Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation

The HEK-293 (human embryonic kidney-293) cell line encoding the simian virus 40 (SV40) large T antigen (heretofore, 293T cells) was used for the production and characterization of lentiviral vectors (both research grade and GMP grade). A master cell bank (MCB) of 293T cells was established at EUFETS GmbH (Idar-Oberstein, Germany). Two randomly picked 293T MCB vials were tested for sterility, endotoxins, mycoplasma and adventitious viruses in full compliance with the GMP requirements of the local regulatory authorities. These safety tests have shown that the MCB was devoid of bovine, porcine and human viruses. HT1080 (human fibrosarcoma cell line) was used for titration of the lentivirus produced with GMP grade materials. 293T cells and HT1080 cells were cultured at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS, HyClone, Fischer Scientific GmbH, Bonn, Germany). The reference K562 cells co-expressing HLA-A02^*01 and pp65 were cultured in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin and 1 mg/mL geneticin (Biochrom AG, Berlin, Germany).

ICLV-pp65 used for control experiments was produced as previously described [21]. IDLV-G2α2pp65 was produced by transient transfection of four plasmids containing the transfer plasmid RRL-CMV-G2α2pp65, the packaging plasmid pCDNA3.g/pD64V.4xCTE encoding the D64V mutation in the integrase gene, the packaging plasmid expressing Rev and the pMDG plasmids encoding for the vesicular stomatitis virus G glycoprotein (VSV-G) as described [20]. For GMP production of the virus, all plasmids were fully sequenced and produced by PlasmidFactory GmbH (Bielefeld, Germany) as ccc-supercoiled Grade plasmids (enzyme free and devoid of bovine derived material and certified for purity). IDLV-G2α2pp65 was produced under GMP conditions following standard operation procedure (SOP) established at EUFETS GmbH. On day 0, 40 stack cell factories were seeded with 293T cells and transfected with qPEI (Polyethylenimine) transfection reagent. 24 h after transfection, medium change and Benzonase treatment were performed. Supernatant containing virus was harvested 48 h after transfection (total volume 2,500 mL), filtered through 0.8 and 0.45 µm filters and purified by chromatography (CEX). Tangential flow filtration and dialysis was performed and the viral supernatant was concentrated approximately 33-fold (to 74 mL). The purified virus was filtered with 0.45 and 0.2 µm filters, the final product was aliquoted as 1 mL/vial and stored at −80°C.

Physical titers of the vectors produced (Research grade and GMP grade) were determined by quantifying the p24 HIV-I core protein by ELISA (QuickTiter™ HIV Lentivirus Quantitation Kit, BioCat, Heidelberg, Germany).

For virus titration, HT1080 cells were transduced and genomic DNA was extracted from using the QiaAmp DNA blood mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For analyses of IDLV copy numbers in transduced monocytes, total DNA (tDNA) was extracted using the Epicenter Masterpure DNA isolation kit (Madison, WI, USA), with adaptations [24]. Cells lysates with incubated with proteinase K treatment, (45 min at 65°C), RNase A treatment (10 µg, 37°C for 30 min) and proteins removed by precipitation. The tDNA was precipitated from supernatants with isopropanol and solubilized in 85 µL of tris-buffer according to the manufacturer’s instructions. IDLV copy numbers were determined by real-time PCR as previously described [22, 25]. Shortly, 2 µL containing 100 ng of genomic DNA were added to 13 µL of RT-q-PCR mix [containing 7.5 µL of SYBRTaq mix with 1 µL of wPRE/PTB2 primer mix (wPRE forward: 5′-GAGGAGTTGTGGCCCGTTGT, wPRE reverse: 5′-TGACAGGTGGTGGCAATGCC or PTBP2 (polypyrimidine tract binding protein 2; PTBP2 forward: 5′-TCTCCATTCCCTATGTTCATGC, PTBP2 reverse: 5′-GTTCCCGCAGAATGGTGAGGTG) and 4.5 µL PCR grade, nuclease free water]. All samples were analyzed with StepOnePlus™ Real time PCR system (Applied Biosystems, Life Technologies, Darmstadt, Germany). The cycling conditions were 10 min at 95°C, 40 cycles of 15 s at 95°C, 20 s at 56°C and 30 s at 65°C. Results were quantified by making use of primer pair-specific real-time PCR efficiencies and by comparing sample CT values to a standard curve generated with the plasmid vector (pCR4-TOPO) containing the wPRE and PTB2 sequences. Data was analyzed by StepOnePlus™ software (Applied Biosystems).

