Induction of Neoantigen-Specific Cytotoxic T Cells and Construction of T-Cell Receptor-Engineered T Cells for Ovarian Cancer

Author Manuscript Published OnlineFirst on May 2, 2018; DOI: 10.1158/1078-0432.CCR-18-0142 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Induction of Neoantigen-specific Cytotoxic T Cells and Construction of T-cell Receptor-engineered T cells for Ovarian Cancer

Tatsuo Matsuda1, Matthias Leisegang2,3, Jae-Hyun Park1, Lili Ren1, Taigo Kato1, Yuji Ikeda1, Makiko Harada1, Kazuma Kiyotani1,4, Ernst Lengyel5, Gini F Fleming1, Yusuke Nakamura1,6

1 Department of Medicine, The University of Chicago, Chicago, IL 60637, USA 2 Institute of Immunology, Charité-Universitätsmedizin Berlin, Campus Buch, Berlin 13125, Germany. 3 Berlin Institute of Health, Berlin 10117, Germany. 4 Project for Immunogenomics, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan 5 Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, The University of Chicago, Chicago, IL 60637, USA 6 Department of Surgery, The University of Chicago, Chicago, IL 60637, USA

Running title: Neoantigen-specific-TCR-engineered T cells for Ovarian Cancer

Keywords: T cell receptor (TCR), neoantigen, adoptive T cell therapy, cytotoxic T lymphocyte (CTL), ovarian cancer

Financial support: This work was supported in part by a Team Science Award of UCCCC (The University of Chicago Medicine Comprehensive Cancer Center) and by a research grant from OncoTherapy Science Inc.

Corresponding author Yusuke Nakamura, M.D., Ph.D. Department of Medicine and Surgery, The University of Chicago 900 E 57th street KCBD 6130, Chicago, IL 60637 Tel: 773-834-1405 Fax: 773-702-9268 E-mail: [email protected]

Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 2, 2018; DOI: 10.1158/1078-0432.CCR-18-0142 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Disclosure of potential conflicts of interest: YN is a stock holder and a scientific advisor of OncoTherapy Science Ltd. J-H P was an advisor of and is now an employee of OncoTherapy Science Ltd.

Number of tables and figures: 4 figures, 2 tables, 2 supplementary figure and 3 supplementary tables

To prepare Translational relevance

Adoptive T cell therapy with neoantigen-specific T cell receptor (TCR)-engineered T cells is considered as a promising novel immunotherapy strategy. It takes four steps to prepare; (1) prediction of neoantigen epitopes, (2) neoantigen peptides synthesis, (3) identification of neoantigen-specific TCR and (4) production of virus vector to express TCR. Among them, the most challenging part is identification of neoantigen-specific TCRs. Our protocol required only two weeks from stimulation of T cells with peptides to the identification of neoantigen-specific TCRs. We conducted a pilot study to validate our time-efficient protocol in solid cancers with relatively lower mutational loads such as ovarian cancer. We successfully induced neoantigen-specific T cells against three neoantigens and established corresponding TCR-engineered T cells. One case of neoantigen-specific TCR-engineered T cells showed cross-reactivity against the corresponding wild-type peptide. These results give an important insight into the clinical application of adoptive T cell therapy with neoantigen-specific TCR-engineered T cells.

ABSTRACT

Purpose: Current evolution of cancer immunotherapies, such as immune checkpoint blockade, has implicated neoantigens as major targets of anti-cancer cytotoxic T cells. Adoptive T cell therapy with neoantigen-specific T cell receptor (TCR)-engineered T cells would be an attractive therapeutic option for advanced cancers where the host antitumor immune function is strongly inhibited. We previously developed a rapid and efficient pipeline for production of neoantigen-specific TCR-engineered T cells using peripheral blood from an HLA-matched healthy donor. Our protocol required only two weeks from stimulation of T cells with neoantigen-loaded dendritic cells to the identification of neoantigen-specific TCRs. We conducted the pilot study to validate our protocol. Experimental Design: We used tumors from 7 ovarian cancer patients to validate our protocol. Results: We chose 14 candidate neoantigens from 7 ovarian tumors (1-3 candidates for each patient), and then successfully induced 3 neoantigen-specific T cells from one healthy donor and identified their TCR sequences. Moreover, we validated functional activity of the three identified TCRs by generating TCR-engineered T cells which recognized the corresponding neoantigens and showed cytotoxic activity in an antigen- dose-dependent manner. However, one case of neoantigen-specific TCR-engineered T cells showed cross-reactivity against the corresponding wild-type peptide. Conclusion/discussions: This pilot study demonstrated the feasibility of our efficient process from identification of neoantigen to production of the neoantigen-targeting cytotoxic TCR-engineered T cells for ovarian cancer and revealed the importance of careful validation of neoantigen-specific-TCR-engineered T cells to avoid severe immune-related adverse events.

