Material and Methods

Ethics Statement

Work performed with primary cells isolated from blood donations was invariably obtained from anonymous donors that had provided written informed consent in full compliance with the requirements of the Ethics Committee of the University Hospital Frankfurt, Germany.

Cell lines and primary cells

HEK293T (ATCC CRL-11268), HT1080 (ATCC CCL-121) and HT1080αHis cells were cultivated in DMEM (Sigma-Aldrich, Munich, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and 2 mM L-glutamine (Sigma-Aldrich, Munich, Germany). Culture medium of HT1080αHis cells was furthermore supplemented with1.2 mg/mL G418 (Thermo Fisher Scientific, Darmstadt, Germany). The cell line HT1080CD62L was generated by LV transduction of the parental HT1080 cell line with LV particles packaging a construct encoding a spleen focus-forming virus (SFFV) promoter, the gene encoding CD62L (UniProt: P14151), an internal ribosome entry site (IRES) element followed by a puromycin resistance gene and a woodchuck posttranscriptional regulatory element (WPRE) (transfer plasmid: pS-CD62L-IPW). Transduced cells were selected using puromycin for 2 weeks. Nalm-6-eBFP-Luc (kindly provided by Prof. Helen Fielding, University College of London), further on called Nalm6, were grown in complete Roswell Park Memorial Institute (RPMI) medium (RPMI 1640, Biowest), supplemented with 10% FCS and 2 mM L-glutamine.
Human PBMC were isolated from fresh blood of healthy donors or buffy coats purchased from the German blood donation center (DRK-Blutspendedienst Hessen, Frankfurt) and cultured in T cell medium (TCM) consisting of RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 0.5% streptomycin/penicillin, 25 mM HEPES (Sigma-Aldrich, Germany), 25 U/mL IL-7 and 50 U/mL IL-15 (all cytokines from Miltenyi Biotec, Germany). For activation, 1x107 PBMC per 6-well were cultured in TCM supplemented with 3 μg/mL anti-CD28 antibody (clone 15E8, Miltenyi Biotec, Germany) for 48 hours. 6-well plates for activation were pre-coated with 1 µg/mL anti-CD3 antibody (clone OKT3, Miltenyi Biotec, Germany).

Generation of CD62L-targeted envelope constructs

To generate the CD62L targeting constructs, the coding sequences of the variable light chain (VL) and heavy chain (VH) of the parental CD62L-specific monoclonal antibody 145/15 were synthesized de novo (GeneArt, Thermo Fisher Scientific) and cloned into the backbone encoding the modified Nipah virus (NiV) glycoprotein G with and without glycine-serine linker (pCG-GNiVΔ34mut-His and pCG-GNiVΔ34mut-L3-His) or the modified measles virus (MV) hemagglutinin protein (pCG-HMVnseΔ18mut-L3-His) via digestion with Sfi I and Not I. DNA sequences were verified by standard sequencing technologies prior to use in LV production.

LV production and characterization

Vector particles were generated by transient transfection of HEK-293T cells using polyethylenimine (PEI) and second generation packaging plasmids as described in detail by Weidner and colleagues. In brief, one day before transfection, 1.5-2x107 cells were seeded into a T175 flask. In total, 35 μg DNA was added to 2.3 mL of DMEM without additives and combined with 2.2 mL DMEM containing 140 μL of 18 mM PEI solution. The transfection solution was mixed and incubated for 20 min at room temperature. The cell medium was replaced by 10 mL DMEM supplemented with 15% FCS and 3 mM L-glutamine, before the transfection solution was added to HEK-293T cells. 4-6 hours later the medium was replaced by DMEM with 10% FCS and 2 mM L-glutamine. Two days after transfection, the cell culture supernatant was collected and filtrated. Released vector particles were concentrated over a 20% sucrose cushion at 4500xg for 24 hours, before supernatant was discarded and pellets were resuspended in 60 µL PBS per T175 flask. The used transfer plasmid encoded a second generation CD19-CAR in conjunction with ΔLNGFR. Notably, based on the co-expression of ΔLNGFR and the CAR construct, detection of ΔLNGFR can be used as surrogate marker for the expression of CAR molecules on the cell surface. Plasmid ratios for the generation of NiV-based and MV-based RT-LV particles as well as particles pseudotyped with VSV-G were described previously and can be found in the supplemental table 1. If not otherwise specified, all concentrated vector stocks were titrated on HT1080αHis cells as described previously using a LNGFR specific antibody for detection. LV particle yields were determined by nanoparticle tracking analysis or p24-specific enzyme-linked immunosorbent assay (HIV type 1 p24 Antigen ELISA; ZeptoMetrix Corporation) according to the manufacturer’s instructions and calculated as described.

