Dendritic cell-based immunotherapy is an approved treatment for prostate cancer and is under clinical evaluation for a variety of human cancers [
Dendritic cells in cancer immunology and immunotherapy.
]. Endogenous dendritic cells are the most proficient antigen-presenting cells to display tumor antigens and play an essential role in anti-tumor immunity [
Regulation of the Cell Biology of Antigen Cross-Presentation.
]. However, it is impractical to isolate a sufficient amount of endogenous dendritic cells for autologous cell therapies [
Engineering dendritic cell vaccines to improve cancer immunotherapy.
]. To manufacture dendritic cells on an industrial scale, the most popular approach is to collect peripheral blood monocytes via leukapheresis. Enriched monocytes can be further purified by elutriation based on the physical properties of monocytes or by the affinity to lineage-specific antibodies. Purified monocytes are then cultured by various methods to generate dendritic cells [
Manufacturing Dendritic Cells for Immunotherapy: Monocyte Enrichment.
]. At the final step of the manufacturing process, different methods are used to load dendritic cells, such as mRNA electroporation and incubation with peptide, recombinant protein or tumor cell lysate [
Antitumour dendritic cell vaccination in a priming and boosting approach.
]. A prominent example is Sipuleucel-T, also known as PROVENGE or Dendreon, the first therapeutic dendritic cell vaccine approved by the US Food and Drug Administration to treat metastatic castration-resistant prostate cancer with no or minimal symptoms [
PROVENGE (Sipuleucel-T) in prostate cancer: the first FDA-approved therapeutic cancer vaccine.
Notably, there is still no consensus on the optimal way to manufacture dendritic cells for cancer therapy [
- Sabado R.L.
- Balan S.
- Bhardwaj N.
Dendritic cell-based immunotherapy.
]. Variations on donor materials and manufacturing methods inevitably result in heterogeneity in cell-type compositions in the final products. For regulatory considerations, it is important to define the cell-type composition to address safety concerns but also to ensure reproducible, high-quality manufacturing of intended cell type. Fluorescence-activated cell sorting (FACS) analysis frequently is used to immunophenotype cell types. However, it requires previous knowledge of the cell types in the products and offers limited information on functional cellular states. Thus, other methodologies are needed to characterize the heterogenous cell types in an unbiased manner.
Ovarian cancer has been the most lethal gynecologic cancer during the past decade and remains a high unmet medical need worldwide. High mortality is partly the result of a long-standing paucity of effective therapies other than platinum-based chemotherapy [
Epithelial ovarian cancer.
]. Interestingly, two decades ago, tumor-infiltrating T cells were demonstrated in many patients with ovarian cancer [
Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer.
]. However, recent large-scale phase 2 and 3 clinical trials with immune checkpoint inhibitors including anti-programmed cell death protein 1 (anti-PD-1, pembrolizumab) and anti-programmed death-ligand 1 monoclonal antibodies (anti-PD-L1, atezolizumab and avelumab) failed to achieve meaningful efficacy in patients with ovarian cancer, either as standalone or combined with stand-of-care therapeutic agents [
Antitumor activity and safety of pembrolizumab in patients with advanced recurrent ovarian cancer: results from the phase II KEYNOTE-100 study.
Atezolizumab, Bevacizumab, and Chemotherapy for Newly Diagnosed Stage III or IV Ovarian Cancer: Placebo-Controlled Randomized Phase III Trial (IMagyn050/GOG 3015/ENGOT-OV39).
Chemotherapy with or without avelumab followed by avelumab maintenance versus chemotherapy alone in patients with previously untreated epithelial ovarian cancer (JAVELIN Ovarian 100): an open-label, randomised, phase 3 trial.
]. These observations indicate that the priming phase of anti-tumor immunity by dendritic cells may be deficient in ovarian cancer [
Immunological control of ovarian carcinoma by chemotherapy and targeted anticancer agents.
The Immune Revolution: A Case for Priming, Not Checkpoint.
We recently developed a versatile dendritic cell-based platform, CUD-002, consisting of Good Manufacturing Practice (GMP)-grade dendritic cells loaded with in vitro–transcribed mRNA encoding personalized neoantigens. CUD-002 exhibits favorable efficacy in pre-clinical cellular and animal models to treat ovarian cancer and is currently under evaluation in phase I clinical trial (NCT05270720). Here, we demonstrate a reproducible production of high-quality CUD-002 product and use single-cell transcriptomic sequencing as an unbiased approach to characterize cell type compositions in CUD-002 and to guide the development of FACS-based method as quality assurance and control steps in the manufacturing process.
