Identification of HLA-B*3502-restricted epitope of MCPyV

Tumor infiltrating lymphocytes (TIL) from a testicular metastasis of a 64-year-old man with advanced MCC (separate from featured case) were non-specifically expanded for 2 weeks with IL-2 and IL-15 cytokine support and screened against peptide pools (13 mers overlapping by 9) tiling across the entirety of expressed portion of MCPyV T antigens by Elispot20. Confirmation of reactivity to the small T antigen pool (containing peptides spanning the C-terminal domain of MCPyV small T antigen), identification of minimal epitope as MCPyV-sT 83-91 (Supplementary Fig. 1), and restriction to HLA-B*3502 was determined by interferon-gamma intracellular cytokine stain, using autologous irradiated PBMC pulsed with peptide at final concentration of 1 microgram per milliliter as antigen presenting cells. This peptide was then confirmed to also be held by HLA-B*3501 and HLA-B*3503 alleles, which are highly similar to HLA-B*3502. For sequence comparison analyses, all Merkel cell polyomavirus small T antigen protein sequences available in NCBI were downloaded on August 21, 2017 in FASTA format and aligned using MUSCLE37.

Clinical protocols

All clinical investigations were performed in compliance with the Declaration of Helsinki principles. The first treated patient (primary focus of manuscript, 2586-4) was enrolled on protocols #2586/NCT01758458 (T cell infusion treatment), #6585 and #1765 (biological sample collection) at the Fred Hutchinson Cancer Research Center (FHCRC, Seattle, WA), all research activities were approved by the FHCRC Institutional Review Board and the Food and Drug Administration. The patient provided written informed consent. The second, “validation” patient was enrolled on protocol #9245/NCT0258482 as well as the sample collection protocols as above (9245-3). The third patient from whom the HLA-B*3502-restricted epitope was identified was enrolled on protocol #6585 (biological sample collection) and also provided written informed consent.

The primary patient (2586-4) received hypofractionated radiation for HLA upregulation to some but not all disease sites: 21 Gy in 6 fractions to pelvic nodes, 8 Gy in 1 fraction to retroperitoneum (previously heavily irradiated and recurrent post radiation), 8 Gy in 1 fraction to one of the thigh tumors. Other sites of disease, including orbit, mediastinum (bulky), bladder, lumbar spine, and other lower extremity tumors were not irradiated. Twenty-four hours thereafter the first of his two infusions of HLA-B3502 restricted CD8+ T cells targeting Merkel cell polyomavirus was administered. The second infusion was 33 days after the first. Cell dose was at 10 billion cells per m2 for a total dose of 26 billion cells. After initial progression, pembrolizumab followed by ipilimumab were added. Please see Fig. 1 for schematic of infusions. Tumor volumes were determined by mWHO criteria38.

The second validation patient (9245-3) is a 59-year-old man with metastatic MCC that had initially presented as stage IIIB disease, now metastatic at multiple sites. He had developed multiple relapses that had previously been treated with radiation; trial interventions were first systemic therapy. He received avelumab (anti-PD-L1) 10 mg/kg every two weeks15, and four infusions of MCPyV-specific T cells at dosages ranging from 0.8-3.9 billion (dose target of 1010 cells per m2 (23.5 billion) was not met for technical reasons). Two HLA-upregulation interventions were performed as detailed in Supplementary Fig. 10 including injection of intralesional interferon-beta 0.1 mg once into a single tumor lesion prior to first cycle of infusions, and irradiation of a single pelvic lymph node with an 8 Gy radiation fraction prior to second infusion. Cells were of two specificities: HLA-B35/FPW, and HLA-A0201 recognizing the “KLL” epitope39, however only HLA-A0201 cells persisted.

Generation of tetramer

Allophycocyanin-conjugated MCPyV-specific antigen pMHC multimers (FHCRC Immune Montoring Core Facility) were used to confirm purity of the cellular product as well as detect transferred CTL in PBMCs, with a staining sensitivity of 0.05% of total CD8+ T cells19.

