 against 4098-F9 revealed that RAPTORD654G and CES1S12A were likely the immunogenic neoantigens as measured by 41BB expression on CD4+ T cells (Fig. 3B). Coculture of 4098-F9 with highly purified peptides (>95% by HPLC) demonstrated that mutated RAPTORD654G, but not CES1S12A or wild-type RAPTORD654, was responsible for the 41BB expression on CD4+ T cells (Fig. 3C). Pairing of the most frequent TCRβ chain with the one of two most frequent TCRα from 4098-F9 led to the identification of 4098-F9-RAPTOR-TCRa2 (second TCRα), which was responsive to the RAPTORD654G mutation after expression in PBL with the Sleeping Beauty transposon/transposase system. T cells expressing 4098-F9-RAPTOR-TCRa2 and 4098-F9 TILs were cocultured with overlapping 15 amino acid RAPTORD654G peptides and showed that the immunogenic neoepitope was present in the N-terminal 15 amino acids (Supplementary Fig. S4). COS7 cells were coelectroporated with autologous HLA Class-II alleles, pulsed with the 15 amino acid RAPTORD654G peptide (HNVAMMLAQLVSGGS), and cocultured with 4098-F9-RAPTOR-TCRa2, which demonstrated that RAPTORD654G neoantigen was restricted by HLA-DPB1*10:01:01 in combination with either HLA-DPA1*01:03:01 or HLA-DPA1*02:01:01 (Fig. 3D). Therefore, TILs from one physically and temporally distinct tumor could recognize a mutation identified in another autologous tumor.

Figure 3.

Recognition of RAPTORD654G neoantigen by TILs from a metastasis. A, Screening of TIL fragments from FrTu#4098 and FrTu#4173 against autologous APCs pulsed with DMSO or 4173 peptide pools (PP). No target (media) and PMA and ionomycin were negative and positive controls, respectively. Secretion of IFNγ was evaluated by ELISPOT. B, Expression of 41BB in CD4+ T cells after coculture of 4098-F9 with autologous APCs pulsed with individual peptides from 4173-PP4. C, Expression of 41BB in CD4+ T cells in cocultures of 4098-F9 with autologous APCs pulsed with DMSO, 4149-PP4, mutated (mut) CES1S12A, wild-type (wt) RAPTORD654, or mut RAPTORD654G LPs. D, Transfection of HLA genes into COS7 cell line, pulsing of DMSO or HNVAMMLAQLVSGGS RAPTORD654G peptide, and coculture with 4098-F9-RAPTOR-TCRa2–transduced T cells. Secretion of IFNγ into coculture supernatants was evaluated by ELISA (mean ± SEM; n = 3). E, Exome sequencing coverage (reads) and variant allele frequency (VAF) for RAPTORD654G at chromosome 17:78,858,926 (c.1961A>G).

Figure 3.

Recognition of RAPTORD654G neoantigen by TILs from a metastasis. A, Screening of TIL fragments from FrTu#4098 and FrTu#4173 against autologous APCs pulsed with DMSO or 4173 peptide pools (PP). No target (media) and PMA and ionomycin were negative and positive controls, respectively. Secretion of IFNγ was evaluated by ELISPOT. B, Expression of 41BB in CD4+ T cells after coculture of 4098-F9 with autologous APCs pulsed with individual peptides from 4173-PP4. C, Expression of 41BB in CD4+ T cells in cocultures of 4098-F9 with autologous APCs pulsed with DMSO, 4149-PP4, mutated (mut) CES1S12A, wild-type (wt) RAPTORD654, or mut RAPTORD654G LPs. D, Transfection of HLA genes into COS7 cell line, pulsing of DMSO or HNVAMMLAQLVSGGS RAPTORD654G peptide, and coculture with 4098-F9-RAPTOR-TCRa2–transduced T cells. Secretion of IFNγ into coculture supernatants was evaluated by ELISA (mean ± SEM; n = 3). E, Exome sequencing coverage (reads) and variant allele frequency (VAF) for RAPTORD654G at chromosome 17:78,858,926 (c.1961A>G).