INTRODUCTION Ovarian cancer is the fifth leading cause of cancer-related death among women and patients are often diagnosed in an advanced stage (1). The majority of ovarian cancer patients experience relapse/recurrence after primary therapy and develop resistance to chemotherapy. Although the overall survival of ovarian cancer has improved due to advances in chemotherapy and surgery, a cure for metastatic ovarian cancer is still elusive (2-5). Immune checkpoint inhibitors (anti-programmed cell death 1 (PD-1) antibodies and anti-programmed cell death ligand 1 (PD-L1) inhibitors) have achieved great success for several cancer types (6-9). Accumulating evidences from patients who responded well to immune checkpoint inhibitors imply that tumor regression is achieved by activation of cytotoxic T cells targeting neoantigens, which are generated mostly by non-synonymous mutations in cancer cells (10-12). In addition, cytotoxic T cells targeting neoantigens were found to be enriched in tumor-infiltrating lymphocytes (TILs) in patients whose tumors responded well to adoptive TIL infusion therapy (13). However, the majority of cancer patients have had no clinical benefit from either immune checkpoint inhibitors or adoptive TIL infusion therapy. Therefore, it is critically essential to develop new strategies to further enhance host immune response. Adoptive T cell therapy with T cell receptor (TCR)-engineered T cells has received attention as a promising new strategy (12,14). We reported TCR-engineered T cells targeting neoantigens could eradicate even a very large solid tumor in a mouse syngeneic tumor model (15). To generate neoantigen-specific TCR-engineered T cells, it is imperative to establish an efficient pipeline to isolate neoantigen-specific T cells and to obtain their TCR sequence information. We established an in-house pipeline to identify neoantigen- specific TCRs using HLA-matched healthy donor-derived peripheral blood mononuclear cells (PBMCs), particularly focusing on the efficiency and timeline for development (16). Our approach takes 2 weeks to induce the neoantigen-specific T cells and obtain TCR information from neoantigen-specific T cells sorted by peptide-loaded HLA-dextramers. Although ovarian cancer has not been regarded as an immunogenic cancer, the presence of CD8+ T cells in ovarian cancers correlates with better prognosis (17,18). Hamanishi et al. reported the clinical efficacy of anti-PD-1 antibody (Nivolumab) in patients with platinum-resistant ovarian cancer (19). Wick et al. reported the presence of a neoantigen-reactive T cell subset in TILs from ovarian cancer patients (20). These reports suggest that cancer neoantigens can be a promising therapeutic target for ovarian

cancer (21). Therefore, we have chosen ovarian cancer for this pilot study to validate our in-house pipeline for induction of neoantigen specific T cells and identification of neoantigen-specific TCRs. We successfully induced neoantigen-specific T cells against a mutation found in ovarian tumor samples and established corresponding TCR- engineered T cells. We propose this method as a promising new therapy for ovarian cancer patients.

MATERIALS AND METHODS Study design Seven patients with ovarian cancer who received surgery and are still being followed at the University of Chicago Medical Center were enrolled after obtaining written informed consent. Tumor blocks and blood samples were obtained from each patient. The study protocol was approved by the Institutional Review Board of the University of Chicago (approval number 15-1738 and 16-0402). This study was conducted in accordance with declaration of Helsinki.

Identification of potential neoantigens Tumor genomic DNA was extracted from paraffin-embedded tumor tissues using QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). Genomic DNA from the patients’ blood was extracted using AllPrep DNA/RNA mini kit (Qiagen) as germline control DNA. Whole-exome libraries were prepared from genomic DNAs using SureSelectXT Human All Exon V5 kit (Agilent Technologies, Santa Clara, CA) and sequenced by 100-bp paired-end reads on a HiSeq2500 Sequencer (Illumina, San Diego, CA). Sequence alignment and mutation calling were performed as described previously (22). Briefly, sequence reads were mapped to the human reference genome GRCh37/hg19 using Burrows-Wheeler Aligner (BWA) (v0.7.10) (23). Possible PCR duplicated reads were excluded using Picard v1.91 (http:// broadinstitute.github.io/picard/). Read pairs with mismatches more than 5% of read length and with a mapping quality of < 30 were also excluded. Finally, single nucleotide variations were called using Fisher’s exact test-based method with the following parameters, (i) base quality of ≥ 15, (ii) sequence depth of ≥ 10, (iii) variant depth of ≥ 2, (iv) variant frequency in tumor of ≥ 10%, (v) variant frequency in normal of < 2%, and (vi) Fisher p value of < 0.05 (24). HLA class I genotypes were determined by OptiType algorithm (25) using whole-exome data of normal controls. We then examined the binding affinities of all

possible 8- to 11-mer peptides harboring each amino acid substitution to HLA-A molecule and filtered out with the predicted binding affinity to HLA-A of <500 nM, using NetMHCv3.4 software (22,26,27). To exclude the no or low expression genes in ovarian cancer, we downloaded RNA sequencing data of The Cancer Genome Atlas (TCGA) (28) ovarian serous cystadenocarcinoma from GDC Data Portal (https://portal.gdc.cancer.gov). The genes with RPKM less than 100 were excluded from the neoantigen list for testing of ability to induce neoantigen specific T cells. We could not examine RNASeq data quantifying transcript expression in the patient's tumor samples, because we could get only paraffin-embedded tumor tissues for DNA extraction, but could not obtain the high quality tumor RNA for RNAseq analysis.