Transduction of cell lines and primary cells

Parental HT1080, HT1080αHis, and HT1080CD62L cells were seeded at 8x103cells per 96-well and incubated with serial dilutions of vector stocks. Transgene expression was analyzed 72 to 96 hours later by flow cytometry. Activated PBMC were seeded at 4x104 or 8x104 cells per 96-well, respectively, in TCM medium before CD62L-LV (5 µL or 10 µL) or VSV-LV (0.05 µL or 0.5 µL) were added. Where indicated, CD62L-LV transduction of PBMC was carried out in presence of Vectofusin-1 (Miltenyi Biotec, Germany) as described previously. Cells were centrifuged at 850g, 32°C for 90 minutes, followed by addition of TCM. Medium was replenished every 2 to 3 days. Transgene expression was assessed by flow cytometry.

Quantification of shed CD62L by ELISA

Activated PBMC of three donors were cultured without medium change or cell passaging for up to 10 days. At the indicated time points, cell suspension was collected and centrifuged for 5 minutes at 5,000 rpm and either stored at -80°C or 4°C. Concentration of sCD62L in supernatant was determined by ELISA (Human L-Selectin/CD62L DuoSet ELISA, R&D Systems) following the manufacturer’s protocol with the exception that heat inactivated FBS was used instead of inactivated goat serum. Quantification of the fluorescent signals was performed with a microplate reader (EmaxPlus, Molecular Devices).

Blocking assay with shed CD62L or antibodies

For the blocking assay with antibodies, 4x104activated PBMC were preincubated with the indicated concentrations of a CD62L-specific antibody (clone 145/15, Miltenyi Biotec) or a CD45-specific antibody (clone 5B1, Miltenyi Biotec) either conjugated to the fluorophore phycoerythrin (PE)-Vio770 or to biotin for 1 h at 4°C. Before and after antibody incubation, cells were washed twice with wash buffer (phosphate-buffered saline (PBS) supplemented with 2% FBS and 0.1% NaN3). Afterwards, either 10 µL of 62L-LV or PBS were added to cells pre-incubated with biotin conjugated antibodies, while PBS was added to cells pre-incubated with fluorophore-conjugated antibodies. All samples were incubated at 4°C for 30 min. Cells pre-incubated with biotin conjugated antibodies were further stained with a PE-labeled anti-LNGFR antibody (clone ME20.4-1.H4, Miltenyi Biotec). After two additional washing steps, antibody and vector bound cells were determined by flow cytometry analysis respectively.
For the sCD62L blocking assays, 10 µL of 62L-LV or VSV-LV vector particles were pre-incubated with 90 µL fresh or frozen supernatant containing sCD62L derived from six days of PBMC culture or TCM only for 1 h at 4°C. Vector/sCD62L-containing supernatant was then added to 4x104 activated PBMC of various donors in 96-wells. Staining for vector bound cells was performed after an incubation for 30 minutes at 4°C by flow cytometry detecting ΔLNGFR.