Close modal

The CD4+ T-cell response to RAPTORD654G was not observed by 4098-F9 in the original screen, because RAPTORD654G was not detected in sequencing the original FrTu#4098. Thus, FrTu#4098 was sequenced again at greater depth (FrTu#4098 seq#2), but the RAPTORD654G mutation was again only seen in FrTu#4173-A (Fig. 3E). Furthermore, RAPTORD654G was also absent in another metastasis sequenced from the latter resection (FrTu#4173-B). The variant allele frequency was 15 in FrTu#4173-A, which suggested that only a fraction of the tumor cells contained the RAPTORD654G neoantigen (Fig. 3E, red bars). Other studies have also shown that different ovarian cancer tumor deposits harbor distinct mutational and immunologic profiles (25). This reactivity showed evidence of T-cell responsiveness to only a fraction of the patient's tumor population, and suggested that multiple reactivities such as this one would be likely needed to effectively treat her disease.

Identification and characterization of TP53 “hotspot” mutation-reactive TCRs

Patient 6 had a metastasis resected from the axilla from which a TP53 “hotspot” mutation (c.733G>A; p.G245S) was detected. Screening of TILs from fragments (n = 24) against 176 putative neoantigens assembled in 10 TMGs and 11 PPs revealed 3% 41BB+ T cells in response to p53G245S by TILs from fragment 11, which was enriched for reactive T cells by p53G245S peptide coculture, 41BB+ T-cell sorting and REP. The resultant T-cell culture displayed highly avid and specific recognition of the mutated p53G245S compared with wild-type counterpart as measured by IFNγ secretion (Fig. 4A). This response was mediated by CD4+ T cells as measured by 41BB expression in cocultures (Fig. 4B).

Figure 4.

Identification and characterization of p53G245S neoantigen-reactive TCRs. A, Coculture of 4127-F11 p53G245S-enriched TIL and autologous APCs pulsed with decreasing concentrations of p53G245S mutated (mut) or wild-type (wt) peptides. Secretion of IFNγ was evaluated by ELISPOT. B, Expression of 41BB in CD4+ T cells after coculture with autologous APCs pulsed with p53G245S LPs. C, Expression of 41BB in T cells expressing transduced TCRs (mTCR+) following coculture with autologous APCs pulsed with p53G245S LPs. D, Transfection of HLA genes into COS7 cell line, pulsing of DMSO or HYNYMCNSSCMGSMN p53G245S peptide, and coculture with 4127-F11 p53G245S-enriched TILs or mock-, 4127-TCR1–, or 4127-TCR4–transduced T cells. Expression of 41BB in gated CD4+ T cells is displayed (mean ± SEM; n = 3).

Figure 4.

Identification and characterization of p53G245S neoantigen-reactive TCRs. A, Coculture of 4127-F11 p53G245S-enriched TIL and autologous APCs pulsed with decreasing concentrations of p53G245S mutated (mut) or wild-type (wt) peptides. Secretion of IFNγ was evaluated by ELISPOT. B, Expression of 41BB in CD4+ T cells after coculture with autologous APCs pulsed with p53G245S LPs. C, Expression of 41BB in T cells expressing transduced TCRs (mTCR+) following coculture with autologous APCs pulsed with p53G245S LPs. D, Transfection of HLA genes into COS7 cell line, pulsing of DMSO or HYNYMCNSSCMGSMN p53G245S peptide, and coculture with 4127-F11 p53G245S-enriched TILs or mock-, 4127-TCR1–, or 4127-TCR4–transduced T cells. Expression of 41BB in gated CD4+ T cells is displayed (mean ± SEM; n = 3).

Close modal

Because TP53 (c.733G>A; p.G245S) was a “hotspot” expressed by tumors from unrelated individuals (∼2.8% of cancers; ref. 39), it was prudent to identify the TCRs for future research and clinical applications. Putative p53G245S reactive TCRs were identified from 4127-F11 by single-cell RT-PCR from 41BB+ T cells following coculture with p53G245S peptide. T cells transduced with 4127-TCR1 and 4127-TCR4 (as measured by mTCRβ expression) demonstrated 41BB upregulation in response to p53G245S peptide (HYNYMCNSSCMGSMN) in the N-terminal 15 amino acids of the mutated p53G245S long peptide (Supplementary Fig. S5). Open repertoire activated T cells (mock transduced) did not express 41BB in response to p53G245S peptide supporting the specificity of the transduced TCR response, whereas 4127-TCR1 and 4127-TCR4–transduced T cells (mTCRβ+) expressed 41BB only in response to mutated p53G245S peptide (Fig. 4C). The p53G245S neoepitope was restricted by HLA-DRB3*02:02:01 (Fig. 4D). HLA-DRB3*02:02:01 was present in 1,367 of 3,719 patients (37%) seen at the NCI. Thus, TIL and TCR responses specific for a frequent TP53 “hotspot” mutation (c.733G>A; p.G245S) in the context of a common HLA-DR allele were observed and characterized from patient 6.

