Doctoral Dissertation Tumor antigen development for dendritic cell-based cancer immunotherapy against multiple myeloma Department of Molecular Medicine Graduate School, Chonnam National University HOANG My Dung August 2015 Tumor antigen development for dendritic cell-based cancer immunotherapy against multiple myeloma Department of Molecular Medicine Graduate School, Chonnam National University HOANG My Dung Supervised by Professor LEE Je-Jung A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy in Science, has been deemed acceptable by the individuals below. Committee in Charge : RHEE Joon Haeng NAM Jong-Hee CHUNG Ik-Joo PARK In-Kyu LEE Je-Jung August 2015 CONTENTS LIST OF FIGURES.1 PART I: Branched polyethylenimine-superparamagnetic iron oxide nanoparticles (bPEI-SPIONs) improve the immunogenicity of tumor antigens and enhance Th1 polarization of dendritic cells ································· 3 I. Materials and Methods ······················································································ 5 I.1) Synthesis and characterization of bPEI-SPION ······························ 5 I.2) Intracellular ferric iron measurement ················································ 5 I.4) Assays of ROS generation ····································································· 6 I.5) Generation of monocyte-derived DCs ··············································· 6 I.6) Tumor antigen preparation ····································································· 7 I.7) Surface heat shock protein (Hsp) expression on tumor antigens ······················································································································ 7 I.9) Antigen uptake assay ··············································································· 8 I.10)Phenotypic analysis of DCs ··································································· 8 I.12)Human IL-12p70 and IL-10 production ··········································· 9 I.13)Allogeneic naïve CD4+ T cell polarization assay ························· 9 I.14)Intracellular staining for cytokine expression ································ 9 I.1) Optimal bPEI-SPION concentration for uptake by U266 cells.2) bPEI-SPION pretreatment accelerates apoptotic cell death after UVB irradiation with induction of ROS production ······················ 11 I.3) Eating-me signal surface Hsp70 and Hsp90 expression and danger signal release were observed in 2-h post-irradiated and bPEI-SPION 2-h post-irradiated cells ························································ 13 I.4) bPEI-SPION 2-h post-irradiated cells enhance Th1 polarization without altering DC surface marker expression and DC migration ················································································································· 15 I. Discussion ············································································································ 17 PART II: Evaluation of tumor antigen expression in Korean multiple myeloma patients and development of recombinant protein for dendritic cell-based vaccine against multiple myeloma.
Materials and Methods ·················································································· 25 II.1) Patient samples ······················································································· 25 II.2) RNA isolation, cDNA synthesis and quantitative real-time PCR ··························································································································· 26 II.3) Expression data and statistic analysis ··········································· 26 II.1) Target gene expression pattern in MM patients of light chain type ··························································································································· 27 II.2) Target gene expression pattern in MM patient of IgG type 28 ii II.3) Target gene expression pattern in MM patientsof IgA type.4) BCMA and hTERT are candidates for MM vaccine development ··········································································································· 30 II. Discussion ·········································································································· 31 Part III: Dendritic cell-based cancer immunotherapy against multiple myeloma: from bench to clinic ·················································································· 38 III. Dendritic cell-based cancer immunotherapy ······································· 39 III. Multiple myeloma immunity ········································································ 41 III.
Dendritic cell-based immunotherapy against multiple myeloma ·· 41 III.1) Id-pulsed DCs ······················································································ 41 III.2) MM-associated antigen-loaded DCs ············································ 42 III.3) Whole tumor antigen-loaded DCs ················································ 43 III. Improvement of DC-based cancer immunotherapy ··························· 44 III.1) Type 1-polarized DCs ······································································ 44 III.2) Tumor antigens to load onto DCs ················································ 47 III.3) Regulation of tumor suppressive microenvironment ·············· 48 III. Future perspectives of DC-based cancer immunotherapy ············· 49 ABSTRACT (KOREAN).60 iii LIST OF FIGURES Figure I- 1: Uptake of branched polyethylenimine-superparamagnetic iron oxide nanoparticles (bPEI-SPION) by U266 cells ············································· 10 Figure I- 2: Characterization of U266 cells after UVB irradiation in the presence or absence of bPEI-SPIONs. ·································································· 12 Figure I- 3: Damage-associated molecular pattern (DAMP) production by dying tumor cells induced by apoptotic pathway ··············································· 14 Figure I- 4: Characterization of DCs loaded with U266 cells ····················· 16 Figure I- 5: T cell polarization of DCs.
