Preparation and Characterization of PEGylated TRAIL for Improved Biological Potency via Introtumoral Route = 종양 내 투여 경로를 이용한 향상된 효과의 PEG접합 TRAIL의 제조 및 특성화 연구

다국어 초록 (Multilingual Abstract)

TRAIL/Apo2L is a novel cytokine since it induces apoptosis in a number of tumor cells without affecting normal cells. The cancer-directed, apoptotic potential of TRAIL makes it a promising candidate for therapeutic interventions. TRAIL itself, however, is inactivated rapidly in vivo and thus does not appear to be useful as a therapeutic agent in the long-term treatment. To solve this problem, we suggest the local injection of PEG modified TRAIL at its N-terminus as a cancer therapy, which is able to enhance the biological activity by reducing renal ultrafiltrayion, preventing the approach of antibodies or antigen processing cells and reducing the enzymatic degradation. As systemic delivery of therapeutic agent to tumor is limited because of the physiologic barriers, local drug delivery is more useful for cancer therapy.
In the present study, we report here that the administration of TRAIL and PEG modified TRAIL. TRAIL and PEG modified TRAIL resulted in remarkable concentration-dependent cytotoxicity in human head and neck cancer cell. In regard to the side effect, both of two drugs showed no cytotoxicity against the normal epitherial cell compared with human head and neck cancer cell. Consequently, effective antitumor activity by PEG modified TRAIL can be accomplished without any apparent toxicity to normal tissues and organs. In addition, the tumor growth was significantly suppressed in balb/c nude mice intratumorally administered PEG-TRAIL versus TRAIL.
The study demonstrates that polyethylene glycol (PEG) can be a useful chemical enhancer to increase intatumoral bioavailability of protein treatments like TRAIL.

다국어 초록 (Multilingual Abstract)

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL)은 종양세포에 대한 선택적인 세포사멸작용으로 인해 매력적인 항암치료제로 각광을 받고 있다. 그러나, TRAIL은 생체 내에서 빠르게 그 활성이 감소되기 때문에 Long-term 치료제로서의 단점을 가지고 있다. 이러한 문제점을 극복하기 위해, N 말단 PEG 접합 TRAIL의 종양 내 투여를 모색하였다. PEG 접합을 통하여 신배설을 감소시키고 항체의 접근을 막으며, 효소적 분해를 감소시켜 생체활성을 증가시킬 수 있다. 또한 약물의 전신순환적 전달은 다양한 신체상 장벽에 의해 극히 제한될 수 있으므로 국소 전달은 항암치료에 매우 유용하다.
본 연구에서는 TRAIL과 PEG 접합 TRAIL의 투여를 보고하였다. TRAIL과 PEG 접합 TRAIL은 구경부암 세포에 뚜렷한 농도 의존적 세포독성을 나타내었다. 항암 약물의 부작용을 고려해보았을 때, 두 약물은 정상 상피세포에서 세포독성을 나타내지 않았다. 따라서, TRAIL과 PEG 접합 TRAIL은 생체 내 정상 조직과 기관에 뚜렷한 독성을 나타내지 않는 효과적인 항암제라고 할 수 있다. 또한, PEG 접합 TRAIL은 balb/c 누드 마우스에 종양 내 투여를 실시하였을 때 TRAIL에 비해 더 증가된 암 성장 억제를 보였다.
이 연구에서는 TRAIL과 같은 단백질 약물의 PEG 접합을 통한 제형화가 약물 국소 전달을 위한 유용한 방법이 될 수 있음을 제시하고 있다.

목차 (Table of Contents)

• Ⅰ. Introduction = 1
• 1. Tumor Nectosis Factor-Related Apoptosis Inducing Ligand = 1
• 2. Local delivery = 8
• 3. PEGylation = 11
• 4. Nickel affinity Chromatography for Protein purification = 20
• 5. MALDI-TOF mass spectrometry = 27
• 6. The scope of this study = 31
• Ⅱ. Materials = 32
• 1. Chemicals = 32
• 2. Instruments = 34
• Ⅲ. Methods = 36
• 1. Expression and Purification of His-ILZ- TRAIL (114-281) = 36
• 1.1. Expression of His-ILZ- TRAIL (114-281) = 36
• 1.2. Purification of His-ILZ- TRAIL (114-281) = 37
• 2. Preparation and Purification of Polyethylene Glycol-modified TRAIL (114-281) = 38
• 2.1. Preparation of polyethylene glycol-modified TRAIL (114-281) = 38
• 2.2. Separation and purification of polyethylene glycol-modified TRAIL (114-281) = 38
• 3. Characterization of PEG-TRAIL = 39
• 3.1. MALDI-TOF mass spectrometry of PEG-TRAIL = 39
• 3.2. Endoproteinase Lys-C digestion = 40
• 3.3. SDS-PAGE analysis for identification of TRAIL and PEG-TRAIL = 40
• 3.4. Western blotting analysis for identification of TRAIL and PEG-TRAIL = 41
• 4. Screening of anti-tumor activity in vitro = 43
• 4.1. HN9 cell culture = 43
• 4.2. L-132 cell culture = 43
• 4.3. MTT assay = 44
• 5. Biological stability of TRAIL and PEG-TRAIL based on cytotoxicity = 45
• 5.1. HN9 cell culture = 45
• 5.2. Preparation of TRAIL and PEG-TRAIL samples for stability test = 45
• 5.3. MTT assay = 46
• 6. Biological activity of TRAIL and PEG-TRAIL in vivo = 47
• 6.1. Balb/c nude mice = 47
• 6.2. In vivo anti-tumor activity test of TRAIL and PEG-TRAIL = 47
• Ⅳ. Results and Discussion = 48
• 1. Expression and Purification of His-ILZ- TRAIL (114-281) = 48
• 2. Preparation and Purification of Polyethylene Glycol-modified TRAIL (114-281) = 51
• 3. Characterization of PEG-TRAIL = 54
• 4. Screening of anti-tumor activity in vitro = 58
• 5. Biological stability of TRAIL and PEG-TRAIL based on cytotoxicity = 62
• 6. Biological activity of TRAIL and PEG-TRAIL in vivo = 64
• Ⅴ. Conclusion = 66
• Ⅵ. References = 68
• Abstract = 72
• Abstract in Korean = 74