Both unique and shared neoantigens were detected in a single patient

Three different immunogenic neoantigens were identified for patient 7, including a TP53 “hotspot” mutation (c.659A>G; p.Y220C; 2.4% of ovarian cancers and 1.5% of all cancers; refs. 40, 41), out of only 64 total neoepitopes identified from resected iliac lymph node metastasis. An initial TIL screen with four TMGs and five PPs revealed a strong response to p53Y220C neoantigen in fragments 7, 8, and 11, and these three fragments were expanded in REP for further testing. The 4149-REP-TIL culture demonstrated a polyclonal response as evidenced by IFNγ secretion when cocultured with three of four TMGs (4149-TMG2, 4149-TMG3 and 4149-TMG4) and three of five peptide pools (4149-PP2, 4149-PP3, and 4149-PP4; Fig. 5A). The TMGs and PPs were congruent except for 4149-PP5, which was composed of overlapping peptides from a single frameshift mutation present in TMG3. Parsing the peptides from each of the reactive TMGs or peptide pools revealed that CTAGE5E576V, p53Y220C and HUWE1F4353S were the neoantigens in 4149-TMG2/-PP2, 4149-TMG3/-PP3 and 4149-TMG4/-PP4, respectively (Supplementary Fig. S6). CD4+ T cells demonstrated specific recognition of CTAGE5E576V and p53Y220C, whereas CD8+ T cells responded to HUWE1F4353S as evidenced by 41BB expression in mutated compared with wild-type cocultures (Fig. 5B). Thus, two CD4 and one CD8 T-cell populations were present in 4149-REP-TILs recognizing three unique mutated neoantigens.

Figure 5.

Both unique and shared neoantigens were detected in a single patient. A, 4149-REP-TILs were screened against autologous APC (top) electroporated with TMGs or (bottom) pulsed with DMSO or peptide pools (PP). Secretion of IFNγ was evaluated by ELISPOT. B, Expression of 41BB in T cells from 4149-REP-TIL after coculture with mutated (mut) or wild-type (wt) CTAGE5E576V, HUWE1F4353S, and p53Y220C LPs. C, Coculture of 4149-REP-TIL with autologous APCs pulsed with 15 amino acid mut peptides overlapping 14 amino acids from (left) CTAGE5E576V or (right) p53Y220C neoantigens. Secretion of IFNγ was evaluated by ELISPOT. D, Coculture of 4149-REP-TIL with autologous APCs pulsed with 9 or 10 amino acid wt or mut HUWE1F4353S peptides. Expression of 41BB was displayed for CD8+ T cells. E, T cells expressing introduced TCRs (left: 4149-HUWE1-TCR1 or right: 4149-TP53-TCRa2b2) were cocultured with autologous APCs pulsed with decreasing concentrations of minimal wt or mut minimal neoepitope peptides. Expression of 41BB in CD8+mTCR+ (4149-HUWE1-TCR1) or CD4+mTCR+ (4149-TP53-TCRa2b2) is displayed (mean ± SEM; n = 3). F, Transfection of HLA genes into COS7 cell line, pulsing of DMSO, NERGVSSCDRLTDPH (CTAGE5E576V), SEKLRHMLL (HUWE1F4353S), or NTFRHSVVVPCEPPE (p53Y220C) peptides, and coculture with 4149-REP-TILs. Expression of 41BB in gated (top) CD8+ or (bottom) CD4+ T cells is displayed (mean ± SEM; n = 3).

Figure 5.

Both unique and shared neoantigens were detected in a single patient. A, 4149-REP-TILs were screened against autologous APC (top) electroporated with TMGs or (bottom) pulsed with DMSO or peptide pools (PP). Secretion of IFNγ was evaluated by ELISPOT. B, Expression of 41BB in T cells from 4149-REP-TIL after coculture with mutated (mut) or wild-type (wt) CTAGE5E576V, HUWE1F4353S, and p53Y220C LPs. C, Coculture of 4149-REP-TIL with autologous APCs pulsed with 15 amino acid mut peptides overlapping 14 amino acids from (left) CTAGE5E576V or (right) p53Y220C neoantigens. Secretion of IFNγ was evaluated by ELISPOT. D, Coculture of 4149-REP-TIL with autologous APCs pulsed with 9 or 10 amino acid wt or mut HUWE1F4353S peptides. Expression of 41BB was displayed for CD8+ T cells. E, T cells expressing introduced TCRs (left: 4149-HUWE1-TCR1 or right: 4149-TP53-TCRa2b2) were cocultured with autologous APCs pulsed with decreasing concentrations of minimal wt or mut minimal neoepitope peptides. Expression of 41BB in CD8+mTCR+ (4149-HUWE1-TCR1) or CD4+mTCR+ (4149-TP53-TCRa2b2) is displayed (mean ± SEM; n = 3). F, Transfection of HLA genes into COS7 cell line, pulsing of DMSO, NERGVSSCDRLTDPH (CTAGE5E576V), SEKLRHMLL (HUWE1F4353S), or NTFRHSVVVPCEPPE (p53Y220C) peptides, and coculture with 4149-REP-TILs. Expression of 41BB in gated (top) CD8+ or (bottom) CD4+ T cells is displayed (mean ± SEM; n = 3).

