(B) Transduced T cells were co-cultured with autologous EBV-B cells pulsed with CTBP1Q277R 25-mer, 13-mer identified in (A), and three additional truncations of the latter (white dots). of the bladder is among the ten most common malignancies worldwide, with an estimated 81,190 new cases and 17,240 deaths per year in the United States (1). Although the early stage disease, which constitutes the majority of newly diagnosed cases, is usually curable with surgery, there have been no curative treatments for patients with metastases, whose 5-12 months overall survival remains around 15% (2). Muscle-invasive bladder cancer (MIBC) is managed with radical cystectomy with neoadjuvant cisplatin-based chemotherapy in selected patients. For patients with metastatic disease, systemic chemotherapy is the standard of care, with excellent but short-lived response rates (2, 3). In addition to these modalities, other therapies have been successfully used in treatment of bladder cancer. In high grade non-muscle-invasive bladder cancer (NMIBC), intravesical instillation of Bacillus CalmetteCGurin (BCG), an attenuated strain of expanded and transferred back into the patients (33C37). Although TILs could be successfully produced from bladder tumors in our previous study (38), no study to date has shown whether or not TILs from bladder tumors can recognize neoantigens. To explore this, we first generated polyclonal TIL cultures from five patients with primary bladder tumors and co-cultured them with autologous APCs presenting the products of cancer mutations. In this study, we describe the isolation and characterization of a neoantigen-reactive TIL populace from a patient with primary localized urothelial carcinoma of the bladder. Materials and Methods Patients Five patients with primary localized urothelial carcinoma of the bladder were evaluated and treated at the Urologic Oncology Branch at the National Malignancy Institute (NCI). All patients were enrolled on protocols approved by the NCI Institutional Review Board, and they had provided their written informed consent for this study. Tumor infiltrating lymphocytes Tumor samples were obtained via TURBT or bladder diverticulectomy. TILs were cultured from tumor fragments following a previously described approach (39). Briefly, tumor tissue was dissected free of hemorrhagic and necrotic areas and cut into approximately 11 mm fragments (N=12 or 24), which Dicarbine were then plated individually in 24-well plates and cultured in 2 mL of RPMI medium supplemented with 2 mM L-glutamine, 25 mM HEPES, 10 g/ml gentamicin (all from Life Technologies, Carlsbad, CA), 10% human AB serum and 6000 IU/ml of IL-2 (Prometheus, San Diego, CA) for 6C8 weeks. Medium was replenished twice weekly; the wells were split in 1:2 fashion when fully confluent and cryopreserved until further use. Whole exome sequencing Cancer-specific mutations were identified from tumor samples using whole exome sequencing (WES), as described previously (35). Briefly, genomic DNA was first extracted from tumors and matched normal blood using a Maxwell instrument (Promega, Madison, WI). Next, WES libraries were prepared from genomic DNA (3 g/sample) using SureSelectXT Target Enrichment System coupled with Human All Exon V4 target bait Rabbit Polyclonal to ERGI3 (Agilent Technologies, Santa Clara, CA). Libraries from Patient 2 (1st resection) and Patient 5 were prepared and sequenced on an Illumina HiSeq2000 sequencer (Axeq/Macrogen USA, Rockville, MD). Libraries from Patient 1, 3 and 4 were prepared and sequenced in-house on a NextSeq 500 desktop sequencer following the manufacturers instructions (Illumina, San Diego, CA). Sequencing reads were aligned to human genome build 19 using Novoalign MPI (http://www.novocraft.com/). Duplicates were marked using Picards MarkDuplicates tool; in/del realignment and base recalibration was carried out according to the GATK best practices workflow (https://www.broadinstitute.org/gatk/). After the data cleanup, pileup files were created using samtools mpileup (http://samtools.sourceforge.net). Somatic variants were called using Varscan2 (http://varscan.sourceforge.net) according to the following criteria: tumor and normal read counts of 10 or greater, variant allele frequency of 10% or greater, and tumor variant reads of 4 or more. Finally, variants were annotated using Annovar (http://annovar.openbioinformatics.org). Tumor-specific mutations for each patient are listed in Supplementary Table 1. Tandem minigene and peptide libraries TMGs were constructed as described previously (40). For non-synonymous point mutations, each mutated Dicarbine amino acid was flanked bilaterally by a sequence encoding 12 wild type (WT) amino acids to generate an individual minigene. For each frameshift mutation, a minigene was designed to contain preceding 12 WT amino acids followed by mutated amino acids in the new reading frame, which terminated at the new stop codon. Next, up to twelve minigenes were linked together to generate tandem minigenes, which were codon-optimized, synthesized and ligated into a pcDNA3.1 vector using an In-Fusion HD EcoDry Cloning Kit (Clontech/Takara, Mountain