To develop a GMP manufacturing process, peripheral blood mononuclear cells were collected by leukapheresis at West China Second University Hospital of Sichuan University. Informed consent was obtained from all volunteers for apheresis donation, and this study was approved by the Ethical Committees of West China Second University Hospital at Sichuan University. A single collection from health donors (median age: 33 years; range: 19–56 years; 66 male and 19 female donors) using Spectra Optia system (Terumo BCT, Lakewood, CO, USA) with the mononuclear cell collection procedure was made.
Enrichment of monocytes by elutriation
Continuous-counterflow elutriation was performed with the Elutra system (Terumo BCT) in Hanks' buffered salt solution (Lonza, Basel, Switzerland) supplemented with 1% human albumin. After priming with Hanks' buffered salt solution, the leukapheresis product was loaded via the inlet pump into the constantly rotating elutriation chamber, using a constant centrifugation speed of 2400 rpm and increasing the cell medium flow rate step-by-step (37 mL/min, 97.5 mL/min, 97.5 mL/min, 97.5 mL/min, 103.4 mL/min and 100 mL/min). The total elutriation time was 1 h. Six fractions were collected, and the percentage of various immune cells was analyzed by ABX Pentra 60 hematology analyzer (HORIBA, Irvine, CA, USA). The cellular viability was measured using flow cytometry (Accuri C6 Plus; Beckman Coulter Life Sciences, Indianapolis, IN, USA) by staining with propidium iodide and anti-CD45 antibody (BD Biosciences, San Diego). The percentage of monocytes was determined using flow cytometry (Accuri C6 Plus; Beckman Coulter Life Sciences) by labeling with propidium iodide and anti-CD14 antibody (BD Biosciences). In most circumstances, monocytes were enriched in fractions 4, 5 or 6. At least 109 monocytes were cultured to generate dendritic cells. Due to variations on donor materials, monocytes from single-fraction or combined fractions were used. Of 85 batches produced, 42 were manufactured using faction 6 alone, 35 from combined fractions 5 and 6, 4 from combined fractions 4, 5 and 6, 3 from fraction 5 alone and 1 from combined fractions 4 and 5.
Generation of dendritic cells
Monocytes were cultured in gas-permeable plastic bags (Corning, Corning, NY, USA) at a density of 5 × 105 cells/mL in AIM-V (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 800 U/mL granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA) and 500 U/mL interleukin-4 (R&D Systems) for 5 days, followed by the addition of 10 ng/mL recombinant human tumor necrosis factor-α (R&D Systems), 5 ng/mL recombinant human interferon (IFN)-γ (R&D Systems) and 2.5 µg/mL PGE2 (Cayman Chemical Company, Ann Arbor, MI, USA) into the culture bag. Mature dendritic cells were collected by centrifugation (300 x g) at day 7. For electroporation with in vitro transcribed mRNA, dendritic cells were resuspended in 4-mm gap cuvettes using the Gene Pulser X cell Electroporation System (Bio-Rad, Hercules, CA, USA) with proprietary parameters. After electroporation, cells were cultured for 4 h, collected by centrifugation, resuspended in CS10 (STEMCELL Technologies) and aliquoted in Daikyo Crystal Zenith Vials (West, Exton, PA, USA; 107 cells/vial). Each vial was placed in a CryoMed controlled-rate freezer (Thermo Fisher Scientific) until temperature reached to −130°C, then transferred to the liquid phase of liquid nitrogen for long-term storage until the day of distribution.
Cell counting and immunophenotyping analyses
The quantity and percentage of various immune cell types in leukapheresis product and elutriation fractions were analyzed by an ABX Pentra 60 hematology analyzer (HORIBA). For other experiments, FACS analysis (Accuri C6 Plus; Beckman Coulter Life Sciences) was used. Monocytes were enumerated by CD45+/CD14+ and propidium iodide–negative population (Beckman Coulter Life Sciences). Dendritic cells were gated by forward scatter and side scatter on the basis of cell size and cellular complexity, and further gated by anti-CD45 and propidium iodide. Phenotypic analyses of dendritic cells were performed using anti-CD209-APC, CD80-PE, CD83-PE, CD86-PE, CD11c-PE, HLA-DR-APC, HLA-ABC-APC and CCR7-APC (Beckman Coulter Life Sciences). To analyze cell type compositions in CUD-002 product, anti-CD45-APC, CD19-PE, CD20-PE, CD3-Percp-Cy5.5, CD56-PE, CD34-PE, lineage cocktail (CD3/CD14/CD16/CD19/CD20/CD56)-FITC, CD13-PE, CD33-PE and S100A9-PE (Beckman Coulter Life Sciences) were used.