Isolation and expansion of MCC-specific CTLs

For the primary patient described (2586-4), peripheral blood mononuclear cells (PBMCs) were collected by leukapheresis, and all ensuing ex vivo manipulations were performed in the clinical Good Manufacturing Practices Cell Processing Facility at the FHCRC. Patient PBMCs were collected by leukapheresis and depleted of CD25+ T cells to eliminate regulatory T cells31. Cells were cultured with cytokines (IL-2, IL-7, IL-15 and IL-21) stimulated twice for 7–10 day cycles with autologous DCs pulsed with the HLA B*3502-restricted Merkel cell polyomavirus (MCPyV) MCPyV-sT 83-91 epitope peptide FPWEEYGTL21,23. Cultures containing >5% specific CD8+ cells (as assessed by tetramer binding) were GMP flow-sorted and then expanded to sufficient numbers for infusion using two cycles of the Rapid Expansion Protocol21,23,40. Cell products bound the MCPyV sT-Ag epitope MCPyV-sT 83-91 peptide-HLA tetramer and secreted IFN-gamma when exposed to the cognate antigen (Supplementary Fig. 2).

For the validation patient (9245-3), identical methods were used, with the exception that a second culture was also performed using the MCPyV-T antigen (sT 15-23 and LT 15-23 ) peptide KLLEIAPNC as stimulus. This is restricted to HLA A*0201, as previously described39. Cells of both specificities were mixed together immediately prior to infusion in a 1:1 ratio.

Flow cytometry on patient peripheral blood samples

Blood samples were collected at the indicated time points, PBMCs isolated through the research cell bank at FHCRC by standard Ficoll-Hypaque gradient, and viably cryopreserved. Cells were analyzed by flow cytometry after permeabilization, fixation and staining with fluorochrome-conjugated monoclonal antibodies to CD4 (SK3; Becton Dickinson), CD8 (3B5; Invitrogen), CD19 (H1B19; Becton Dickinson), CD16 (3G8; Becton Dickinson), CCR7 (G043H7; Biolegend), CD45RO (UCHL1; Becton Dickinson), CD28 (CD28.2; Biolegend), KLRG1 (SA231A2; Biolegend), CD27 (L128; Biolegend), CXCR3 (G025H7; Biolegend), CD127 (A019D5; Biolegend), PD1 (ED12.2H7; Biolegend), Lag3 (2DS223H; eBioscience), 4-1BB (4B4-1; Biolegend), BCL2 (100; Biolegend), CTLA-4 (L3D10) and the above described tetramers. Cells were analyzed on a Fortessa cytometer (Becton Dickinson) and data analysis performed with FlowJo. Staining, acquisition and analyses were performed on all samples in a batch on same day and negative controls included.

Epitope spreading assessments

For the analyses of epitope spreading in Supplementary Fig. 5, PBMCs were cultured for 72 h with MCPyV peptide pools (13 mers overlapping by 9)20, and responses detected with interferon gamma intracellular cytokine stain.

TCR beta CDR3 sequencing

DNA was extracted from tetramer-sorted infusion product and unsorted PBMCs using a Qiagen DNEasy Blood and Tissue kit per manufacturers instruction. These samples and tumor biopsies were submitted to Adaptive Biotechnologies (Seattle, WA) for DNA amplification and sequencing of TCRB CDR3 using the immunoSEQ platform41,42.

Immunohistochemistry

Formalin-fixed paraffin-embedded tissues were sectioned and baked per standard protocol.