Induction of neoantigen-specific cytotoxic T lymphocytes (CTL)s using PBMCs from healthy donor. Induction of neoantigen-specific T cells were performed following the protocol we developed previously (16). To examine the HLA-A genotype of healthy donors, PCR amplicon-based high-resolution HLA-A genotyping on MiSeq (Illumina) was performed in Scisco Genetics, Inc. (Seattle, WA). Briefly, PBMCs from healthy donors were collected using Vacutainer CPT Cell Preparation Tube (BD Biosciences, San Jose, CA). CD8+ T cells were isolated from PBMCs using Dynabeads CD8 Positive Isolation Kit (Thermo Fisher Scientific, Carlsbad, CA). CD8- cells were used to generate monocyte-derived dendritic cells (DCs) using plastic adherence methods, and were cultured in CellGro DC (Cellgenix, Freiburg, Germany) containing 1% human AB serum (ABS), 500 U/mL IL-4 (R&D Systems, Minneapolis, MN) and 1,000 U/mL GM- CSF (R&D Systems) for 72 h in Primaria 6-well plate (Corning, Inc., Corning, NY). Then, 100 U/mL IFN-γ (PeproTech, Rocky Hill, NJ) and 10 ng/mL LPS (Sigma- Aldrich, ST. Louis, MO) were added in the culture medium to induce the maturation of DCs. DCs were pulsed with 20 µg/mL of the respective neoantigen peptides (Innopep, San Diego, CA) for 16 h at 37°C. After the peptide pulse, DCs were treated with 30 µg/mL of mitomycin C (Sigma-Aldrich) at 37°C for 30 min, and then, were co-cultured with autologous CD8+ T cells in CellGro DC/5% ABS with 30 ng/mL IL-21 (R&D Systems) on day 1 (each well contained 1.0 X 105 peptide pulsed DCs, 5 X 105 CD8+ T cells). We examined the single to quintuple scales for each neoantigen candidates based on the total number of PBMCs from healthy donor. Three days later (day 4), 5 ng/mL IL-7 (R&D Systems) and 5 ng/mL IL-15 (Novoprotein, Summit, NJ) were added in the culture media. On day 6, the cultures were transferred to 12-well plate with CellGro DC/5% ABS with 5 ng/mL IL-7 and 5 ng/mL IL-15. On day 8, cultures were

supplemented with CellGro DC/5% ABS with 10 ng/mL IL-7 and 10 ng/mL IL-15. On day 11, neoantigen-specific T cells were assessed using peptide-HLA dextramers (Immudex, Copenhagen, Denmark) for each neoantigen peptide by flow cytometry analysis. CD8+Dextramer+ T cells were sorted out and used for the following TCR sequencing analysis.

Flow cytometry analysis and antibodies To assess the neoantigen-specific T cells, the cells were incubated with each neoantigen-specific dextramer for 10 min at room temperature and then incubated with anti-human CD8 antibody (clone HIT8a, BD Biosciences) at 4°C for 20 min. Negative control dextramer (WI3233, Immudex) was used to examine general background or unspecific staining on the donor analyzed. Anti-human CD137 antibody (clone 4B4-1, Miltenyi Biotec, Bergisch Gladbach, Germany) was used to examine the cell surface expression of CD137. Anti-mouse TCR beta monoclonal antibody (H57-597, eBioscience, San Diego, CA) was used to assess the cell surface expression of engineered TCRs. CD8+Dextramer+ T cells were analyzed and sorted by flow cytometry (FACS LSRII, Aria Fusion; Becton Dickinson, San Jose, CA). Data analysis was performed using Flow Jo software (Treestar, Ashland, OR).

TCR sequencing analysis TCR sequencing was performed using the methods described previously (26,29,30). In brief, we extracted total RNAs from sorted CD8+Dextramer+ T cells by flow cytometry. The cDNAs with common 5'-RACE adapter were synthesized from total RNA using SMART library construction kit (Clontech, Mountain View, CA). The TCRA and TCRB cDNAs were amplified by PCR using a forward primer for the SMART adapter and reverse primers corresponding to the constant region of each of TCRA and TCRB. After adding the Illumina index sequences with barcode using the Nextera XT Index kit (Illumina), the prepared libraries were sequenced by 300-bp paired-end reads on Illumina MiSeq platform, using MiSeq Reagent v3 600-cycels kit (Illumina). Obtained sequence reads were analyzed using Tcrip software (30).

TCR-engineered T cells Both TCRA and TCRB sequences were codon-optimized, synthesized by GeneArt (Life Technologies, Carlsbad, CA) and cloned into pMP71-PRE as described previously (31). To increase TCR surface expression, we used TCRs with mouse constant regions (32). Transient retroviral supernatants were generated and PBMCs

from healthy donors were transduced as described previously (33).