Single-cell transcriptomic analysis
Cell suspension (20 µL, 106/mL, viability >95%) was loaded into Chromium microfluidic chips with 3´ reagent kit (v3.1 chemistry), and barcoded with a 10x Chromium Controller (10X Genomics, Pleasanton, CA, USA). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3´ v3.1 reagent kit (10X Genomics) following manufacturer's instructions. Sequencing was performed using Illumina NovaSeq platform following manufacturer's instructions. In total, 25,718 single cells from two healthy donors were analyzed. On average, approximately 45,000 reads were generated per cell by Illumina NovaSeq PE150. Raw binary base call files were converted to FASTQ files, aligned to human GRCh38 reference genome to produce sparse gene expression matrix by CellRanger v3.0 pipeline [
Massively parallel digital transcriptional profiling of single cells.
]. To filter out low-quality data, cells with percentage of counts in mitochondrial genes over 30% and with less than 100 genes or 200 UMI were removed from downstream analyses [
Single-cell RNA sequencing reveals cell heterogeneity and transcriptome profile of breast cancer lymph node metastasis.
]. Potential cell doublets were identified by DoubletFinder and removed downstream analyses [
- McGinnis C.S.
- Murrow L.M.
- Gartner Z.J.
DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors.
]. The expression matrix generated by CellRanger v3.0 was further processed and analyzed by Seurat v4.0. SCTransform to normalize UMI counts in each cell [
Integrated analysis of multimodal single-cell data.
]. Percentage of counts in mitochondrial genes and cell cycle scores were regressed out using vars.to.regress. variable.features.n was set to 5000 for the following analysis. Fifty principal components were calculated and their significances were tested using ElbowPlot [
Integrated analysis of multimodal single-cell data.
]. The top 30 principal components were analyzed by Seurat v4.0. FindNeighbors and uniform manifold approximation and projection (UMAP) visualization [
Dimensionality reduction for visualizing single-cell data using UMAP.
]. Batch effects were corrected using the Harmony package in UMAP visualization. Differentially expressed genes in each cell cluster were identified by Seurat v4.0 FindAllMarkers (log2
(fold change) > 0.5, min.pct = 0.5), and visualized using Seurat v4.0 DoHeatmap. Wilcoxon rank-sum test was used to compute P
-values adjusted for multiple testing using the Benjamini–Hochberg correction. To assign cell identity for each cluster, SingleR package was used to annotate the most plausible cell types, and marker genes were used to confirm their identities manually.
Functional analyses of the efficacy of CUD-002
CUD-002 products were co-cultured with autologous CD8+ T cells in AIM-V (Gibco/Thermo Fisher Scientific) supplemented with interleukin-2 (20 U/mL) for 14 days. The functionality of CUD-002-stimulated T cells was analyzed by three assays. First, the percentage of neoantigen-specific CD8+ T cells was determined by the major histocompatibility complex tetramer staining assay following manufacturer's instruction (MBL, Tokyo, Japan). To summarize, the stimulated CD8+ T cells were harvested and stained with PE-conjugated MART-1 peptide/HLA-A0201 tetramer (MBL) as well as fluorescein isothiocyanate–conjugated anti-human CD8 (BD Biosciences), and then were analyzed by flow cytometry (BD Biosciences). Second, the ability of neoantigen-specific CD8+ T cells to secret IFN-γ was measured by an automated ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH, USA). Third, neoantigen-specific CD8+ T cells were examined for their abilities to kill OVCAR-8 ovarian cancer cells in a cytotoxicity assay. To summarize, CD8+ T cells were incubated with OVCAR-8 ovarian cancer cells loaded with MART-1 peptide for 24 h. OVCAR-8 were also stably transfected with red fluorescent protein (mCherry) to facilitate its detection by FACS analysis.
To examine the tumorigenic potential of CUD-002 products, NOD/SCID mice (Beijing Vitalstar Biotechnology Co., Ltd., Beijing, China) were used with equal number of 7- to 8-week-old male and female mice. In accordance with the China National Medical Products Administration (NMPA) guidelines, this experiment was carried out by an independent contractor, JOINN Laboratories, Suzhou, China, under the Organisation for Economic Co-operation and Development (OECD) principles of Good Laboratory Practice in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)–accredited animal research facility. Each mouse was subcutaneously injected with a single dose of CUD-002 product containing 1 × 107 dendritic cells (4 × 108 cells/kg for a 25 g mouse) (n = 24). This quantity of dendritic cells will be used in human patients in the clinical trial (NCT05270720). As a positive control, six male and six female mice were subcutaneously injected with Hela cells (single dose of 1 × 106 Hela cells per mouse). Subsequently, all animals were assessed for body weight, macroscopic and microscopic changes, and tumor masses. Animals injected with HeLa cells were sacrificed after 56 days, due to tumor volume approaching 1 cm3. Animals injected with CUD-002 products were observed till 112 days. All masses formed at the site of injection were isolated and assessed by histopathology. None of mice injected with CUD-002 product formed any detectable tumor.