MCPyV large T antigen was detected using the CM2B4 antibody (Santa Cruz, dilution 1 :50) and HLA-ABC using clone EMR8-5 (MBL, dilution 1 :1500 for slides from discovery patient and dilution 1 :8000 for slides from validation patient; antibody lots titered on normal tissues with endothelial cells serving as on-slide positive control)22,43. For multiplex immunohistochemistry, slides were dewaxed and stained on a Leica Bond Rx stainer using Leica Dewax solution, antigen retrieval, and antibody stripping and rinsing after each step (bond wash solution). A high stringency wash was performed after the secondary and tertiary applications using high-salt TBST solution (0.05 M Tris, 0.3 M NaCl, and 0.1% Tween-20, pH 7.2–7.6). OPAL Polymer HRP Mouse plus Rabbit (PerkinElmer, Hopkington, MA) was used for all secondary applications. Antigen retrieval and antibody stripping steps were performed at 100 °C with all other steps at ambient temperature. Endogenous peroxidase was blocked with 3% H 2 O 2 for 8 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 + /- 0.1) for 30 min. The first primary antibody (position 1) was applied for 60 min followed by the secondary antibody application for 10 min and the application of the tertiary amplification reagent (PerkinElmer OPAL fluor) for 10 min. The primary and secondary antibodies were stripped with retrieval solution for 20 min before repeating the process with the second primary antibody (position 2) starting with a new application of 3% H 2 O 2 . Antibodies included CD8 (clone 144B; Dako; opal fluor 520, concentration 0.2 microgram/mL), CD56 (clone 123c3.d5; Bio SB; opal fluor 540, 1 ug/mL), CD3 (clone SP7; Thermo, opal fluor 650, concentration 1 :400), and HLA-DR (clone EP96; Bio SB; opal fluor 690; concentration 0.125 ug/mL). Slides were removed from the stainer and stained with Spectral DAPI (Perkin Elmer) for 5 min, rinsed for 5 min, and coverslipped with Prolong Gold Antifade reagent (Invitrogen/Life Technologies, Grand Island, NY). Slides were cured for 24 h at room temperature, then representative images from each slide were acquired on PerkinElmer Vectra 3.0 Automated Imaging System. Images were spectrally unmixed using PerkinElmer inForm software and exported as multi-image TIFF’s for analysis. Quantitative image analysis was performed in HALO software (Indica Labs, Corrales, NM). For each slide, three representative sections were scored. CD56 staining readily identified tumor. Layers were manually drawn for intratumoral (within tumor borders) and peritumoral (edge of tumor to 100 micron beyond tumor border) regions. Three sections were counted for each slide and comparisons between time points were made using the Student’s t test.

Exome sequencing

For both patients 2586-4 (discovery) and 9245-3 (validation), DNA was isolated from PBMC as germline control, and from tumor time point pre-immunotherapy and at acquired resistance as tumor specimens (DNA from frozen tumor specimens), using a Qiagen QIAAmp DNA extraction kit under manufacturer’s recommended conditions. Quality was confirmed with spectrophotometry and Qubit (Fisher Biosciences). For patient 2586-4 samples were submitted for exome sequencing through the FHCRC Genomics Core. Library prep was performed with standard procedures using Agilent SureSelect Human All Exon V6 and sequencing performed on an illumina HiSeq 2500 per Agilent recommendations. Samples were aligned to hg19 reference using annovar44 and somatic mutations determined using MuTect45. For patient 9245-3, samples were submitted for exome sequencing through the University of Washington Northwest Clinical Genomics Laboratory research sequencing service. Library preparation was with xGen technology and sequencing on an illumina HiSeq 4000 at 100x coverage. Samples were aligned to hg19 with BWA46 and variants identified with GATK47 as per the NCGL standard pipeline.