Close modal

Minimal epitopes were then identified for the three neoantigens from patient 7. Mutations within the minimal 15 amino acid neoepitopes for CTAGE5E576V and p53Y220C were closer to the C- and N- termini, respectively (Fig. 5C). Two HUWE1F4353S peptides were predicted to bind either HLA-B*07:02:01 (LPAYESSEKL) or HLA-B*40:02:01 (SEKLRHMLL), and it was the latter peptide which was specifically recognized over wild-type variants by CD8+ T cells (Fig. 5D). The TCRs targeting HUWE1F4353S and p53Y220C neoantigens were identified and expressed in peripheral blood T cells using the Sleeping Beauty transposon/transposase system. HUWE1F4353S and p53Y220C minimal peptides were specifically recognized by 4149-HUWE1-TCR1 and 4149-TP53-TCRa2b2, respectively, as measured by 41BB coexpression on TCR transposed (mTCRβ+) T cells with minimal response to wild-type peptides (Fig. 5E). Transfection of individual HLA Class-I genes or cotransfection of α and β HLA Class-II genes into COS7 cells followed by mutated peptide pulsing and coculture with the 4149-REP-TILs demonstrated that HUWE1F4353S was restricted by HLA-B*40:02:01, CTAGE5E576V was restricted by HLA-DQA1*01:03:01 and HLA-DQB1*06:03:01 and p53Y220C was restricted by HLA-DRB3*02:02:01 (Fig. 5F). In a patient with only 64 mutations, both shared (p53Y220C) and unique (HUWE1F4353S and CTAGE5E576V) neoantigens were targets of T cells.

In sum, all recognized mutations were unique to each patient. The immunogenic TP53 “hotspot” mutations, G245S in patient 6 and Y220C in patient 7, were presented by the same restriction element (HLA-DRB3*02:02:01), indicating that there may be commonalties for T cells to target oncogenic mutations. With the exception of the TP53 “hotspot” mutations, the neoantigens recognized by T cells arose from seemingly random, somatic, nonsynonymous mutations. Therefore, the universe of potential neoantigens expressed by ovarian cancer was complex and derived from mutations unique to each patient and as well as TP53 “hotspot” mutations present in tumors of unrelated people.

Herein, we identified and characterized T-cell responses to the ovarian cancer neoantigenome. The study of T-cell antigens expressed by autologous epithelial cancers has been hampered by the relative inability to establish autologous tumor cell lines from resected tumors. This problem was circumvented using TMG and LP screening of all autologous cancer mutations such that the tumor cell could be, in essence, recreated in an autologous APC and used for T cell coculture (12–17). Application of this strategy to the study of metastatic ovarian cancers established that T cells expressing TCRs capable of binding mutated neoantigens had infiltrated ovarian tumors.

The identification of T-cell responses to TP53 “hotspot” mutations from patients 6 and 7 was promising because TP53 is the most commonly mutated gene in human cancers (42) and approximately 95% of high-grade serous ovarian cancers will have a mutation in the TP53 gene (20, 21, 23, 24, 39). Mutated p53 is an attractive target for tumor-specific therapies because it is absent on normal tissue. However, tumor-infiltrating T-cell responses to TP53 “hotspot” mutations have not been characterized. The frequencies of G245S and Y220C TP53 “hotspot” mutations in all cancers are 2.8% and 1.5%, respectively, (39, 40) and HLA-DRB3*02:02 was typed in 37% of patients at the NCI, which is consistent with The Allele Frequency Net Database (http://www.allelefrequencies.net). Thus, it is estimated that approximately 1.5% of cancers in general would have both HLA-DRB3*02:02 and either a G245S and Y220C TP53 “hotspot” mutation. In vitro sensitization of an HLA-A*02:01 donor's PBL generated a CD8+ T-cell clone capable of recognizing p53Y220C-mutant epitope, suggesting that circulating T cells may bind mutated p53 epitopes (43). A library of TCRs targeting mutated p53 and other commonly altered driver genes, for example, KRAS, could be generated to treat a wide variety of patients with T cells genetically modified with TCRs.