Leukapheresis of non-mobilized HCMV-seropositive and HLA-A02*01 positive adult healthy donors was performed in accordance with study protocols approved by Hannover Medical School Ethics Review Board. Peripheral blood mononuclear cells (PBMNCs) were purified by sediment centrifugation (Ficol, Biochrome AG) and cryopreserved. CD14⁺ cells were selected using CD14 immunomagnetic microbeads (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) and processed as previously described [20, 22]. In short, CD14⁺ cells were pre-conditioned with hGM-CSF and hIL-4 (50 ng/mL each, CellGenix, Freiburg im Breisgau, Germany) for 8 h followed by transduction. IDLV-G2α (a bicistronic vector expressing GM-CSF and IFN-α, but no antigen) was used to generate “empty” SmyleDCs, while IDLV-G2α2pp65 (a tricistronic vector expressing GM-CSF, IFN-α and pp65) was used to generate SmyleDCpp65. The design and validation of these vectors were described previously [20, 22, 23]. For production of research grade SmyleDC or SmyleDCpp65, 2.5 μg/mL p24 equivalent of the respective vector were used to transduce 5 × 10⁶ monocytes at MOI of 5 in the presence of 5 μg/mL protamine sulfate (Valeant, Eschborn, Germany) for 16 h. After transduction, cells were washed twice with CellGro medium (CellGenix). Cells were cryopreserved in freezing medium containing 15.5% human albumin, 10% DMSO and 5%Glucose. Conventional IFN-α DCs were generated from monocytes by supplementing the medium with hGM-CSF and hIFN-α every 3 days.

Leukapheresis of non-mobilized HCMV sero-positive HLA-A02*01 and/or HLA-B07*02 healthy adult volunteers was performed with a COBE^® Spectra apheresis system. PBMCs were used fresh for selection of CD14⁺ monocytes with a GMP-compliant CliniMACS immunomagnetic separation system (Miltenyi Biotec). Quantitative and qualitative analyses of the selected CD14⁺ fraction and flow through were performed by flow cytometry. From the enriched CD14⁺ fraction, 1.5 × 10⁸ cells were resuspended in 25 mL of serum free CellGro DC medium (CellGenix) and seeded in a 100 mL bag (CellGenix). Cells were preconditioned with 25 mL of medium containing hGM-CSF and hIL-4 cytokines (50 ng/mL each, CellGenix) for 8 h. Cells were transduced with 7.5 × 10⁸ infective particles (multiplicity of infection of 5) in 50 mL medium containing Protamine sulphate (5 µg/mL). The bag was incubated at 37°C and 5% CO₂ for 16 h. Next day, cells were washed three times with CellGro medium. After washing, cell number and viability was determined. Transduced cells were cryopreserved in aliquots of 2 × 10⁶ cell/mL/vial. Surplus, non-transduced monocytes were cryopreserved in aliquots of 2 × 10⁶ cell/mL/vial and 50 × 10⁶ cell/mL/vial and were used as controls for the characterization experiments. Sterility tests were performed with the “Bactec” system (BD Biosciences, Heidelberg, Germany). Three independent production batches were prepared with three independent leukaphereses obtained from three independent healthy adult volunteers.

Three independent cryopreserved vials (2 × 10⁶ cell/mL/vial) of monocytes transduced with IDLV-G2α2pp65 in each production batch were analyzed directly at thaw (AT), or cultured in CellGro for 5 or 7 days at a concentration of 1 × 10⁶ cells/mL. Surface marker expression was analyzed by flow cytometry using the following monoclonal Ab conjugated with fluorochromes: Krome Orange-CD45 (clone J.33), APC750-CD14 (clone RMO52), PC7-CD11c (clone BU15), PE-CD86 (clone HA5.2B7), Pacific Blue-HLA-DR (clone Immu-357; all from Beckman Coulter, Krefeld, Germany), and APC-CD80 (clone 2D10; Miltenyi Biotec). Residual cell analyses were performed with the following Ab: APC700-CD3/CD19 (T and B cells; clones: UCHT1 and J3-119 respectively) and PC5.5-CD56 (NK cells; clone: N901 (HLDA6); all from Beckman Coulter). After staining and prior to acquisition, Flow-Count™ Fluorospheres (Beckman Coulter) were added to the cells to determine the absolute cell counts and to control the flow rate. Expression of pp65 was determined by intracellular staining and flow cytometry. Cells were stained with the following monoclonal antibodies Ab: Krome Orange-CD45 (clone J.33), Pacific Blue-CD14 (clone RMO52), PC7-CD11c (clone BU15), PE-CD86 (clone HA5.2B7), ECD-HLADR (clone Immu-357), and PC5.5-CD56 (clone N901 (HLDA6); all from Beckman Coulter). After surface staining, cells were washed and permeabilized with BD cytofix/cytoperm solution (BD Biosciences). After permeabilization, cells were incubated with FITC-conjugated mAB against HCMV-pp65 (clone: I1010D; Thermo Scientific, Germany) in a 1:20 dilution with BD perm/wash solution. Non-transduced monocytes are used as negative controls and conventional IFN-α DCs were used as positive controls. Acquisitions and analyses were performed by Navios™ Flow Cytometer and Navios™ analysis software (Beckman Coulter).

SmyleDCpp65 produced under GMP-like conditions secreted several endogenous cytokines. We analyzed and quantified the levels of a set of commonly highly expressed cytokines in culture supernatants (GM-CSF, IFN-α, MCP1 and IL-8) by bead array luminex based kit according to the manufacturer’s protocol (Milliplex Milipore, MA, USA).

Integration analyses were performed by using LAM-PCR to identify the lentiviral vector-flanking genomic sequences as described [26]. Briefly, tDNA was extracted from the samples and two 50-cycle linear PCR amplification steps were carried out using biotinylated primers hybridizing to the 3-prime region of the long terminal repeats (LTR) of the vector. The biotinylated PCR-products were further captured with paramagnetic beads followed by second strand DNA synthesis, restriction digestion and ligation of a cohesive double-stranded linker sequence carrying a molecular barcode of 12 nt. Two nested PCR were then performed with linker and vector specific primers each complementary to one of the known ends of the target DNA. In 5′–3′ orientation, LAM-PCR products contained a LTR sequence, a flanking human genomic sequence and a linker cassette (LC) sequence. LAM-PCR amplicons were further prepared for MiSeq sequencing (Illumina, San Diego, CA, USA). Therefore, an additional PCR with special fusion-primers carrying MiSeq specific sequencing adaptors was performed. DNA barcoding was used to allow parallel sequencing of multiple samples in a single sequencing run. Libraries were mixed with an φX bacteriophage genome library to introduce diversity and optimize the sequencing run performance and sequenced using the Illumina MiSeq v2 Reagent Kit. The pair-end runs were initiated for Illunima’s sequencing by synthesis technology, including clustering, paired-end preparation, barcode sequencing and analysis. After completion of the run, base calling was performed on data, sequences were de-multiplexed and φX reads were filtered. Next generation sequencing (NGS) data processing dealt with the management of high-throughput data from Roche 454/Illumina MiSeq sequencing platforms and comprise two main goals: (1) data quality inspection and analysis, in which lentiviral vector sequences and other contaminants were trimmed; (2) integration site identification, in which all valid sequence reads are aligned to the genome of reference and valid ISs were retrieved.

The potency assays were based on activation of autologous T cells with SmyleDCpp65, which were produced with monocytes after CliniMACS selection. GMP grade-SmyleDCpp65 were compared with research grade (RG) SmyleDCpp65 and SmyleDC produced with monocytes from the same donor, but which were produced with IDLVs produced in the laboratory. Autologous CD3⁺ T cells from each of the three GMP-grade batches were isolated from the CD14^neg fraction (MACS positive selection, Miltenyi Biotec). CD3⁺ T cells were co-cultured with RG-SmyleDC, RG-SmyleDCpp65 or GMP-grade SmyleDCpp65 at a ratio of 10–1. Non-stimulated CD3⁺ T cells were used as negative control. CD3⁺ T cells stimulated for 16 h with 10 µg/mL PepTivator CMV-pp65 overlapping peptide pool (Miltenyi Biotec) were used as reference controls for the IFN-γ intracellular detection assay. Protein transport inhibitor cocktail (eBioscience, Frankfurt, Germany) was added to the cells 1 h after stimulation. After 16 h, T cells were harvested, stained with APC-conjugated anti-human CD3, Pacific Blue-conjugated anti-human CD4 and PECy7-conjugated anti-human CD8 antibodies. After fixation/permeabilization with Cyofix/perm (BD Biosciences) for 20 min at 4°C and washing, anti-human PE-IFNγ (eBioscience) was used for staining for 30 min. The cells were acquired by flow cytometry using LSRII (BD Biosciences) and analyzed by Flowjo^® software (Treestar Inc., Ashland, OR, USA).

We have previously demonstrated and validated a tricistronic vector IDLV-G2α2pp65 co-expressing simultaneously GM-CSF, IFN-α and pp65 (Additional file 1: Figure S1A) for generation of SmyleDCpp65 [23]. Advancing towards clinical grade SmyleDCpp65 for clinical trials, we evaluated the feasibility of vector production and cell generation under GMP compliant conditions. In collaboration with a contract manufacturing organization (CMO, EUFETS GmbH), we validated upstream and downstream processes and established standard-operating-protocols (SOPs) for production of the vector IDLV-G2α2pp65 by transfection of 293T cells. High purity ccc plasmids consisting the transfer vector (LV-G2α2pp65), the gag/pol vector harboring a D64V mutation (pcDNA3g/pD64V.4xCTE), the vector containing codon optimized REV (pRSV-REV), and the envelope vector expressing VSV-G (pMD.G) were used for transfection in 40 stack cell factories (Figure 1a). After transfection, viral supernatant was harvested and subjected to downstream purification (benzonase treatment, filtration, chromatographic purification, tangential flow filtration, sterile filtration, filling and storage). At each step of the purification process, a QC step was added to determine the recovery and yield of IDLV (Figure 1a). Infectious titer of IDLV-G2α2pp65 was determined by RT-q-PCR and the physical titer by quantifying the HIV-I core protein p24. Starting with 2 × 10⁶ infectious particles/mL (ip/mL; 2 L volume) after CEX purification, final product was concentrated by 33-fold in relation to the starting volume with a final titer of (from 2,500 to 74 mL; Figure 1a). The filtration and concentration steps did not alter the infective titer of IDLV. The final product showed a titer of 5.7 × 10⁷ ip/mL in a total of 74 mL (4.2 × 10⁹ infectious particles) (Figure 1b). On the other hand, physical p24 titer of IDLV-G2α2pp65 revealed major reduction after CEX purification, filtration and dialysis steps (Figure 1c), which was probably due to removal of empty particles and cell debris containing p24 during the purification process. Overall, GMP-grade IDLV-G2α2pp65 production and recovery demonstrated that IDLV production was not fundamentally different from ICLV production methods established by the same CMO.

Since cryopreservation of SmyleDCpp65 could facilitate the production logistics, storage and performance of quality control analyses, we performed preliminary tests to evaluate the effects of cryopreservation immediately after IDLV transduction (representative example Additional file 1: Figure S1B-F). After thaw (AT), transduced cells were highly viable 7AAD^neg and pure CD14⁺ monocytes containing detectable IDLV copies. After culture for 7 days, SmyleDCpp65 maintained IDLV copies and the persistent gene transfer was associated with expression of the pp65 antigen. After culture, SmyleDCpp65 were still highly viable, down-regulated the expression of monocytic marker CD14⁺, up-regulated the expression of the DC marker CD11c and co-expressed the relevant molecules HLA-DR/CD86 and HLA-DR/CD80. Therefore, under good research practice, cryopreservation was not detrimental to recovery of self-differentiated SmyleDCpp65 after thawing.