Cell line C1R (lacking HLA-A and HLA-B, B lymphoblast) was purchased from American Type Culture Collection (Manassas, VA). C1R cells stably expressing HLA- A2 (HLA*02:01) (C1R-A02) or HLA-A24 (HLA-A24:02) (C1R-A24) were prepared by the transfection of the vectors encoding HLA-A*02:01 or HLA-A*24:02 gene. C1R cells were cultured under the recommendation of the depositor.

Transfection of RFC5 and BRAP mutated gene into C1R-A02 cells The plasmid DNAs designed to express a part of RFC5 or BRAP mutated proteins (50 amino acid length and the mutation was placed in the center) and GFP were codon-optimized, and synthesized by GeneArt. Linearized plasmid DNA was used as in vitro transcription template to produce mRNA using the mMESSAGE mMACHINE T7 kit and Poly(A) Tailing Kit (Ambion, Austin, TX). Electroporation was done with Gene Pulser Xcell (Bio-Rad, Hercules, CA) at 300 V and 250μF. Immediately after electroporation, cells were returned to culture medium and incubated for 12-16 h at

37°C and 5.0% CO2.

Enzyme-Linked ImmunoSpot (ELISPOT) and Enzyme-Linked ImmunoSorbent Assay (ELISA) assay ELISPOT assay to detect interferon (IFN)-γ secreting T cells, was performed using Human IFN-γ ELISpotPRO kit (MABTECH, Cincinnati, OH) according to the manufacturer’s instruction. Briefly, antigen presenting cells were pulsed with respective peptides at 37°C for 20 h and 5% CO2. T cells were pre-treated with IL-2 (35 U/mL) for 16 h and then cocultured with the peptide-pulsed antigen presenting cells (2 × 104 cells/well) at 37°C for 20 h in 96-well plate. Spots were captured and analyzed by an automated ELISPOT reader, ImmunoSPOT S4 (Cellular Technology Ltd, Shaker Heights, OH) and the ImmunoSpot Professional Software package, Version 5.1 (Cellular Technology Ltd). To measure the secreted cytokine levels in the supernatant, we used OptEIA Human IFN-γ ELISA set (BD Biosciences), OptEIA Human IL2 ELISA set (BD Biosciences), OptEIA Human TNF ELISA set (BD Biosciences) and Human IL-4 ELISA development kit (MABTECH). Briefly, antigen presenting cells were pulsed

with respective peptides at 37°C for 20 h and 5% CO2. T cells were pre-treated with IL- 2 (35 U/mL) for 16 h and then co-cultured with the peptide-pulsed antigen presenting

cells (2 × 104 cells/well) at 37°C for 20 h in 96-well plate. The supernatant was transferred into a new 96-well plate and each protein concentration was measured according to the manufacturer’s instruction.

Cytotoxic assay Cytotoxic assay was performed using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI) according to the manufacturer’s instruction. Briefly, C1R-A02 cells were pulsed with respective peptides (1 µM) at 37°C for 20 h and used as target cells. Effector cells and target cells were incubated in 96- well plate at 5:1, 10:1, 20:1 and 50:1 ratios for 4 h at 37°C, 5% CO2. Experiments were conducted in triplicate. Maximum lactate dehydrogenase (LDH) release from target cells was measured by the addition of lysis solution. The spontaneous LDH release of effector and target cells was measured by separate incubation of the respective population. After 4-hour incubation, plate was centrifuged at 250 × g for 4 min. Supernatant was transferred to a new 96-well plate. The substrate (CytoTox 96 nonradioactive cytotoxicity assay kit; Promega) was added to each well, and the plate was incubated for 30 min in the dark at room temperature. Stop solution was added to terminate the reaction and absorbance at 490 nm was recorded. The percentage of cytotoxic activity was calculated according to the following formula: % Cytotoxicity = [(Experimental-Effector Spontaneous-Target Spontaneous) / (Target Maximum-Target Spontaneous)] × 100.

Statistical analysis The student’s t test was performed for comparison of the percentage of cytotoxic activity between C1R-A02 pulsed with mutant and corresponding wild-type peptides. Statistical analyses were done using GraphPad Prism version 6.0 (GraphPad software, La Jolla, CA). P value of <0.05 was considered to be statistically significant.

RESULTS Whole-exome sequencing and neoantigen prediction Through whole-exome sequencing of genomic DNAs from paraffin-embedded ovarian cancer tissues and corresponding normal cells of 7 ovarian cancer patients, we identified a total of 463 non-synonymous mutations (35-97 non-synonymous mutations in individual patients, Table 1, Supplementary Table 1). We then predicted the binding affinity of peptides including amino-acid substitutions to individual HLA-A molecules that were estimated from the whole-exome sequence data of normal DNAs and obtained

neoantigen candidate epitope sequences which showed IC50 of 500 nM or lower (Table 1, Supplementary Table 2). From these candidate peptides, we focused on neoantigen

candidates which showed IC50 of <50 nM and further filtered potential peptides by high expression levels in the TCGA transcriptome data (median reads per kilobase of exon per million mapped reads (RPKM) >100) (28). We selected 14 neoantigen candidates (1-3 for each tumor sample, Table 2) to examine their ability to induce neoantigen- specific T cells using PBMCs isolated from healthy donors.

Induction of neoantigen-specific T cells using HLA-matched healthy donor blood Among the 14 peptides we synthesized, eleven neoantigens were expected to be recognized by HLA-A2-restricted CTL and the remaining three were by HLA-A24- restricted CTL. We obtained PBMCs from two healthy donors, one having HLA- A*02:01 and the other having HLA-A*24:02. After 11 days of co-culturing CD8+ T cells with autologous DCs with/without each neoantigen peptide, we searched for CD8+ T cells binding to each peptide-HLA-dextramer complex by flow cytometry. We detected CD8+HLA-dextramer+ T cells for three neoantigens were recognized by HLA-A2- restricted CTL (Fig. 1A). The proportions of CD8+HLA-dextramer+ T cells were

0.088% (1348 cells) for RFC5K160N, 0.0097% (248 cells) for BRAPR543C and 0.20%

(300 cells) for GINS1I87V.

TCR sequencing of sorted CD8+ HLA-dextramer+ T cells We extracted total RNAs from sorted CD8+HLA-dextramer+ T cells for three peptides and performed TCRα and β sequencing using our previously published method (30) (Fig. 1B). We observed dominant TCRA and TCRB sequences that accounted for >50 % in all three cases (Supplementary Table 3). Although the total cell numbers were very small, the dominant TCR frequencies of 50% or higher may imply that these T cells were likely to be expanded by neoantigen stimulation. We speculated that T cells with these dominant TCRA and TCRB pairs were likely to be neoantigen-specific and generated TCR-engineered T cells with these TCRA and TCRB-paired sequences for further functional analysis. Low-frequency TCR sequences might derive from impurities arising from sorting a low frequency population. It is also possible that low-frequency TCR sequences may also be neoantigen-specific. However, in this study, we focused on the validation of the most dominant TCR clones from a very small number of sorted T cells.

TCR-engineered T cells recognized the neoantigens

We constructed the retroviral vectors encoding for the RFC5K160N-specific

TCR, BRAPR543C-specific TCR, GINS1I87V-specific TCR cDNAs, and transduced each of them into PBMCs from a healthy donor. Subsequently we examined whether these TCR-engineered T cells bound to the HLA-dextramer loaded with the mutant or wild-

type peptide. As shown in Figure 2, RFC5K160N and BRAPR543CTCR-engineered T cells bound to the HLA-dextramer with the mutant peptide, but not to that with the wild-type

peptide. On the other hand, GINS1I87V TCR-engineered T cells bound to both mutant and wild-type peptide-HLA dextramers (Fig. 2). As predicted from results of the HLA-dextramer-binding assay, ELISPOT

assay showed IFN-γ secretion in RFC5K160N and BRAPR543C TCR-engineered T cells in a mutant-specific and peptide-dose-dependent manner using C1R-A02 cells with HLA- A2 (HLA-A*02:01) as antigen presenting cells (APCs) (Fig. 3A), but IFN-γ secretion was observed in both mutant-peptide- and wild-type-peptide-pulsed C1R-A02 cells

when we used GINS1I87V TCR-engineered T cells. Furthermore, CD137 upregulation

(Fig. 3B) was observed in RFC5K160N and BRAPR543C TCR-engineered T cells upon co- culture with C1R-A02 cells loaded with graded amounts of the mutant peptide. On the other hand, CD137 upregulation was not observed when the engineered T cells were co- cultured with C1R-A02 cells loaded with physiologically relevant amounts of the corresponding wild-type peptide. However, BRAPR543C TCR-engineered T cells responded to 10-5M of the wild-type peptide. As expected, CD137 upregulation in

GINS1I87V TCR-engineered T cells was observed when C1R-A02 cells were pulsed with both wild-type and mutant peptides. We also quantified the levels of several cytokines (IFNγ, TNF alpha, IL-2 and IL-4) with an ELISA assay and all results are comparable with the IFNγ ELISPOT and CD137 assays (Fig. 3C and Supplementary Figure 1). To further validate these TCR-engineered T cells, we explored peptide- dependent cytotoxic activity of T cells against C1R-A02 cells loaded with either a

mutant or a corresponding wild-type peptide. RFC5K160N TCR-engineered T cells revealed cytotoxic activity exclusively against mutant-peptide-pulsed C1R-A02 cells

(Fig. 4A). However, BRAPR543C TCR-engineered T cells showed some levels of cytotoxicity against wild-type-peptide-loaded C1R2-A02 cells when the ratios of engineered T cells/C1R-A02 cells were very high. As we expected from other functional

analysis, GINS1I87V TCR-engineered T cells also showed cytotoxicity to C1R-A02 cells pulsed with the mutant peptide as well as the wild-type peptide. To verify that the mutant epitopes are endogenously processed and presented on the cell with MHC molecules, we transfected each of the plasmid constructs designed to express a part of RFC5 and BRAP proteins including the amino-acid

substitution into C1R-A02 cells. When RFC5K160N and BRAPR543C TCR-engineered T

cells were mixed with these transfected C1R-A02 cells, we observed high levels of IFNγ secretion, indicating that the mutant epitope was expectedly processed and presented on the cell surface (Fig. 4B).

HLA-restricted activity of TCR-engineered T cells Finally, we examined HLA-restricted activity of three TCR-engineered T cells. We used C1R-A24 cells that express HLA-A24 (HLA-A*24:02) as APCs and evaluated IFN-γ secretion levels. As shown in Supplementary Figure 2, IFN-γ secretion was almost exclusively observed when these three TCR-engineered T cells were co-cultured with C1R-A02 cells pulsed with mutant peptides, and not when these three TCR- engineered T cells were co-cultured with C1R-A24 cells pulsed with mutant peptides or with both cell lines without peptides, clearly indicating that the mutant peptides were recognized by TCRs through the presentation on HLA-A02 molecules.

DISCUSSION In this study, we validated our previously-developed time-efficient protocol for identification of specific TCRs against neoantigens which were identified from whole- exome sequence results of 7 clinical ovarian cancers. We stimulated CD8+ cells derived from HLA-matched healthy donors with 14 candidate neoantigen peptides and obtained T cells reacting with the peptide-HLA complex for three neoantigen peptides. We sequenced TCRs of these sorted cells and identified possible TCR α and β pairs. We generated three neoantigen-specific TCR-engineered T cells and confirmed their reactivity to the respective neoantigens although one of them showed cross-reactivity to the wild-type peptide. Strønen et al. initially reported the successful application of healthy donor- derived T cells for targeting tumor-specific neoantigens (34). They selected 57 neoantigen candidates from three melanoma patients and succeeded in inducing neoantigen-specific T cells for 11 of the 57 peptides. They concluded that T cells obtained from healthy donors have a broad T cell repertoire, which could increase a chance of inducing neoantigen-specific T cells. The use of HLA-matched healthy-donor T cells may be effective because advanced cancer patients often suffer from myelosuppression caused by multiple regimens of chemotherapy and have a smaller T cell diversity than healthy donors. Moreover, we intended to shorten the process of isolating neoantigen-specific T cells; this is critically important since advanced cancer patients are usually unable to wait a long period to have access to new treatments. Our

protocol requires only two weeks from the priming of T cells with neoantigens to the identification of neoantigen-specific TCRs (16). We previously reported that our next- generation sequencer-based TCR repertoire analysis is able to accurately identify V- (D)-J combinations including CDR3 sequences in various types of tumor samples (26,29,30,35-41). In this study, our TCR repertoire analysis facilitated the rapid identification of neoantigen-specific TCRs and allowed cloning of TCRαβ pairs and subsequent production of TCR-engineered T cells. Most of the neoantigen-targeting studies have been performed using melanoma samples, because melanoma has the highest somatic mutation burden among human cancers and hundreds of neoantigen candidates can be identified for each patient (34,42- 46). Only few studies have been performed in cancer types with a relatively lower number of somatic mutations, although a large body of evidence has suggested the importance of neoantigens. We selected ovarian cancer as a pilot study to validate our protocol because ovarian cancer is one of the common female cancers and harbors a number of somatic mutations that is average (1 mutation/Mb compared to 12 mutations/Mb for melanoma) among various cancer types (1,45). In addition, the presence of neoantigen-reactive T cell subtypes in TILs from ovarian cancer patients has been reported (20). In this study, we successfully identified 3 neoantigen-specific TCRαβ pairs from 14 predicted neoantigen candidates. Although our sample size was small, the success rate of obtaining neoantigen-specific T cells was similar to that in previous report for melanoma (34). To exclude the risk of severe autoimmune adverse events, the neoantigen- specific TCR should recognize the mutant peptide (neoantigen peptide) exclusively, and not the corresponding wild-type epitope peptide. Our findings of cross-reactivity in

GINS1I87V TCR-engineered T cells as well as BRAPR543C TCR-engineered T cells indicate the importance of careful validation for the specificity of TCRs against neoantigens. The neoantigens can be distinguished from corresponding wild-type peptides by TCR either when the mutation changes the motif (structure) recognized by TCR or when the mutation changes an amino acid at the HLA anchor position and significantly enhances the affinity to HLA molecules (47). The anchor positions for HLA-A2 (HLA-A*02:01) are usually at the position-2 (P2) and P9. Both the wild-type and mutant GINS1 peptide (I87V is located at the P9, Table 2) are predicted to have similar and high affinity to the HLA molecule. Probably these two peptides bound equally to HLA-A2 and did not influence the recognition by TCR. We have previously observed cross-reactivity to wild and mutant peptides in a case of a different amino acid substitution in the anchor position by another TCR-engineered T cell clone (the affinity

to HLA-A was also similar between mutant and wild-type peptides) (16). On the other hand, amino acid substitutions in RFC5 and BRAP proteins occurred in P4 and P3 positions of the epitope peptides, respectively. Since these P3 and P4 positions are

critical positions for recognition by TCR, RFC5K160N- and BRAPR543C-specific TCR engineered T cells revealed recognition specifically to the mutated peptides, but not to the wild-type peptides. These results suggest we should consider the position of the amino substitution in neoantigen peptides. When the amino acid change occurs at the anchor position, and the affinity of the wild-type- and mutant- peptides to HLA molecules are predicted to be similar, these neoantigen peptides might not be a feasible target. Furthermore, we have to be cautious about TCR-engineered T cells like

BRAPR543C TCR-engineered T cells. Under the very high peptide concentration (probably non-physiological condition) of the wild-type peptide, we observed CD137

upregulation in BRAPR543C TCR-engineered T cells. Since the affinities of mutant- and wild-type-peptides to HLA-A2 were predicted to be 5 nM and 25 nM, respectively, it is very likely that the wild-type peptide still binds very effectively. It is also possible that

BRAPR543C TCR may have a weak affinity for the wild-type peptide sequence. Hence, under the conditions of a higher ratio of TCR-engineered T cells/APCs with high

concentration of the wild-type peptide (Fig. 4A), BRAPR543C TCR-engineered T cells could show the some level of cytotoxicity to C1R-A02 cells with the wild-type peptide. Although the high level of antigen presentation and the high ratio of effector/target cells used in this study are not physiologic, we have to be extremely careful to the safety of the TCR-engineered T cells in a clinical setting. If somatic mutations are observed in a part of tumor tissues, a subset of tumors may not be targeted by TCR-engineered T cells. Hence, it would be ideal to identify and target neoantigens that are commonly presented in all the tumor cells. The future clinical application of this approach might need to collect multiple biopsies from a patient’s tumor(s) to identify such trunk mutations. However, we think that it is practically very difficult to obtain the multiple portions of an original tumor and metastatic regions from advanced cancer patients because of the risk of complications during the biopsy processes. Although we focused MHC Class I restricted neoantigens in this study, our strategy can be applied to MHC Class II restricted neoantigens. The binding affinities of each peptide to MHC Class II molecules can be examined (48-50). Recently the adoptive CD4+ T cell therapy using an MHC class II–restricted TCR that recognized MAGE-A3 (cancer germline antigen) was investigated in a clinical trial (51). We would also like to identify MHC Class II-restricted neoantigen specific TCRs and apply them

to adoptive TCR engineered CD4+ T cell therapy. In conclusion, for the data using 7 ovarian cancer cases, we successfully induced 3 neoantigen-specific T cells and identified their TCR sequences within two weeks (from peptide stimulation to TCR identification). This pilot study supports the feasibility of our time-efficient protocol for the use in solid cancers with relatively lower mutational loads such as ovarian cancer. In addition, we suggest some considerations for the selection of neoantigen candidates as well as the specificity of TCR against the mutant peptide versus the wild-type-peptide.

ACKNOWLEDGMENTS We appreciate Drs. Rui Yamaguchi, Seiya Imoto and Satoru Miyano at the University of Tokyo for developing the algorithm of TCR repertoire analysis and helpful support in data management. The super-computing resource (http://sc.hgc.jp/shirokane.html) was provided by Human Genome Center, the Institute of Medical Science, the University of Tokyo. We also thank Kimberley Borutta for excellent technical support.

This work was supported in part by a Team Science Award of UCCCC (The University of Chicago Medicine Comprehensive Cancer Center), Einstein Foundation Berlin and by a research grant from OncoTherapy Science Inc.

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MAGE-A3. J Clin Oncol 2017;35:3322-9.

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clincancerres.aacrjournals.org Table 1. Summary of patients' predicted neoantigens

Number of predicted neoantigens nonsynonymous affinity affinity Patient ≦10nM ≦50nM ≦100nM ≦500nM ≦10nM ≦50nM ≦100nM ≦500nM mutation (IC50) (IC50) 12066 97 A03:01 0 3 12 38 A24:02 1 10 16 23 12183 70 A02:01 3 6 13 40 A02:01 homozygous on September 25, 2021. © 2018American Association for Cancer 12231 60 A02:01 1 8 8 22 A29:02 0 2 4 9 Research. 12475 35 A01:01 0 0 0 1 A02:01 0 4 5 17 12705 41 A02:01 1 6 9 24 A68:02 3 10 22 58 12832 82 A02:01 2 14 20 48 A31:01 2 26 55 119 12912 78 A01:01 0 0 1 4 A02:01 2 12 22 56

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clincancerres.aacrjournals.org Table 2. List of tested neoantigen peptide

Mutated Peptide Wild-type Peptide Affinity Affinity Amino acid Peptide Median Patient Gene Sequence (IC , Sequence (IC , HLA-A substitution length 50 50 RPKM nM) nM) 12066 FGD2 F164Y 9 IYQFHSQYF 10 IYQFHSQFF 8 HLA-A24:02 178.2 XK P349H 10 IYMYVCAHLL 13 IYMYVCAPLL 18 HLA-A24:02 141.6 BRAF L711F 9 LFPQIFASI 43 LFPQILASI 156 HLA-A24:02 232.7 on September 25, 2021. © 2018American Association for Cancer

Research. 12183 BRAP R534C 9 QLCDVMFYL 5 QLRDVMFYL 25 HLA-A02:01 596.6 ARFGEF2 D809Y 10 YLPEEYLSSI 10 DLPEEYLSSI 2338 HLA-A02:01 1314 12231 BSCL2 T423M 9 LLMEANLPA 5 LLTEANLPA 31 HLA-A02:01 2335.3 EHMT1 A275V 10 YMATTKSQTV 28 YMATTKSQTA 287 HLA-A02:01 1375.3 12475 TYMS A231T 10 TLLTYMIAHI 20 ALLTYMIAHI 16 HLA-A02:01 852.3 12705 MAP1S S569F 10 STFHSGFPPV 34 STSHSGFPPV 224 HLA-A02:01 2349.7 12832 GINS1 I87V 9 YLYDRLLRV 3 YLYDRLLRI 5 HLA-A02:01 461.4 RFC5 K160N 9 YLSNIIPAL 4 YLSKIIPAL 4 HLA-A02:01 565.6 BAP1 E20K 9 TLLVKDFGV 23 TLLVEDFGV 21 HLA-A02:01 1914.2 12912 ATP13A3 R54P 10 LLYWMPEWPV 5 LLYWMPEWRV 8 HLA-A02:01 2375.7 GFOD2 G11V 10 KMLPGVGVFV 9 KMLPGVGVFG 1465 HLA-A02:01 402.1 RPKM: reads per kilobase of exon per million mapped reads RPKM data was obtained from RNA sequencing data of The Cancer Genome Atlas (TCGA)28 ovarian serous cystadenocarcinoma

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Figure Legends Fig. 1 Induction of neoantigen-specific T cells and identification of TCRαβ sequences of sorted CD8+Dextramer+ T cells (A) Peptide-HLA dextramer assay for CD8+ T cells co-cultured with autologous DCs

with/without mutant peptides (RFC5K160N, BRAPR543C and GINS1I87V). Each figure shows the record of first 10,000 cells at the time of cell sorting. (B) Each pie chart illustrates the frequency of unique TCRA and TCRB sequences of sorted CD8+Dextramer+ T cells.

Fig. 2 Peptide-HLA dextramer staining for TCR-engineered T cells. Flow cytometric analysis of dextramer loaded with wild-type or mutant peptide on,

RFC5K160N (top panels), BRAPR543C (middle panels) and GINS1I87V (bottom panels) TCR- engineered T cells. The expression of engineered TCRs was confirmed using anti-mouse TCR beta antibody.

Fig. 3 Functional assay of TCR-engineered T cells (A) IFN-γ ELISPOT assay on TCR-engineered T cells co-cultured with C1R-A02 cells loaded with graded amounts of mutant or wild-type peptide. (B) CD137 staining on TCR-engineered T cells co-cultured with C1R-A02 cells loaded with graded amounts of mutant or wild-type peptide. (C) IFN-γ ELISA assay on TCR-engineered T cells co-cultured with C1R-A02 cells loaded with graded amounts of the mutant or wild-type peptide.

Fig. 4 Cytotoxic activity of TCR engineered T cells and TCR-engineered T cells can recognize the endogenously processed mutated peptide on C1R-A02 cells.

(A) RFC5K160N and BRAPR543C TCR-engineered T cells exerted significantly higher cytotoxic activity against mutant-peptide-pulsed C1R-A02 cells than wild-type-peptide-pulsed ones.

GINS1I87V TCR-engineered T cells led to cytotoxicity of C1R-A02 cells pulsed with that mutant peptide as well as the wild-type peptide. Four different ratios (5:1, 10:1, 20:1 and 50:1) of effector (TCR-engineered T cells) to target cells (E:T ratio) were tested. The figure between brackets indicates the CD8+engineered TCR+ T cells to target cells ratio. The proportion of the CD8+engineered TCR+ T cells was calculated based on the percentage of CD8+ and mouse TCR beta+ cells (shown in Figure 2). The asterisks indicate the statistically significant difference (p < 0.05) between C1R-A02 pulsed with mutant and corresponding

wild-type peptides. (B) IFN-γ ELISA assay on TCR-engineered T cells co-cultured with C1R-A02 cells transfected with/without plasmids designed to express corresponding mutated peptides. The asterisks indicate the statistically significant difference (p < 0.05) between two groups.

Induction of Neoantigen-specific Cytotoxic T Cells and Construction of T-cell Receptor-engineered T cells for Ovarian Cancer

Tatsuo Matsuda, Matthias Leisegang, Jae-Hyun Park, et al.

Clin Cancer Res Published OnlineFirst May 2, 2018.

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