PRAME and CTCFL-reactive TCRs for the treatment of ovarian cancer

Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic ‘cold’ ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2′-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.


Dissociation primary OVCA patient samples
Solid OVCA patient samples were sliced into small pieces and dead, clotted or non-tumor material was removed. The small tumor-pieces were added to a C-tube (Miltenyi Biotec) with ice cold buffer without detergent and cOmplete Protease Inhibitor (Sigma-Aldrich), to prevent protein degradation. Using a gentleMACS (Miltenyi Biotec) procedure small tumor-pieces were dissociated until an almost homogenous cell solution. Benzonase (Merck) was added in a concentration of 125 IU/mL to remove DNA/RNA complexes during lysis.

Gene expression by Quantitative Polymerase Chain Reaction
Gene expression was quantified by Quantitative Polymerase Chain Reaction (qPCR). Total RNA was isolated using the RNAqueous-Micro Kit (Ambion) or ReliaPrep RNA Cell Miniprep System (Promega). First strand cDNA synthesis was performed with Moloney murine leukemia virus reverse transcriptase and Oligo (dT) primers (Invitrogen by Thermo Fisher Scientific). qPCR was performed using Fast Start TaqDNA Polymerase (Roche) and EvaGreen (Biotium), and gene expression was measured on the Lightcycler 480 (Roche). All samples and genes were run in triplicate with 10 ng cDNA per reaction. Expression was calculated as percentage relative to the average of housekeeping genes GUSB, VPS29 and PSMB4, which was set at 100%. The following primers were used: PRAME (forward: GTTGCTCAGGCACGTGAT, reverse:

HLA class I-peptide elution procedure, fractionation and mass spectrometry
Peptide elution was performed as outlined previously. 1,2 In short, the cell pellets were lysed and subjected to an immunoaffinity column to collect bound peptide-HLA complexes, with either an HLA class-I antibody (W6/32, ATCC) or an HLA-A*02:01 antibody (BB7.2, ATCC). To separate the peptides, bound peptide-HLA complexes were dissociated with 10% acetic acid and filtrated using a 10 kDa membrane. Eluted peptide pools were either fractionated by strong cation exchange chromatography (SCX) 1 or by high pH reversed phase fractionation (High pH-RP) 3 . SCX and high pH-RP peptide fractions were lyophilized, dissolved in 95/3/0.1 water/ acetonitrile/formic acid v/v/v and subsequently analysed by data-dependent MS/MS on either an LTQ FT Ultra equipped with a nanoflow liquid chromatography 1100 HPLC system (Agilent Technologies) or a Q Exactive mass spectrometer equipped with an easy-nLC 1000 (Thermo Fisher Scientific). Proteome Discoverer version 2.1 (Thermo Fisher Scientific) was used for peptide and protein identification, using the mascot search node for identification (mascot version 2.2.04) and the UniProt Homo Sapiens database (UP000005640; Jan 2015; 67,911 entries). All unique PRAME, CTCFL and CLDN6derived peptides with a length between 8 and 14 amino acids, preferably a minimal Best Mascot Ion (BMI) score of 20, a mass accuracy of 10 ppm and predicted to bind to a common HLA molecule according to the netMHC peptide binding algorithm 4 were selected as candidate for peptide synthesis and validation.

Peptide synthesis and pMHC-multimer production
In total 34 synthetic peptides were in-house synthesized using standard Fmoc chemistry. By mass spectrometry the tandem mass spectra of the eluted peptides were validated with synthetic peptides. In total 17 pMHC-multimer complexes were generated with minor modifications. 5 In short, monomers consisting of the selected HLA allele heavy chain, human beta-2 microglobulin (B2M) light chain and selected peptide were purified by gel-filtration high-performance liquid chromatography and biotinylated.

TCR identification and production of retroviral supernatants
TCR α and β chains of the selected T-cell clones were identified by sequencing with minor modifications, as previously described. 6 mRNA was isolated by the Dynabeads mRNA DIRECT Kit (Invitrogen) or total RNA was isolated by the ReliaPrep RNA cell Miniprep System (Promega). TCR cDNA was generated using TCR constant α and β primers, a SA.rt anchor template-switching oligonucleotide (TSO), and SMARTScribe Reverse Transcriptase (Takara, Clontech). 7 The TCR α and β products were generated in a first PCR using Phusion Flash (Thermo Fisher Scientific), followed by a second PCR that was used to include 2-sided barcode sequences for the different T-cell clones. Barcoded TCR PCR products were pooled and TCR sequences were identified by HiSeq or NovaSeq (GenomeScan). The Vα and Vβ families were determined of the NGS data using the MiXCR software and ImMunoGeneTics (IMGT) database. 8 The TCR α (VJ) and β