Therefore, we proceeded with upscaling the production of cryopreserved SmyleDCpp65 with SOPs using the vector generated under GMP-like conditions. The standardized production was validated in three independent runs. Ex vivo monocyte manipulation starting with leukapheresis at the clinical center and proceeding with transportation under controlled conditions to the CMO for CD14⁺ isolation, cytokine preconditioning, transduction, wash and cryopreservation could be fully achieved in only 3 days (Figure 2a). In process QC steps were included after each step in the production process to assess the cell viability and recovery (Figure 2a). In order to assess the purity, viability and recovery of monocytes from the leukapheresis until thaw, in process QC steps were added after each step in the production process. Three independent CD14⁺ CliniMACS^® enrichments were performed with leukapheresis obtained from donors, resulting in >80% viable CD14⁺ purity (Table 1). A range of 0.47–1.00 × 10⁹ CD14⁺ cells were recovered from 1.47–2.29 × 10⁹ PBMNCs (Figure 2b). 1.5 × 10⁸ CD14⁺ monocytes were used in transductions with GMP-grade IDLV-G2α2pp65 at an MOI of 5 (7.5 × 10⁸ infectious particles). After transduction and washing, 0.60–1.23 × 10⁸ viable monocytes were recovered (Figure 2b). SmyleDCpp65 were cryopreserved as 2 × 10⁶ cells in 1 mL per vial as the final cell product. A fraction of the CD14⁺ cells that were not transduced were equally cryopreserved at 2 × 10⁶ cells per mL per vial and were used as negative controls for the QC analyses. Sterility tests were negative for bacterial and fungal contamination (data not shown). After cryopreservation, samples were transported to our clinical center under controlled conditions for further QC and characterization analyses. At independent time points, three vials from each of the three SmyleDCpp65 production batches were thawed and characterized for sterility, viability, identity and purity. At thaw (AT), a range of 0.60–1.73 × 10⁶ cells corresponding to 30.0–86.5% of the cryopreserved cells were recovered (Figure 2c). Cell counts performed at the CMO demonstrated that 30% of the cells seeded AT maintained viable after ex vivo culture for 5–7 days (Figure 2c).

The most relevant criteria to ensure quality of the cell product after filling, cryopreservation and thawing are purity and viability. Thus, standardized quality control analyses to define these parameters were performed in collaboration with a GMP development unit in our institution. After thawing, SmyleDCpp65 showed undifferentiated monocyte morphology and high viability (>90% 7AAD), expressed monocyte markers (>90% CD45⁺ and CD14⁺) and showed low frequency of residual T and B cells (<1% CD45⁺/CD19⁺/CD3⁺) (Figure 3a–c). Unexpectedly, as we intended to evaluate the frequency of residual natural killer cells with the CD56 marker, we observed a consistent population (8%) of CD56⁺ cells, which may relate to the recently reported CD56⁺ IFN-α-induced dendritic cells shown to efficiently stimulate autologous Vγ9γδT cells [27].

As the biological activity of SmyleDCpp65 depends on the successful transduction, detection of IDLV copies is a primary criterion for QC of the process. For the first batch of transduced monocytes (“GMP1”), there was a technical problem with the liquid nitrogen storage of samples at the CMO site. Thus, some of the samples of GMP1 were lost and could not be further analyzed regarding IDLV integration. AT, tDNA was isolated for GMP2 and GMP3 batches. Non-transduced monocytes obtained from the same donors were used as baseline control. IDLV copies could be detected in the GMP batch 2 (0.66 copies per cell, 11× above assay baseline) and in the GMP batch 3 (1.31 copies per cell, 6× above assay baseline) (Figure 3d). All together, these results demonstrated a robust and consistent 3-day manufacturing method for generation of cryopreserved SmyleDCpp65 under GMP.

Analytical validation methods were established to demonstrate parameters of the cells after thawing and in vitro culture such as morphology, high viability, self-differentiation into an activated DC phenotype, expression of pp65 and cytokines. Cells were thawed, seeded at 10⁶ cells/mL, and re-analyzed immediately after thaw (day 0) or after 5 and 7 days of culture. Three independent cryopreserved SmyleDCpp65 vials from each production batch were thawed and analyzed and corresponding triplicates of freshly thawed monocytes and conventional monocyte-derived DCs from the same donor were used as control parameters for the flow cytometry analyses. While non-transduced monocytes showed clear viability loss after 3 days of culture in absence of added cytokines, SmyleDCpp65 maintained viable and progressively showed morphologic changes towards DC differentiation from days 3–7 of culture (Figure 4a). Two panels of antibodies were developed for flow cytometry characterization of the activation status of SmyleDCpp65 (Panel I; viable), and to detect the expression of the pp65 antigen in DCs (Panel II; permeabilized) (Figure 4b; Table 2). For the activated DC panel, CD45⁺ hematopoietic cells were stained for viability (7AAD), the monocyte marker (CD14, down-regulated) and DC markers (CD11c, up-regulated). Activated SmyleDCpp65 were identified as 7AAD^neg, CD45⁺, CD14^neg, CD11c^high, HLA-DR⁺, CD86⁺ or 7AAD^neg, CD45⁺, CD14^neg, CD11c^high, HLA-DR⁺, CD80⁺ (Figures 4c, d and 5a). Hence, SmyleDCpp65 cultured for 5 and 7 days, down-regulated CD14 expression (AT: >95%; day 5: 4%; day 7: <1%), expressed high levels of DC markers CD11c (12.2% AT; day 5: 94%; day 7: 97%; Figure 4c), co-expressed HLA-DR/CD86 (36% AT; day 5: 65.58%; day 7: 75.45%; Figure 4d) and highly expressed HLA-DR/CD80 (16.6% AT; day 5: 88.25%; day 7: 91.71%; Figure 5a). Although both CD86 (B7-2) and CD80 (B7-1) are key co-stimulatory molecules both providing signaling to T cells through binding to the co-stimulatory receptor CD28 or the co-inhibitory receptor CTLA-4, expression of CD86 is stable and can be found in immature DCs, whereas CD80 expression is upregulated and a hallmark of active DCs [28]. Residual cells were identified by gating on viable cells (7AAD^neg) and stained for CD3, CD19 and CD56. For qualitative analyses of pp65 expression in DCs, non-transduced monocytes were used as for setting up the negative controls gates, and freshly thawed K562/pp65 were used as positive controls for pp65 expression (Additional file 2: Figure S2). Cells were stained with surface markers, permeabilized and stained for pp65. Frequency of pp65⁺ expressing DCs was represented as CD45⁺, CD14^neg, CD11c^high and pp65⁺ (AT: 4.28%; day 5: 16.4%; day 7: 33.71%; Figure 5b). Thus, expression of pp65 continuously increased during culture. Bead array analyses of SmyleDCpp65 cell culture supernatants demonstrated only baseline levels of secreted transgenic cytokines AT, whereas detectable levels of GM-CSF (day 5: 7.26 and day 7: 7.02 pg/10⁶ cells/mL) and IFN-α (day 5: 32.93 and day 7: 33.25 pg/10⁶ cells/mL) could be measured in the cell supernatant only after 5 or 7 days of culture (Figure 5c). In addition, high levels (>10⁴ pg/10⁶ cells/mL) of endogenous up-regulated monocyte chemotactic protein-I (MCP-I) and IL-8 were detectable in cell culture supernatants harvested on days 5 and 7 (Figure 5d). MCP-I and IL-8 are chemokines and we have previously demonstrated dramatic up-regulation of these cytokines in lentivirus-induced DCs [22, 29]. Therefore, analyses of quality control by these flow cytometry and luminex

systems provided a consistent pattern of the product and confirmed identity and immunologic potency of

SmyleDCpp65.