All of the immunogenic mutations discovered in this study, except the p53G245S and p53Y220C neoantigens, were unique and were not reported in the COSMIC database. The Histone H1.5A71D mutation, which was in a linker H1 histone that tethers nucleosomes composed of a H2A, H2B, H3, and H4 core histones, could have impacted the chromatin structure and epigenetics (44). Linker Histone H1.3 and HUWE1, a ubiquitin E3 ligase that targets proteins for proteolysis, worked together to promote the pathogenesis of ovarian cancer in mice (45). The USP9X protein is a deubiquitinase that reverses ubiquitin-targeted protein degradation and is a central component of the centrosome (46). Transposon-mediated mutagenesis murine models of pancreatic and hepatocellular cancers were linked to USP9X and HUWE1, respectively, suggesting that these may be driver genes (38, 47). CTAGE5 transports proteins from the endoplasmic reticulum and the E576V mutation could also impact the HLA Class-I pathway because this is within the proline-rich domain, which is used to bind other proteins (48). Mutations in proteins involved with intracellular signaling pathways were also immunogenic. For example, RAPTOR is a critical member of the mTOR complex and the D654G mutation resides in the HEAT-3 (Huntington-EF3-PP2A-TOR1) domain that serves as vital structural support (49). The catalytic domain of was altered in the INPP5KL272V mutation and may have impacted on inositol 5-phosphate signaling and could have contributed to pathogenesis in line with the effects of other INPP5K mutations (50). A more complete understanding of the relationship between the functional consequences of a mutation and the generation of an immunogenic neoepitope may yield insights useful for T-cell immunotherapy and tumor biology.

Knowledge of the identity of mutated neoantigens as described in this article could potentially be used to improve ovarian cancer T-cell immunotherapy. The effectiveness of immunotherapy using the direct infusion of mutation-reactive TILs is likely limited by the existing differentiation status of TIL, that is, likely senescent and/or terminally differentiated with limited proliferative potential. High frequencies of mutation-reactive T cells with improved in vivo proliferative potential could thus be infused by transducing or transposing TCRs into autologous naïve or central memory T cells. Personalized TCR gene therapy targeting a mutation expressed by the autologous tumor could be developed by transducing PBL with a TCR from transiently produced γ-retroviral supernatant or by electroporating TCR transposons in the nonviral Sleeping Beauty transposon/transposase system (37). The landscape of somatic mutations in ovarian cancer neoantigens is large and the elucidation of their functional roles, participation in oncogenesis and metastasis, neoepitope formation, T-cell responsiveness, and molecular structural determinants could enable deeper understanding and the development of effective treatments for ovarian cancer.

No potential conflicts of interest were disclosed.

Conception and design: D.C. Deniger, A. Pasetto, P.F. Robbins

Development of methodology: D.C. Deniger, A. Pasetto, P.F. Robbins, L. Jia

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.C. Deniger, A. Pasetto, T.D. Prickett, B.C. Paria, P. Malekzadeh, R. Yossef, M.M. Langhan, J.R. Wunderlich, R.P.T. Somerville

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.C. Deniger, A. Pasetto, P.F. Robbins, J.J. Gartner, P. Malekzadeh, L. Jia, R. Yossef, S.A. Rosenberg

Writing, review, and/or revision of the manuscript: D.C. Deniger, A. Pasetto, P.F. Robbins, J.J. Gartner, P. Malekzadeh, D.N. Danforth, S.A. Rosenberg

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.C. Deniger, T.D. Prickett, P. Malekzadeh, D.N. Danforth, S.A. Rosenberg

Study supervision: D.C. Deniger, S.A. Rosenberg

The authors thank the TIL lab for its efforts growing the TILs. The authors thank the FACS core members Arnold Mixon and Shawn Farid for their assistance. Thanks to Eric Tran and Sanja Stevanovic for academic and technical discussions about this study. This work was supported by intramural funding of the Center for Cancer Research, NCI.