······························································· 17 Figure II- 1: Target gene expression in MM patients of light chain type ······································································································································ 27 Figure II- 2: Target gene expression in lgG type MM patient ··················· 28 Figure II- 3: Target gene expression in IgA type MM patient. ·················· 29 Figure II- 4: Target gene expression analysis in total 9 MM patients. ··· 30 Figure III- 1: Critical points to improve cancer immunotherapy using dendritic cells in cancer patients ············································································· 40 iv ABBREVIATIONS BCMA B-Cell Maturation Antigen bPEI-SPION branch PolyEthylImine-SuperParamagnetic Iron Oxide Nanoparticles Cox-2 Cyclo-oxygenase-2 CTA Cancer Testis Antigen CTL Cytotoxic T Lymphocytes DC Dendritic Cell DCFH-DA 2',7'-Dichlorofluorescein-diacetate DEPDC1A Disheveled, EGL-10, Pleckstrin Domain Contained protein 1A DKK1 Dickkopf-1 DNA Deoxyribo Nucleic Acid ER Endoplasmic Reticulum FasL Fas ligand FBS Fetal Bovine Serum GM-CSF Granulocyte Macrophage Colony Stimulating Factor HMGB1 High-Mobility Group Box 1 Hsp Heat shock protein hTERT human Telomerase Id Idiotype IFN Interferon IL Interleukin IMDM Iscove's Modified Dulbecco's Media IMiD Immunomodulatory Drug MACS Magnetic Activating Cell Sorting MDSC Myeloid Derived Suppressor Cells MFI Mean Fluorescence Intensity v MHC Major Histocompatibility Complex MM Mutiple Meloma MMP Matrix MetalloProteinases NAC N-acetyl cysteine NK Natural Killer qPCR Quantitative real-time Polymerase Chain Reaction RNA RiboNucleic Acid ROS Reactive Oxygen Species RPMI Roswell Park Memorial Institute PBMC Peripheral Blood Mononuclear Cells PBS Phosphate Buffered Saline pDC plasmacytoid Dendritic Cell PMA PhorbolMyristate Acetate P/S Penicillin/Streptomycin PTD Protein Transduction Domain URP Unfolded Protein Response UVB Ultraviolet B VEGF Vascular Endothelial Growth Factor SDS Sodium Dodecyl Sulfate siRNA small interference RiboNucleotide Acid STEAP1 Six Transmembrane Epithelial Antigen of Prostate 1 TAA Tumor Associated Antigens TEM Transmission Electron Microscopy TGF Transforming Growth Factor TNF Tumor Necrosis Factor Tregs regulatory T cells vi Tumor antigen development for dendritic cell-based cancer immunotherapy against multiple myeloma ABSTRACT: Although introduction of stem cell transplantation and novel agents has improved in survival, MM is still difficult to cure. Alternative approaches are clearly needed to prolong survival of patients with MM.
DC therapy is a very promising tool to improve MM treatment. Several approaches could be employed to generate effective DC vaccine. Among them, development of tumor antigen is an important goal. In part I, the thesis presents effect of bPEI-SPIONs on U266 tumor antigen preparation which was loaded onto DCs.
The tumor antigens were prepared as follows: 1) apoptotic U266 cells with UVB-irradiation followed by a 2-h incubation in the absence (2-h post-irradiated cells) or 2) presence of bPEI-SPIONs (bPEI-SPION 2-h post-irradiated cells) and 3) apoptotic U266 cells with UVB-irradiation followed by an overnight 16-h incubation (16-h post-irradiated cells). bPEI-SPIONs render U266 cells sensitive to UVB-irradiation through reactive oxygen species production to accelerate apoptotic death. The 2-h post-irradiated cells and bPEI-SPION 2-h post-irradiated cells released immunogenic proteins, including Hsp70, Hsp90, and HMGB1. The DCs loaded with bPEI-SPION 2-h post-irradiated cells showed the highest IL-12p70 production and Th1 polarization compared with other DCs.
These results suggest that bPEI-SPIONs are a promising method of enhancing the immunogenicity of tumor cells and promoting Th1 polarization of DCs loaded with these tumor cells. 1 In part II, certain tumor antigen, related to poor overall survival, expression was evaluated in Korean MM patients. Results showed that BCMA and hTERT are good candidates for tumor antigen development. These mentioned results promise effective DC vaccine generations against MM.
In part III, we described several approaches of DC-based vaccine development for application of this method to clinical setting in MM. PART I: Branched polyethylenimine-superparamagnetic iron oxide nanoparticles (bPEI-SPIONs) improve the immunogenicity of tumor antigens and enhance Th1 polarization of dendritic cells Abstract Nanoparticles in the field of DC research are emerging as a promising method of enhancing the efficacy of cancer immunotherapy. We investigated the effect of branched (bPEI-SPIONs) on tumor cells loaded onto DCs. The tumor antigens were prepared as follows: 1) apoptotic U266 cells with ultraviolet B (UVB)-irradiation followed by a 2-h incubation in the absence (2-h post-irradiated cells) or 2) presence of bPEI-SPIONs (bPEI-SPION 2-h post-irradiated cells) and 3) apoptotic U266 cells with UVB-irradiation followed by an overnight 16-h incubation (16-h post-irradiated cells).
bPEI-SPIONs render U266 cells sensitive to UVB-irradiation through ROS production to accelerate apoptotic death. The 2-h post-irradiated cells and bPEI-SPION 2-h post-irradiated cells released immunogenic proteins, including Hsp70, Hsp90, and HMGB1. The DCs loaded with bPEI-SPION 2-h post-irradiated cells showed the highest IL-12p70 production and Th1 polarization compared with other DCs. These results suggest that bPEI-SPIONs are a promising method of enhancing the immunogenicity of tumor cells and promoting Th1 polarization of DCs loaded with these tumor cells.