Although IDLVs are currently explored in experimental animal models to be used as recombinant viral vaccines against infections, cancer and parasites [16‐18], there is still a lack of studies in human preclinical studies correlating transgene expression, vector copy numbers and pattern of residual integration. Due to the potential risk of insertional mutagenesis, this is a serious preclinical requirement to be observed. Residual lentiviral integration of IDLV was characterized in hepatocytes of mice infused with IDLV, demonstrating that the risk was greatly reduced but not totally eliminated [30]. At day 7 after culture, we could estimate based on detection of pp65 antigen as a qualitative method, that at least 30–40% of the cells produced under GMP were transduced (Figure 6a). Analyses of different batches of cells by quantitative RT-PCR demonstrated that IDLV copy content per cell ranged between 0.5 and 1.2 copies per cell (Figure 6b). We subsequently compared the LV integration pattern observed in day 7 SmyleDCpp65 transduced with vectors produced as research grade (ICLV versus IDLV) and as GMP grade (GMP-2 and GMP-3). DNA was extracted and analyzed by LAM-PCR and NGS and the clonal frequencies of the transduced monocytes maintained in culture for 7 days was monitored with a high throughput IS analysis [26]. The number of total vector sequences that could be retrieved for the four groups were in similar orders of magnitude (0.5–1.0 × 10⁴ sequences, Figure 6c). However, the detectable number of unique insertion sequences (IS) was much higher for RG-ICLV (>1,000) than RG-IDLV (335). Unique IS detectable for GMP2-IDLV and GMP3-IDLV combined were even lower, in total 99 sequences (Figure 6d). This indicated that only residual genomic lentiviral integration sites could be observed after IDLV gene transfer of monocytes under GMP-compliant conditions. Whereas for the research-grade vectors IDLV integrations were found in all chromosomes and frequencies were roughly correlated with chromosome size, interestingly no integrations were found in chromosome 22 for the two GMP-grade batches of SmyleDCpp65 (Figure 6d). As expected, ICLV integrated sequences were found in higher frequency in genes than upstream of the transcription start site (TSS) (Figure 6e). Surprisingly, for the RG IDLV sample, vector integrations were most frequent up to 5 kb upstream of the TTS. In turn, integrations detectable in the two GMP SmyleDCpp65 batches show a random distribution, but mostly in genes (Figure 6e). Although one could speculate that IDLV pattern of integration may indeed be more random than ICLV in monocytes, we would have to compare more samples for further analyses. Nevertheless, a relevant information was that IDLV genome integrations in gene loci were random and highly diverse among all RG and GMP samples analyzed. The research grade vectors showed higher polyclonality, but this may be due to the fact that there were more sequences to be analyzed. The 10 most frequent identified gene loci among the four samples were discordant and did not occur near oncogenes (Figure 6f). Tracking of the most common clones based on a Common Integration Site (CIS) statistical models [31‐33] showed that, for RG-IDLV transduction, only one integration could be observed as 4th order (4 integrations within 100 kb genomic region, one of them in TRIM27), four integrations as 3rd order (3 integrations within 50 kb, with integration in WDR74 among others) and 9 integrations as 2nd order (2 integrations within 30 kb, with integration in DENND1B). Overall, these analyses confirmed previous observations that LV integrations are random showing a non-biased integration profile not matching a known biased integration sequences assigned for gamma-retroviral genotoxicity such as LMO2 [34]. Long-term cultures (30 days) of research-grade SmyleDCpp65 (produced with IDLV or ICLV without the cryopreservation step) were longitudinally harvested on days 10, 20 and 30, showing progressive reductions of LV copies (Additional File 3: Figure S3A). Pooling these time-points for each vector type, we observed that most of the ICLV and IDLV integrations combined were in gene ±10 kb outside genes (Additional File 3: Figure S3B-D). Analyses of the cultures maintained for up to 30 days showed also a consistent random integration in genes and polyclonality (Additional File 3: Figure S3C-D). Integration sites that showed up more than once in these analyses were WD Repeat-Containing Protein 74 (WDR74), ZNF37A, SEL1L, ILF2, but these genes are not known as proto-oncogenic. Notwithstanding, a common insertion of IDLV in the analyses of GMP batches were observed in WDR74 genes (Figure 6f, g), but insertional mutagenesis involved with the human locus was not described (NCBI Gene ID: 54663, updated on 17-Mar-2015). Furthermore, since monocytes and DCs are mostly post-mitotic and non-replicating cells, and it is unlikely that a biased integration pattern could predispose to insertional mutagenesis and genotoxic effects.

Ultimately, in order to demonstrate that SmyleDCpp65 produced under GMP-compliant conditions were functional in stimulating T cells, SmyleDCpp65 produced from the three donors were thawed, cultured for 7 days and used to stimulate autologous selected CD3⁺ T cells (Figure 7a). The donors used for these studies were all HCMV sero-positive and thus contained memory T cells reactive against pp65 epitopes. Non-stimulated T cells and cells pulsed with a pp65 peptide mix were included as experimental parameters. RG SmyleDC and SmyleDCpp65 produced with CD14⁺ cells from the same donor were used as positive controls. 16 h after stimulation, intracellular staining of IFN-γ and flow cytometry analyses were performed (Figure 7b, representative example). T cells stimulated with SmyleDCpp65 produced as GMP resulted in significant increases in frequencies of IFN-γ producing CD8⁺ T cells (SmyleDCpp65: 9.8%; versus unstimulated, p < 0.05) and CD4⁺ T cells (SmyleDCpp65: 7.5%; versus unstimulated p < 0.05) (Figure 7d). The T cell stimulation was modestly higher for SmyleDCpp65 (GMP or RG) than for SmyleDC (which can still activate T cells homeostatically, but lack the antigen). Under these assay conditions, the pp65 peptide mix loading on the T cells did not stimulate IFN-γ production in CD4⁺ or CD8⁺ T cells above the non-stimulated T cell control group. Therefore, these assays confirmed the functionality of GMP-grade SmyleDCpp65 to stimulate T cells in vitro.