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

1.
American Cancer Society
. 
American Cancer Society facts & figures
; 
2017
.
Available from:
https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2017.html.
2.
Ovarian Tumor Tissue Analysis Consortium
,
Goode
EL
,
Block
MS
,
Kalli
KR
,
Vierkant
RA
,
Chen
W
, et al
Dose-response association of CD8+ tumor-infiltrating lymphocytes and survival time in high-grade serous ovarian cancer
.
JAMA Oncol
2017
;
3
:
e173290
.
3.
Hwang
WT
,
Adams
SF
,
Tahirovic
E
,
Hagemann
IS
,
Coukos
G
. 
Prognostic significance of tumor-infiltrating T cells in ovarian cancer: a meta-analysis
.
Gynecol Oncol
2012
;
124
:
192
8
.
4.
Zhang
L
,
Conejo-Garcia
JR
,
Katsaros
D
,
Gimotty
PA
,
Massobrio
M
,
Regnani
G
, et al
Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer
.
N Engl J Med
2003
;
348
:
203
13
.
5.
Lo
CS
,
Sanii
S
,
Kroeger
DR
,
Milne
K
,
Talhouk
A
,
Chiu
DS
, et al
Neoadjuvant chemotherapy of ovarian cancer results in three patterns of tumor-infiltrating lymphocyte response with distinct implications for immunotherapy
.
Clin Cancer Res
2017
;
23
:
925
34
.
6.
Aoki
Y
,
Takakuwa
K
,
Kodama
S
,
Tanaka
K
,
Takahashi
M
,
Tokunaga
A
, et al
Use of adoptive transfer of tumor-infiltrating lymphocytes alone or in combination with cisplatin-containing chemotherapy in patients with epithelial ovarian cancer
.
Cancer Res
1991
;
51
:
1934
9
.
7.
Ikarashi
H
,
Fujita
K
,
Takakuwa
K
,
Kodama
S
,
Tokunaga
A
,
Takahashi
T
, et al
Immunomodulation in patients with epithelial ovarian cancer after adoptive transfer of tumor-infiltrating lymphocytes
.
Cancer Res
1994
;
54
:
190
6
.
8.
Besser
MJ
,
Shapira-Frommer
R
,
Itzhaki
O
,
Treves
AJ
,
Zippel
DB
,
Levy
D
, et al
Adoptive transfer of tumor-infiltrating lymphocytes in patients with metastatic melanoma: intent-to-treat analysis and efficacy after failure to prior immunotherapies
.
Clin Cancer Res
2013
;
19
:
4792
800
.
9.
Goff
SL
,
Dudley
ME
,
Citrin
DE
,
Somerville
RP
,
Wunderlich
JR
,
Danforth
DN
, et al
Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma
.
J Clin Oncol
2016
;
34
:
2389
97
.
10.
Radvanyi
LG
,
Bernatchez
C
,
Zhang
M
,
Fox
PS
,
Miller
P
,
Chacon
J
, et al
Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients
.
Clin Cancer Res
2012
;
18
:
6758
70
.
11.
Rosenberg
SA
,
Yang
JC
,
Sherry
RM
,
Kammula
US
,
Hughes
MS
,
Phan
GQ
, et al
Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy
.
Clin Cancer Res
2011
;
17
:
4550
7
.
12.
Robbins
PF
,
Lu
YC
,
El-Gamil
M
,
Li
YF
,
Gross
C
,
Gartner
J
, et al
Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells
.
Nat Med
2013
;
19
:
747
52
.
13.
Lu
YC
,
Yao
X
,
Crystal
JS
,
Li
YF
,
El-Gamil
M
,
Gross
C
, et al
Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions
.
Clin Cancer Res
2014
;
20
:
3401
10
.
14.
Lu
YC
,
Yao
X
,
Li
YF
,
El-Gamil
M
,
Dudley
ME
,
Yang
JC
, et al
Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression
.
J Immunol
2013
;
190
:
6034
42
.
15.
Tran
E
,
Ahmadzadeh
M
,
Lu
YC
,
Gros
A
,
Turcotte
S
,
Robbins
PF
, et al
Immunogenicity of somatic mutations in human gastrointestinal cancers
.
Science
2015
;
350
:
1387
90
.
16.
Tran
E
,
Robbins
PF
,
Lu
YC
,
Prickett
TD
,
Gartner
JJ
,
Jia
L
, et al
T-cell transfer therapy targeting mutant KRAS in cancer
.
N Engl J Med
2016
;
375
:
2255
62
.
17.
Tran
E
,
Turcotte
S
,
Gros
A
,
Robbins
PF
,
Lu
YC
,
Dudley
ME
, et al
Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer
.
Science
2014
;
344
:
641
5
.
18.
Lawrence
MS
,
Stojanov
P
,
Polak
P
,
Kryukov
GV
,
Cibulskis
K
,
Sivachenko
A
, et al
Mutational heterogeneity in cancer and the search for new cancer-associated genes
.
Nature
2013
;
499
:
214
8
.
19.
Vogelstein
B
,
Papadopoulos
N
,
Velculescu
VE
,
Zhou
S
,
Diaz
LA
 Jr
,
Kinzler
KW
. 
Cancer genome landscapes
.
Science
2013
;
339
:
1546
58
.
20.
Kanchi
KL
,
Johnson
KJ
,
Lu
C
,
McLellan
MD
,
Leiserson
MD
,
Wendl
MC
, et al
Integrated analysis of germline and somatic variants in ovarian cancer
.
Nat Commun
2014
;
5
:
3156
.
21.
Cancer Genome Atlas Research Network
. 
Integrated genomic analyses of ovarian carcinoma
.
Nature
2011
;
474
:
609
15
.
22.
Ahmed
AA
,
Etemadmoghadam
D
,
Temple
J
,
Lynch
AG
,
Riad
M
,
Sharma
R
, et al
Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary
.
J Pathol
2010
;
221
:
49
56
.
23.
Cole
AJ
,
Dwight
T
,
Gill
AJ
,
Dickson
KA
,
Zhu
Y
,
Clarkson
A
, et al
Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing
.
Sci Rep
2016
;
6
:
26191
.
24.
Ab Mutalib
NS
,
Syafruddin
SE
,
Md Zain
RR
,
Mohd Dali
AZ
,
Mohd Yunos
RI
,
Saidin
S
, et al
Molecular characterization of serous ovarian carcinoma using a multigene next generation sequencing cancer panel approach
.
BMC Res Notes
2014
;
7
:
805
.
25.
Bashashati
A
,
Ha
G
,
Tone
A
,
Ding
J
,
Prentice
LM
,
Roth
A
, et al
Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling
.
J Pathol
2013
;
231
:
21
34
.
26.
Xu
J
,
Qian
J
,
Hu
Y
,
Wang
J
,
Zhou
X
,
Chen
H
, et al
Heterogeneity of Li-Fraumeni syndrome links to unequal gain-of-function effects of p53 mutations
.
Sci Rep
2014
;
4
:
4223
.
27.
Mello
SS
,
Attardi
LD
. 
Not all p53 gain-of-function mutants are created equal
.
Cell Death Differ
2013
;
20
:
855
7
.
28.
Yue
X
,
Zhao
Y
,
Xu
Y
,
Zheng
M
,
Feng
Z
,
Hu
W
. 
Mutant p53 in cancer: accumulation, gain-of-function, and therapy
.
J Mol Biol
2017
;
429
:
1595
606
.
29.
Hoadley
KA
,
Yau
C
,
Wolf
DM
,
Cherniack
AD
,
Tamborero
D
,
Ng
S
, et al
Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin
.
Cell
2014
;
158
:
929
44
.
30.
Wick
DA
,
Webb
JR
,
Nielsen
JS
,
Martin
SD
,
Kroeger
DR
,
Milne
K
, et al
Surveillance of the tumor mutanome by T cells during progression from primary to recurrent ovarian cancer
.
Clin Cancer Res
2014
;
20
:
1125
34
.
31.
Martin
SD
,
Brown
SD
,
Wick
DA
,
Nielsen
JS
,
Kroeger
DR
,
Twumasi-Boateng
K
, et al
Low mutation burden in ovarian cancer may limit the utility of neoantigen-targeted vaccines
.
PLoS One
2016
;
11
:
e0155189
.
32.
Bobisse
S
,
Genolet
R
,
Roberti
A
,
Tanyi
JL
,
Racle
J
,
Stevenson
BJ
, et al
Sensitive and frequent identification of high avidity neo-epitope specific CD8 (+) T cells in immunotherapy-naive ovarian cancer
.
Nat Commun
2018
;
9
:
1092
.
33.
Tanyi
JL
,
Bobisse
S
,
Ophir
E
,
Tuyaerts
S
,
Roberti
A
,
Genolet
R
, et al
Personalized cancer vaccine effectively mobilizes antitumor T cell immunity in ovarian cancer
.
Sci Transl Med
2018
;
10
.
pii: eaao5931.
34.
Howie
B
,
Sherwood
AM
,
Berkebile
AD
,
Berka
J
,
Emerson
RO
,
Williamson
DW
, et al
