(A) study of enhancing the therapeutic efficacy of adoptive T cell therapy in cancer treatment

다국어 초록 (Multilingual Abstract)

In recent years, immunotherapy has emerged as a fourth pillar of cancer treatment other than conventional three pillars of cancer therapy: chemotherapy, and radiotherapy, and surgery. There has been increasing interest in optimizing this immunotherapy due to a unique approach for cancer treatment, using the patient’s immune system to recognize and kill tumor cells. Among many modalities in immunotherapy, adoptive T cell transfer (ACT) is the primary modality that uses tumor specific cytotoxic T lymphocytes to recognize and destroy tumor cells. ACT including chimeric antigen receptor (CAR) T cell therapy and T cell receptor (TCR) T cell therapy, have achieved significant improvements but the therapeutic result is varying among patients with same treatment. To minimize the therapeutic gap between the patients, it important to track the adoptively transferred T cells to understand the transfer route, in vivo biodistribution and tumor-targeting ability. Moreover, the ACT has been showed potent therapeutic in liquid tumor and melanomas, however therapeutic outcome of patients with solid tumor is relatively poor due to immunosuppressive tumor microenvironment (TME). TME possess variety of factors that influence the therapeutic result of ACT such as think layer extracellular membrane that impedes the T cell infiltration, immune checkpoint receptors on tumor cells, and low-oxygen and acidic environment that causes T cell exhaustion or senescence of T cell. In order to resolve the issues that hamper the outcome of ACT, we need to develop 1) noninvasive and stable cell tracing strategy for understanding the T cell biodistribution and evaluating the efficacy of T cell therapy, 2) remodeling of thick ECM surrounding tumor tissue to enhance the T cell accumulation in tumor region, 3) combinational therapy with other conventional therapy to establish synergetic effect for complete tumor eradication.

목차 (Table of Contents)

• TABLE OF CONTENTS
• List of Figures i
• List of abbreviations iii
• Abstracts v
• Chapter I. Introduction………………………………………………………….….1
• 1.1. Cancer immunotherapy…………………………………………………..…2
• 1.2. Research Rationale…………………………………………………………5
• 1.2.1. Significance of study………………………………………………...5
• 1.2.2. Aim of study…………………………………………………………6
• Chapter II. In Vivo Tracking of Bioorthogonally Labeled T-cells for Predicting Therapeutic Efficacy of Adoptive T-cell Therapy…………………………………9
• 2.1. Introduction………………………………………………………………...9
• 2.2. Materials ………………………………………………………………….14
• 2.2.2. T cell isolation, activation, and expansion …………………………15
• 2.2.3. In vitro cytotoxicity.………………………………………………...16
• 2.2.4. In vitro azide generation and bioorthgonal labeling of CTLs………17
• 2.2.5. In vitro proliferation and functional assays of Cy5.5-CTLs…………19
• 2.2.6. NIRF imaging-based non-invasive tracking of Cy5.5-CTLs and evaluation of therapeutic efficacy in immune-deficient tumor-bearing mouse model……………………………………………………………………...21
• 2.2.7 NIRF imaging-based non-invasive tracking of Cy5.5-CTLs for prediction of therapeutic response using immune-competent tumor-bearing mouse model………………………………………………………………23
• 2.2.8. Immunofluorescence (IF) staining of tumor tissues……………..….24
• 2.2.9. Statistical analysis…………………………………………………..24
• 2.3. Result and discussion…………………………………………………….. 26
• 2.3.1. Incorporating azide groups in cultured CTLs via metabolic glycoengineering………………………………………………………….26
• 2.3.2. In vitro proliferation and functional assays of Cy5.5-CTLs………....32
• 2.3.3. In vivo tracking of Cy5.5-CTLs in immune-deficient tumor-bearing mice.………………………………………………………………………37
• 2.3.4. Correlation between tumor-specific homing and therapeutic efficacy of Cy5.5-CTLs in an immune-deficient tumor-bearing mouse model…….41
• 2.3.5. NIRF imaging-based prediction of therapeutic response of Cy5.5-CTLs in immune-competent tumor-bearing mouse model………………..47
• 2.4. Conclusion………………………………………………………………...52
• Chapter III. Remodeling of Tumor microenvironment using PH20 hyaluronidase to enhance adoptive T cell therapy ....……………………………………………54
• 3.1. Introduction……………………………………………………………….55
• 3.2. Materials and Methods……………………………………………………58
• 3.2.1. Materials……………………………………………………………58
• 3.2.2. Transfection of PH20 DNA plasmid to HEK293T…………………59
• 3.2.3. Characterization of PH20 exosome ………………………………...59
• 3.2.4. T cell isolation, activation, and expansion………………………….60
• 3.2.5. In vitro azide generation and bioorthgonal labeling of CTLs………61
• 3.2.6. In vivo tracking of Cy5.5-CTLs…………………………………….61
• 3.2.7. Immunofluorescence (IF) staining of tumor tissues………………...62
• 3.2.8. Statistical analysis…………………………………………………..63
• 3.3. Results ……………..……………………………………………………...64
• 3.3.1. Preparation and characterization of PH20 exosome………………..64
• 3.3.2. Tumor accumulation Cy5.5-CTLs in B16F10 OVA tumor-bearing mice……...…………………………………………………………….….68
• 3.4. Conclusion………………………………………………………...............72
• Chapter IV. Adoptive transfer of Temoporfin loaded cytotoxic T lymphocytes for combination of immuno- and photodynamic therapy……………………...…...73
• 4.1. Introduction…………………………………………………………….....74
• 4.2. Materials and Methods……………………………………………………78
• 4.2.1. Materials……………………………………………………………78
• 4.2.2. T cell isolation, activation, and expansion………………………….79
• 4.2.3. Preparation and loading of mTHPC to OT-1 CTLs…………………80
• 4.2.4. In vitro proliferation and functional assays of Cy5.5-CTLs…………81
• 4.2.5. Cellular uptake of mTHPC by OT-I CTLs via flow cytometry and confocal laser scanning microscopy (CLSM)………………………...…...81
• 4.2.6. Quantitative analysis of mTHPC loaded OT-I cells using UV/Vis spectroscopy…...…………………………………………………....…….82
• 4.2.7. Photodynamic therapy of tumor-bearing mice……………………...83
• 4.2.8. Statistical analysis……………………………………………...…...83
• 4.3. Results……………………………………..……………………………...85
• 4.3.1. mTHPC uptake by OT-1 CTLs………………………………….….85
• 4.3.2. Antigen specific transfer and in vitro cytotoxic function of loaded OT-1 CTLs……………………………………………………………….........91
• 4.3.3. In vitro cytotoxic function of loaded OT-1 CTLs…………………...94
• 4.3.4. In vivo anti-tumor effect of loaded OT-1 CTLs…………………….97
• 4.4. Conclusion……………………………………………………………….100
• Chapter V. Reference………………...…………………………………………...101
• Chapter VI. Summary…………………………………………………………....118
• Abstract in Korean…………………………………………………………………120