Generation of single cell tumor digests

Tumor material not required for clinical pathology analysis was placed in RPMI. Tumor was mechanically minced with scissors and forceps into small pieces and then resuspended in 20 mL freshly prepared digestion medium (20 mL RPMI plus 0.002 g DNAse (Worthington Biochemical) plus 0.008 g collagenase (Worthington Biochemical) plus 0.002 g hyaluronidase (Worthington Biochemical) in a 10 cm dish. Digesting tumor was incubated in 37 degree tissue culture incubator for 3 h with occasional manual rocking. After incubation, tumor digest was strained through a 70 micron cell strainer, cells were centrifuged, counted, and assessed for single cellularity and viability, and resuspended in freeze medium (50% human serum, 45% RPMI, 5% DMSO). Cells were frozen overnight at -80C in a styrofoam sandwich then transferred to liquid nitrogen for long-term storage.

Single cell RNA sequencing (scRNAseq)

For samples from patient 2586-4, cells were thawed, washed, and labeled in a single cell fashion using the 10x genomics 3’ Chromium v2.0 platform24 as per manufacturer’s instructions. Library preparation was performed as per manufacturer’s protocol with no modifications. Library quality was confirmed with illumina TapeStation high sensitivity (evaluates library size), qubit (evaluates dsDNA quantity), and KAPA qPCR analysis (KAPA Biosystems, evaluates quantity of amplifiable transcript). Samples were mixed in equimolar fashion and sequenced on an illumina hiSeq 2500 “rapid run” mode according to standard 10x genomics protocol.

For samples from patient 9245-3, scRNAseq was performed using the 10x Genomics 5’ V(D)J and gene expression chromium platform, with cell washing, barcoding, and library preparation as per manufactures instruction. Library quality was confirmed as above. Samples were sequenced on an Illumina NovaSeq 6000 (gene expression) and HiSeq 4000 (V(D)J) as per 10x genomics protocol for this instrument.

Transcriptome alignment, barcode assignment and UMI counting

The Cell Ranger Single-Cell Software Suite (versions 2.0.0 and 2.1.0 for the discovery and validation patients respectively) were used to perform sample demultiplexing, barcode processing and single-cell gene counting (http://10xgenomics.com/). First, raw base BCL files were demultiplexed using the Cell Ranger mkfastq pipeline into sample-specific FASTQ files. Second, these FASTQ files were processed individually using the Cell Ranger count pipeline, which made use of the STAR software48 to align cDNA reads to the hg38 human reference genome (Ensembl) and the Merkel cell polyomavirus sequence (HM011556.1). Aligned reads were then filtered for valid cell barcodes and unique molecular identifiers (UMIs). Cell barcodes with 1-Hamming-distance from a list of known barcodes were considered. UMIs with sequencing quality score >10% and not homopolymers were retained as valid UMIs. A UMI with 1-Hamming-distance from another UMI with more reads, for a same gene and a same cell was corrected to this UMI with more reads. Tumor and PBMC samples were, respectively, aggregated together using the Cell Ranger aggr pipeline resulting in two gene-barcode count matrices (tumor and PBMC) to be used for downstream analyses. A correction for sequencing depth was also performed during the aggregation19.

Biostatistical analysis—data normalization and correction

UMI normalization was performed as in Zheng et al.19. Only genes with at least one UMI count detected in at least one cell were retained for analysis. A library-size normalization was performed for each cell. UMI counts were scaled by the total number of UMI in each cell and multiplied by the median of the total UMI counts across cells. The data were then log2-transformed and corrected for unwanted sources of variation (number of detected UMIs) using the ScaleData R function as described in the Seurat manual49. The corrected-normalized gene-barcode matrix was used as input for dimension reduction and clustering analysis, whereas the normalized gene-cell barcode matrix was used for the MAST analysis as described below.

Gene expression analysis: discovery patient tumor

Following sequence alignment and filtering, a total of 7431 tumor cells (2243 cells before and 5188 cells after T cell therapy) were analyzed. The corrected-normalized gene-barcode matrix was used to perform principal component analysis (PCA) and t-distributed stochastic neighbor embedding (tSNE) analyses. First, the top 873 most variable genes selected by Seurat (log-mean expression values greater than 0.0125 and dispersion (variance/mean) greater than 0.5) were kept for PCA. The first top 10 principal components (PCs) were then down selected for tSNE visualization. One thousand iterations of tSNE using a perplexity value of 30 were performed. Cell classification and clustering were done according to the expression of established MCC tumor markers: NCAM1, ENO2, CHGA and KRT20 and also TILs markers. There were a total of 1984 cancer cells before, and 5131 cancer cells at the acquired resistance time point. Differential expression analysis between tumor cells before and after T cell therapy was performed using the R package MAST50. The normalized gene-cell barcode was used as input. The model included the cellular detection rate (CDR) as a covariate to correct for biological and technical nuisance factors that can affect the number of genes detected in a cell (e.g., cell size and amplification bias). Genes were declared significantly differentially expressed at a false discovery rate (FDR) of 5% and a fold-change >1.3.

Gene expression analysis: discovery patient PBMC

Following sequence alignment and filtering, a total of 12,874 cells were analyzed. As for the tumor samples, the corrected-normalized gene-barcode matrix was used to run PCA and t-distributed stochastic neighbor embedding (tSNE) analyses. First, the top 1203 most variable genes selected by Seurat (log-mean expression values greater than 0.0125 and dispersion (variance/mean) greater than 0.5) were kept for PCA. Again, the first top 10 PCs were then down selected for tSNE visualization. One thousand iterations of tSNE using a perplexity value of 30 were performed. Cell clustering was performed using a graph-based clustering method implemented in Seurat (FindClusters R function—share nearest neighbor (SNN) modularity optimization based clustering algorithm). Thirteen distinct clusters of cells were identified using the top 10 PCs with a neighborhood size of 40 and resolution of 0.6. Based on the clustering results, we removed cells belonging to three different distinct clusters enriched for expression of red blood cell and megakaryocyte markers that were likely the result of blood contamination (leaving total of 11,021 cells for analysis). Clusters were labeled according to enrichment of specific markers (FindMarkers R function implemented in Seurat). This analysis resulted in nine distinct clusters: CD4+ T cells, CD8+ T cells, CD8+ effector T cells, B cells, NK cells, CD14+ monocytes, CD16+ monocytes, myeloid cells and dendritic cells. Differential expression analysis between CD8+ and CD8+ effector T cells for the third time point was performed using the R package MAST50 as described in the previous section. R code is attached in Supplementary Data 3. Data submitted to NCBI Gene Expression Omnibus (GEO), accession GSE 117988.

Gene expression analysis: validation patient

The aggregated corrected-normalized gene-barcode matrix was used to first run PCA and t-distributed stochastic neighbor embedding (tSNE) analyses. The top 2450 most variable genes selected by Seurat (log-mean expression values greater than 0.05 and dispersion (variance/mean) greater than 0.5) were kept for PCA. The top 10 PCs were used for tSNE visualization. One thousand iterations of tSNE using a perplexity value of 50 were performed. As described in the previous section, cell clustering was performed using a graph-based clustering method implemented in Seurat (FindClusters R function). Nineteen distinct clusters of cells were identified using the top 10 PCs with a neighborhood size of 20 and resolution of 0.6. Clusters were labeled according to enrichment of specific markers. R code is attached in Supplementary Data 4, and data submitted to NCBI GEO, accession 118056.

Treatment with hypomethylating agents

Ex vivo tumor was mechanically dissociated, filtered and cultured for 48 h in RPMI supplemented with 20% FBS and pen/strep antibiotics. Untreated tumor was compared to tumor treated with 5-azacytidine (Toronto Research Chemicals, concentration 1 μM final) or gamma-interferon (final concentration 1000 IU/mL). RNA was isolated using directzol and reverse transcription performed. qPCR was performed using RT2 SYBR mastermix and commercially available primer sets to the indicated genes (Qiagen). Each condition was run in triplicate and qPCR repeated twice.