Introduction Lack of specific hallmark of cancer is reason for using of whole tumor cells (tumor apoptotic bodies, tumor cell lysates, or tumor cell-derived RNA), which represent full characteristics of tumor identity, as common source of tumor antigens in clinical trials of DC-based cancer [1, 2] vaccines. Among these antigen preparation procedures, UVB irradiation is a safe, inexpensive, and easy method of inducing a mixed population of viable, early apoptotic, and late apoptotic/necrotic cells with various proportions during tumor antigen preparation [3, 4]. However, the immunogenic properties of prepared tumor antigens depend on the cell death stage. Engulfment of the early apoptotic body leads to silent phagocytosis with anti-inflammatory activity, whereas phagocytes are activated when encountering late apoptotic/necrotic cells; as a result, the [5, 6] latter gives rise to an inflammatory response.
In our previous studies, apoptotic cells or dying tumor cells, used as a tumor antigen source, showed high anti-tumor induction efficacy of DCs to T cells [7, 8]. To develop novel techniques for tumor antigen preparation, we induced immunogenic cell death using JSI-124 combined with bortezomib in MM [4]. [9] Recently, SPIONs have been reported to enhance ROS production. Based on our previous studies on DCs, we suppose that SPIONs accelerate tumor cell death to an immunogenic induction stage; hence, the antigen can be more highly immunogenic than UVB-irradiated tumor antigens.
SPIONs are an interesting tool for cell labeling, cell therapy, and diagnostic imaging. However, uncoated SPIONs can cause toxicity to living cells, and coating materials have been developed to stabilize [10] aqueous SPION suspensions and reduce toxicity. bPEI-SPIONs, iron oxide nanoparticles coated with bPEI, are less toxic than SPIONs and [11] readily bind to the cell membrane to enhance their uptake. 4 Here, we investigated the immunogenicity of tumor antigen sources prepared from UVB-irradiated tumor cells in the presence of bPEI-SPIONs during T cell responses elicited by DCs loaded with these tumor antigens.
We showed that bPEI-SPIONs accelerated UVB-irradiated cell death to the late apoptotic/necrotic stage after 2-h incubation. Furthermore, prepared antigen with bPEI-SPIONs induced the highest production of IL-12p70 of DCs, and these DCs favored Th1 polarization during the T cell response. Materials and Method I.1) Synthesis and characterization of bPEI-SPION bPEI-SPION was synthesized by conjugation of low molecular weight bPEI (Mw 1,800 Da, Aldrich) onto thermally cross-linked SPION [12] (TCL-SPION) via amide linkage. The physic-chemical properties of bPEI-SPION were further characterized by using Zetasizer Nano Z (Malven Instruments, Malvern, UK), TEM (JEOL JEM-2000 FXII, Japan) and TGA analysis (Mettler-Toledo, SDT851, Columbus,USA) in order to confirm its successful synthesis.2) Intracellular ferric iron measurement bPEI-SPION uptake by the U266 MM cell line was evaluated using [13] 5 a quantitative spectrophotometric method.
Briefly, 5 × 10 U266 cells were put in contact with different amount of bPEI-SPIONs in shaking for 1-h at room temperature. Cells were collected and washed three times in 1×PBS (Sigma Aldrich, St.Louis, MO, USA). The pellet was resuspended in 30% HCl (Sigma–Aldrich) for 2-h at 60°C.08% potassic persulfate, 8% potassium thiocyanate, and 3.6% HCl (Sigma Aldrich) were added to form the iron-thiocyanate complex. The absorbance at 490 nm was measured using a microplate reader (TECAN Infinite M200 PRO, Tecan, Männedorf, Switzerland) after 10-min incubation.6H2O (SigmaAldrich) solution was treated in the same manner to create the standard curve.3) Confocal microscopy U266 cells were put in contact with bPEI-SPIONs conjugated with FNR-675 dye (BioActs, Namdong-gu, Incheon, Korea), which appears as a red color under confocal microscopy (Carl Zeiss, Jena, Germany).
Cells were fixed on a glass slide and the nuclei were stained with DAPI (Thermo Scientific Pierce, Rockford, USA), which appears as a blue color.4) Assays of ROS generation DCFH-DA (Sigma–Aldrich) and NAC (Sigma–Aldrich), which blocks ROS production, were used to determine intracellular ROS levels based on fluorescence measurements. Briefly, cells were incubated in warm RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% FBS (PAA, Murarrie, Australia) and 1% P/S (Lonza, Walkersville, MD, USA) with 6 DCFH-DA at 37°C for 1h[14]. The probe was then removed and cells were used for preparation of several types of antigen.