NOG-dKO mice (Charles River Laboratories, Beijing, China) were used with an equal number of 9-week-old male and female mice. The experiment was carried out by an independent contractor, JOINN Laboratories, Suzhou, China, under the OECD principles of Good Laboratory Practice in an AAALAC-accredited animal research facility. The potential toxicity profiles of CUD-002 product were assessed after repeated intravenous slow bolus injections in NOG-dKO mice (n = 60). Two doses were examined, containing 3 × 105 or 3 × 106 dendritic cells, respectively (1.2 × 107 or 1.2 × 108 cells/kg for a 25-g mouse, respectively). Dendritic cells were injected weekly for eight consecutive weeks. Eight doses were chosen because human patient will receive eight doses of CUD-002 (NCT05270720). During a period of 64 days, animals were constantly monitored and assessed for clinical signs, body weight, food consumption, functional and behavioral changes, clinical pathology such as clinical chemistry parameters (A/G ratio (calculated), alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, bilirubin (total), calcium, chloride, cholesterol (total), creatinine, globulin (calculated), glucose, phosphorus (inorganic), potassium, protein (total), sodium, triglycerides, urea; urinalysis: blood, pH, glucose, protein, urobilinogen, ketones, bilirubin, color and appearance, specific gravity, volume). At the end of the observation time, macroscopic changes and organ weights were recorded, and organs were subjected to histopathology examination.
Successful anti-tumor immunity largely depends on the cross-presentation of tumor-derived antigens by dendritic cells to cytotoxic T cells. This principle has been exploited to develop novel therapeutics that have achieved remarkable clinical efficacies in human cancers. The first cell-based immunotherapy approved by the US Food and Drug Administration to treat cancer is Sipuleucel-T, also known as PROVENGE or Dendreon, a dendritic cell–based cytotherapy. Since then, dendritic cells have been used as vessels to load tumor-specific antigens to treat cancer patients in many clinical trials. These trials have collectively demonstrated that dendritic cell–based therapies are usually safe in humans. However, the clinical efficacies vary among patients, suggesting that several key aspects need further optimization. In fact, there is currently no census on the best way to manufacture dendritic cells on an industrial scale.
In the present study, we demonstrate a fully developed process to reproducibly manufacture GMP-grade, therapeutic dendritic cell product (named CUD-002). CUD-002 is a versatile platform developed to load in vitro
–transcribed mRNA encoding any tumor-specific antigens of interest, and its efficacy is under clinical evaluation (NCT05270720). To manufacture CUD-002, peripheral blood monocytes are first collected by leukapheresis and then enriched by elutriation. Alternatively, antibody-based affinity purification strategy also can be used to enrich monocytes after leukapheresis. Elutriation is cheaper and requires fewer handing steps, although the resulting monocyte purity may be slightly lower than affinity purification with anti-CD14 antibodies. However, anti-CD14 antibodies may have the potential to activate monocytes [
Manufacturing Dendritic Cells for Immunotherapy: Monocyte Enrichment.
CD14: Biology and role in the pathogenesis of disease.
]. Here, we demonstrate that we have established a robust manufacturing process to produce dendritic cells with high efficiency using materials from more than a hundred donors. Regardless of elutriation or affinity purification, the final product inevitably contains cell types other than dendritic cells. The heterogeneity in cell type composition may elicit regulatory concerns. Using single-cell transcriptomic profiling, we identify lymphocyte, myelocyte and trace amount of NK and HSC present in CUD-002. These cells likely originate from leukapheresis product because we can detect their presence by FACS analysis. Single-cell sequencing and FACS analysis also demonstrate a complete absence of monocytes and granulocytes in CUD-002. These two cell types are the most abundant ones in leukapheresis product, further demonstrating a highly efficient manufacturing process. We anticipate that single-cell sequencing will become an indispensable tool to guide the development of manufacturing process to ensure reproducibility and high quality for cytotherapies.
Published online: November 25, 2022
© 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc.