第一部分：以液相層析串聯質譜儀搭配磁珠上酵素消化法建立高效率的單株抗體定量平台 第二部分：評估使用BEEP療法之乳癌腦或腦膜轉移病患bevacizumab濃度與臨床反應之關聯性

Part I: Development of an efficient mAb quantification assay by LC-MS/MS using rapid on-bead digestion Part II: Evaluation of association between bevacizumab concentration and clinical response in patients with breast cancer brain or leptomeningeal metastasis receiving BEEP regimen

蔡昀蓉(Yun-Jung Tsai)

指導教授 ： 郭錦樺

國立臺灣大學/醫學院/藥學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102718

摘要

隨著生物醫療科技進步，單株抗體藥物的開發日漸蓬勃，並且廣泛地被運用在各種疾病治療，如癌症、自體免疫疾病及代謝性疾病之上。臨床上，單株抗體體內血漿濃度因人而異，可能隨之影響其對病人之治療反應。近年來，精準醫療備受關注，然而有關單株抗體個人化療法的劑量知之甚少，若能提供有效率且準確的定量方法，將對單株抗體的藥物濃度-療效反應研究大有助益。但目前定量單株抗體的方法通常很耗時，且需要繁瑣的樣品製備。本研究使用液相層析串聯質譜儀搭配磁珠上酵素消化開發快速且高效率的單株抗體定量平台，並進一步進行臨床研究，探討人體血漿樣品內bevacizumab濃度及臨床反應之關係，期望可以提供臨床研究廣用的分析方法，並了解bevacizumab濃度之臨床意義，促進單株抗體藥物個人化醫療之發展。
本論文分成兩個部分，第一部份，我們開發了磁珠上酵素消化搭配高溫縮時的純化、消化方法，搭配液相層析質譜儀作為定量平台。此研究選用五種不同適應症及不同IgG子分類的單株抗體藥物：bevacizumab、evolocumab、nivolumab、pembrolizumab、trastuzumab進行方法開發及測試，並以tocilizumab做為內標準品，校正製備過程中可能產生的誤差。本方法以Protein G磁珠純化單株抗體藥物，並改良以酸性溶液從磁珠分離純化完成的抗體之過程，直接於磁珠上進行酵素消化，同時搭配高溫加速消化過程，使製備過程更為簡便及快速。此方法大幅改善先前樣品製備方法再現性不佳的問題，同時也提升多數單株抗體藥物的測量強度。確效結果顯示，五種單株抗體藥物的定量準確度均在 94.5 ± 5.2% 至 111.6 ± 3.7% 之內，在日內和日間的再現性評估，相對標準差均分別低於 6.1% 和 9.5%。此方法也成功應用於在不同治療周期下對 6 名乳癌患者的trastuzumab血中最低濃度測量，濃度範圍為 67.3 至 171.8 μg mL-1。此方法對於大量臨床樣本的真實世界分析更有效、更實用，可用於常規治療藥物監測，並有助於發展個人化單株抗體藥物治療。
本論文第二部份將液相層析串聯質譜平台的單株抗體定量方法運用在22位使用bevacizumab、etoposide、cisplatin合併療法 (BEEP療法)的乳癌腦轉移病人血漿bevacizumab濃度測量，我們分別測量療程第一周期及最後周期(第六周期)之最低bevacizumab血中濃度，同時評估bevacizumab於此類病人中體內濃度與臨床反應療效、疾病無惡化存活期(Progression-free survival, PFS)、整體存活率(Overall survival, OS)之關係。為了驗證bevacizumab能提升化療藥物遞送至腦部的推論，我們同時測量了6位腦膜轉移病人在施打bevacizumab前後血漿中及腦脊髓液中etoposide藥物的濃度用以計算穿透率，同時也測量了bevacizumab的濃度並計算其曲線下面積(Area under curve, AUC)，並評估etoposide穿透率與bevacizumab曲線下面積之關聯性。於bevacizumab濃度與療效研究結果顯示，患者的第一周期、第六周期最低血漿中bevacizumab濃度與臨床反應、PFS 和 OS 無關，且bevacizumab在治療的六個周期間會逐漸累積。而bevacizumab AUC (0-6) 與etoposide穿透率研究結果顯示，bevacizumab AUC (0-6) 在腦膜轉移患者中與etoposide 穿透率呈正相關。考量bevacizumab血漿中濃度並沒有和療效呈現直接的相關性，且在治療期間濃度會逐漸累積，本研究認為監測bevacizumab的治療濃度以緩解藥事經濟的成本是未來可以探討的方向，同時也可避免因藥物累積至高濃度而衍生的潛在副作用，達成精準醫療，實現成本效益。未來需要更進一步研究以提出bevacizumab在乳癌腦轉移病人中的最佳濃度範圍。
綜上所述，本論文開發了高效率的單株抗體分析方法，加速臨床上實施單株抗體藥物療劑監測的便利性及適用性；並以接受BEEP療法之乳癌腦轉移患者血漿檢體評估了bevacizumab 濃度與療效、疾病無惡化存活期、整體存活率之相關性，探討bevacizumab濃度在BEEP療法中的腳色，結果顯示監測bevacizumab治療劑量有其合理性，未來如能結合大型的收案進行驗證，將有助於建立BEEP療法中bevacizumab的劑量使用參考依據。

關鍵字

單株抗體藥物 ； 療劑監測 ； BEEP療法 ； 液相層析質譜法 ； Protein G 純化 ； 磁珠上酵素消化 ； 高溫酵素消化

並列摘要

With the advancement of biomedical technology, the development of monoclonal antibody (mAb) therapeutics is flourishing and mAbs are widely used in the treatment of various diseases including cancer, autoimmune diseases and metabolic diseases. Clinically, the mAb plasma concentration of monoclonal antibodies varies between individuals, which might subsequently affect the treatment response. In recent years, precision medicine has gained increasing attention. However, little is known about the optimal dosage of monoclonal antibody for personalized therapy. An efficient and accurate quantification method could facilitate the concentration-response relationship research of mAbs. However, current methods for quantifying monoclonal antibodies are usually time-consuming and require tedious sample preparation. In this study, we developed a rapid and efficient on-bead digestion method coupled to LC-MS/MS for mAb quantification, and further investigated the relationship between plasma bevacizumab concentration and clinical response. We look forward to providing a general analytical method for clinical researches, understanding the role of bevacizumab concentration in clinical response, and shedding light on personalized medicine of mAb therapy.
This thesis is divided into two parts. In the first part of this thesis, we developed an efficient LC-MS/MS method using an on-bead trypsin digestion procedure at a higher digestion temperature at 60 C. Five mAbs from different IgG subclasses, bevacizumab, evolocumab, nivolumab, pembrolizumab, and trastuzumab, used for treating different diseases, were selected for method development. Tocilizumab was selected as the internal standard to calibrate the potential preparation errors. This method used Protein G magnetic beads to purify mAbs. On-bead digestion method was applied to improve the tedious elution process in conventional low-pH elution method. Moreover, the digestion process was performed under high temperature to improve the trypsin digestion efficiency. Our results indicated the on-bead preparation is more convenient with better reproducibility compared to the conventional low-pH elution method while enhance the signal intensity in LC-MS/MS for most mAbs. The validation results showed that the quantitative accuracies of the five monoclonal antibody drugs were all within 94.5 ± 5.2% to 111.6 ± 3.7%, and the repeatabilities and intermediate precisions were all within 6.1% and 9.5% RSD, respectively. This method was successfully applied to the measurement of plasma trastuzumab trough concentration from six breast cancer patients under different treatment cycles, with a concentration range of 67.3 to 171.8 μg mL-1. This method is more efficient and practical for real-world analysis of a large number of clinical samples and could be used for routine therapeutic drug monitoring.
In the second part, an LC-MS/MS analytical method was used to measure the bevacizumab plasma concentration in 22 patients with brain metastases from breast cancer under the combination therapy of bevacizumab, etoposide, and cisplatin (the BEEP regimen). We measured the trough concentration of the first and the last (the sixth) cycles of the treatment course and evaluated the relationship between the bevacizumab concentration and clinical response, progression-free survival (PFS), and overall survival (OS). To verify the hypothesis that bevacizumab could enhance the penetration of chemotherapeutic drugs to the brain, we further measured the etoposide plasma concentration and etoposide CSF concentration before and after bevacizumab administration to calculate the penetration to brain in 6 patients with leptomeningeal metastasis. Bevacizumab plasma concentration was also measured and the AUC (0-6) was calculated. The correlation between etoposide penetration and bevacizumab AUC (0-6) was evaluated. In the evaluation of bevacizumab concentration-clinical response relationship, the results indicated that the bevacizumab trough concentration in the first and sixth cycles were not related with clinical response, PFS and OS. We also observed that bevacizumab gradually accumulated from cycle one to cycle six. In the evaluation of the improvement in etoposide penetration, the results indicated that bevacizumab AUC (0-6) was positively correlated with etoposide penetration in patients with leptomeningeal metastases. Since bevacizumab plasma concentration was not related to clinical response and bevacizumab could accumulate in body during treatment course, our results raised the potential of monitoring therapeutic dose of bevacizumab which could not only reduce economic burden but also alleviate potential side effect and to achieve precision medicine and cost-effectiveness. Further studies are warranted to propose the optimal concentration range of bevacizumab in patients with breast cancer brain metastases receiving the BEEP regimen.
In conclusion, we developed an efficient analytical method for quantifying mAbs in plasma samples which greatly enhanced analytical convenience and could be applied to therapeutic drug monitoring of mAb in clinical therapy. We also evaluated the relationship between bevacizumab plasma concentration and therapeutic response, PFS, and OS in breast cancer brain metastasis patients receiving the BEEP regimen. Our results provided a useful reference for adjusting bevacizumab therapeutic dose. Future larger scale studies are warranted to verify our observation and to establish optimal BEEP regimen.

並列關鍵字

Monoclonal antibody drugs ； Therapeutic drug monitoring ； BEEP therapy ； Liquid chromatography-mass spectrometry ； Protein G purification ； On-bead digestion ； High temperature trypsin digestion

參考文獻

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