View, CA). TMG RNA was made by in vitro transcription using a HiScribe T7 Quick High Yield.Finally, variants were annotated using Annovar (http://annovar.openbioinformatics.org). year in the United States (1). Although the early stage disease, which constitutes the majority of newly diagnosed cases, is curable with surgery, there have been no curative treatments for patients with metastases, whose 5-year overall survival remains around 15% (2). Muscle-invasive bladder cancer (MIBC) is managed with radical cystectomy with neoadjuvant cisplatin-based chemotherapy in selected patients. For patients with metastatic disease, systemic chemotherapy is the standard of care, with excellent but short-lived response rates Dicarbine (2, 3). In addition to these modalities, other therapies have been successfully used in treatment of bladder cancer. In high grade non-muscle-invasive bladder cancer (NMIBC), intravesical instillation of Bacillus CalmetteCGurin (BCG), an attenuated strain of expanded and transferred back into the patients (33C37). Although TILs could be successfully grown from bladder tumors in our previous study (38), no study to date has shown whether or not TILs from bladder tumors can recognize neoantigens. To explore this, we first generated polyclonal TIL cultures from five patients with primary bladder tumors and co-cultured them with autologous APCs presenting the products of cancer mutations. In this study, we describe the isolation and characterization of a neoantigen-reactive TIL population from a patient with primary localized urothelial carcinoma of the bladder. Materials and Methods Patients Five patients with primary localized urothelial carcinoma of the bladder were evaluated and treated at the Urologic Oncology Branch at the National Cancer Institute (NCI). All patients were enrolled on protocols approved by the NCI Institutional Review Board, and they had provided their written informed consent for this study. Tumor infiltrating lymphocytes Tumor samples were obtained via TURBT or bladder diverticulectomy. TILs were cultured from tumor fragments following a previously described approach (39). Briefly, tumor tissue was dissected free of hemorrhagic and necrotic areas and cut into approximately 11 mm fragments (N=12 or 24), which were then plated individually in 24-well plates and cultured in 2 mL of RPMI medium supplemented with 2 mM L-glutamine, 25 mM HEPES, 10 g/ml gentamicin (all from Life Technologies, Carlsbad, CA), 10% human AB serum and 6000 IU/ml of IL-2 (Prometheus, San Diego, CA) for 6C8 weeks. Medium was replenished twice weekly; the wells were split in 1:2 fashion when fully confluent and cryopreserved until further use. Whole exome sequencing Cancer-specific mutations were identified from tumor samples using whole exome sequencing (WES), as described previously (35). Briefly, genomic DNA was first extracted from tumors and matched normal blood using a Maxwell instrument (Promega, Madison, WI). Next, WES libraries were prepared from genomic DNA (3 g/sample) using SureSelectXT Target Enrichment System coupled with Human All Exon V4 target bait (Agilent Technologies, Santa Clara, CA). Libraries from Patient 2 (1st resection) and Patient 5 were prepared and sequenced on an Illumina HiSeq2000 sequencer (Axeq/Macrogen USA, Rockville, MD). Libraries from Patient 1, 3 and 4 were prepared and sequenced in-house on a NextSeq 500 desktop sequencer following the manufacturers instructions (Illumina, San Diego, CA). Sequencing reads were aligned to human genome build 19 using Novoalign MPI (http://www.novocraft.com/). Duplicates were marked using Picards MarkDuplicates tool; in/del realignment and base recalibration was carried out according to the GATK best practices workflow (https://www.broadinstitute.org/gatk/). After the data cleanup, pileup files were created using samtools mpileup (http://samtools.sourceforge.net). Somatic variants were called using Varscan2 (http://varscan.sourceforge.net) according to the following criteria: tumor and normal read counts of 10 or greater, variant allele frequency of 10% or greater, and tumor variant reads of 4 or more. Finally, variants were annotated using Annovar (http://annovar.openbioinformatics.org). Tumor-specific mutations for each patient are listed in Supplementary Table 1. Tandem minigene and peptide libraries TMGs were constructed as described previously (40). For non-synonymous point mutations, each mutated amino acid was flanked bilaterally by a sequence encoding 12 wild type (WT) amino acids to generate an individual minigene. For each frameshift mutation, a minigene was designed to contain preceding 12 WT amino acids followed by mutated amino acids in the new reading frame, which terminated at the new stop codon. Next, up to twelve minigenes were linked together to generate tandem minigenes, which were codon-optimized, synthesized and ligated into a pcDNA3.1 vector using an In-Fusion HD EcoDry Cloning Kit (Clontech/Takara, Mountain View, CA). TMG RNA was made by in vitro transcription using a HiScribe T7 Quick High Yield RNA Synthesis Kit (New England BioLabs, Ipswich, MA). RNA samples were.