High-throughput pairing of T cell receptor alpha and beta sequences
.
Sci Transl Med
2015
;
7
:
301ra131
.
35.
Pasetto
A
,
Alena
G
,
Robbins
PF
,
Deniger
DC
,
Prickett
TD
,
Matus-Nicodemos
R
, et al
Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor
.
Cancer Immunol Res
2016
;
4
:
734
43
.
36.
Parkhurst
M
,
Gros
A
,
Pasetto
A
,
Prickett
T
,
Crystal
JS
,
Robbins
P
, et al
Isolation of T-cell receptors specifically reactive with mutated tumor-associated antigens from tumor-infiltrating lymphocytes based on CD137 expression
.
Clin Cancer Res
2017
;
23
:
2491
505
.
37.
Deniger
DC
,
Pasetto
A
,
Tran
E
,
Parkhurst
MR
,
Cohen
CJ
,
Robbins
PF
, et al
Stable, nonviral expression of mutated tumor neoantigen-specific T-cell receptors using the sleeping beauty transposon/transposase system
.
Mol Ther
2016
;
24
:
1078
89
.
38.
Perez-Mancera
PA
,
Rust
AG
,
van der Weyden
L
,
Kristiansen
G
,
Li
A
,
Sarver
AL
, et al
The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma
.
Nature
2012
;
486
:
266
70
.
39.
Freed-Pastor
WA
,
Prives
C
. 
Mutant p53: one name, many proteins
.
Genes Dev
2012
;
26
:
1268
86
.
40.
Bouaoun
L
,
Sonkin
D
,
Ardin
M
,
Hollstein
M
,
Byrnes
G
,
Zavadil
J
, et al
TP53 variations in human cancers: new lessons from the IARC TP53 database and genomics data
.
Hum Mutat
2016
;
37
:
865
76
.
41.
Kobel
M
,
Piskorz
AM
,
Lee
S
,
Lui
S
,
LePage
C
,
Marass
F
, et al
Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma
.
J Pathol Clin Res
2016
;
2
:
247
58
.
42.
Bailey
MH
,
Tokheim
C
,
Porta-Pardo
E
,
Sengupta
S
,
Bertrand
D
,
Weerasinghe
A
, et al
Comprehensive characterization of cancer driver genes and mutations
.
Cell
2018
;
173
:
371
85
.
43.
Ito
D
,
Visus
C
,
Hoffmann
TK
,
Balz
V
,
Bier
H
,
Appella
E
, et al
Immunological characterization of missense mutations occurring within cytotoxic T cell-defined p53 epitopes in HLA-A*0201+ squamous cell carcinomas of the head and neck
.
Int J Cancer
2007
;
120
:
2618
24
.
44.
Fyodorov
DV
,
Zhou
BR
,
Skoultchi
AI
,
Bai
Y
. 
Emerging roles of linker histones in regulating chromatin structure and function
.
Nat Rev Mol Cell Biol
2018
;
19
:
192
206
.
45.
Yang
D
,
Sun
B
,
Zhang
X
,
Cheng
D
,
Yu
X
,
Yan
L
, et al
Huwe1 sustains normal ovarian epithelial cell transformation and tumor growth through the histone H1.3-H19 cascade
.
Cancer Res
2017
;
77
:
4773
84
.
46.
Li
X
,
Song
N
,
Liu
L
,
Liu
X
,
Ding
X
,
Song
X
, et al
USP9X regulates centrosome duplication and promotes breast carcinogenesis
.
Nat Commun
2017
;
8
:
14866
.
47.
Kodama
T
,
Newberg
JY
,
Kodama
M
,
Rangel
R
,
Yoshihara
K
,
Tien
JC
, et al
Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma
.
Proc Natl Acad Sci U S A
2016
;
113
:
E3384
93
.
48.
Ma
W
,
Goldberg
J
. 
TANGO1/cTAGE5 receptor as a polyvalent template for assembly of large COPII coats
.
Proc Natl Acad Sci U S A
2016
;
113
:
10061
6
.
49.
Zhou
P
,
Zhang
N
,
Nussinov
R
,
Ma
B
. 
Defining the domain arrangement of the mammalian target of rapamycin complex component rictor protein
.
J Comput Biol
2015
;
22
:
876
86
.
50.
Wiessner
M
,
Roos
A
,
Munn
CJ
,
Viswanathan
R
,
Whyte
T
,
Cox
D
, et al
Mutations in INPP5K, encoding a phosphoinositide 5-phosphatase, cause congenital muscular dystrophy with cataracts and mild cognitive impairment
.
Am J Hum Genet
2017
;
100
:
523
36
.