Animal experiment

All animal experiments were conducted in accordance with the German Animal Protection Law and the respective European Union guidelines.
For short-time generation of CAR T cells, 1.8x106activated PBMCs were seeded in 600 µL TCM per 24 well, mixed with 30.6 µL 62L-LV (equals MOI of 1.3 or ~4x1010 particles) or VSV-LV (equals an MOI of 8.8 or ~3x1010 particles) or equal volume of PBS and centrifuged for 90 min at 850g, 32°C before addition of TCM to a total volume of 1.2 mL per well. 24h after vector incubation, cells were harvested and washed 2x with PBS prior in vivo application. NSG mice (NOD.Cg.PrkdcscidIL2rgtmWjl/SzJ, Jackson Laboratory) were intravenously (i.v.) injected with 2x106 vector bound cells or 1.4x106PBS treated cells. Three days later, 5x105 Nalm-6 were injected i.v. and tumor growth was followed by bioluminescence imaging (BLI). This was performed by injecting D-luciferin (Perkin Elmer) intraperitoneally at 150 µg/kg body weight and imaging luciferase signals 10 minutes after injection using the IVIS Imaging System (Perkin Elmer). CAR T cell engraftment was monitored through regular, blood drawings. Mice were checked regularly for health status and tumor load by IVIS. All mice were sacrificed on day 17 for final analysis of blood and organs (spleen, bone marrow and liver).
Collected blood and organs were prepared to a single cell suspension and analyzed by flow cytometry analysis. Blood was washed with PBS prior and after erythrocyte lysis using BD Pharm Lyse buffer (BD Bioscience). Spleens were minced through a 70 µm cell strainer to obtain a single cell solution and proceeded with erythrocyte lysis. Bone marrow was harvested through centrifugation of long bones cut open with a scalpel in pierced 0.5 mL tubes at 8000 rpm for 5 min. Bone marrow cells were then washed with PBS and singularized through a 70 µm cell strainer and erythrocyte lysis was performed. Liver cells were isolated using the mouse liver dissociation kit (Miltenyi Biotec) according to the manufacturer’s instruction, washed with PBS and erythrocytes were lysed.

Flow cytometry

Flow cytometry analysis was performed using MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) or LSR Fortessa (BD Biosciences) flow cytometers. Data were analyzed by FCS Express 6 (De Novo Software, Glendale, CA, USA) or FlowJo 7 (BD Biosciences). Before and after staining with fluorescently labelled antibodies, cells were washed twice with wash buffer. Before measurement cells were fixed by addition of PBS supplemented with 1% formaldehyde. To determine the percentage of transduced cells or cell-bound vector particles, staining of up to 1x105 cells was performed. The reporter protein ΔLNGFR, which is co-expressed with the CD19-CAR, was detected using the anti-LNGFR-PE antibody. PBMC were further stained with the fixable viability dye eFluor780 (Life Technologies, Darmstadt, Germany), according to the manufacturer’s instructions or with 7-AAD to detect viable cells. To further characterize the PBMC, cells were stained in addition with a CD4-specific antibody (clone VIT4) labeled with VioGreen or PE-Vio770 and a CD8-specific antibody (clone BW135/80) labeled with allophycocyanin (APC) or APCVio770 and if indicated with a CD62L-specific antibody (clone 145/15) labeled with PEVio770. All antibodies were from Miltenyi Biotec (Bergisch Gladbach, Germany). The following antibodies were used for flow cytometry analysis of thein vivo experiment: CD45-BV510 (clone 2D1, BioLegend), CD3-BV605 (clone HIT3a, BD Bioscience), CD8-BV786 (clone RPA-T8, BD Bioscience), LNGFR-PE (clone ME20.4-1.H4, Miltenyi Biotec), CD4-PE-CF594 (clone RPA-T4, BD Bioscience), CD19-Alexa Fluor 700 (clone HIB19, Thermo Fisher), eFluor780 (eBioscience).

Statistical Analysis

Statistical analyses were performed with Prism 7 software (GraphPad). Tests for statistical significance used the unpaired or paired two-tailed Student’s t test, one-way ANOVA (Dunnett multiple comparisons test), two-way ANOVA (Dunnett or Turkey multiple comparisons test) or Fisher’s least significant difference (LSD) test as indicated. Statistical differences in experiments were considered significant atp < 0.05.