建立抗第二型嵌膜絲胺酸蛋白酶II的診斷和治療性抗體及其在攝護腺癌症的應用

Establishment of anti-TMPRSS2 antibody for diagnostic and treatment in prostate cancer

閻大立(Ta-Li Yen)

指導教授 ： 李明學

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902200

摘要

雄性激素的訊息傳遞及細胞周圍的蛋白水解作用在攝護腺癌的進程及轉移過程中，扮演重要的角色。其中我們對第二型嵌膜絲胺酸蛋白酶II (TMPRSS2)有高度的興趣，因為先前的研究中發現第二型嵌膜絲胺酸蛋白酶II可以透過活化它的蛋白酶疉層和細胞外基質的水解，促進攝護腺癌細胞侵襲、腫瘤生長及轉移。此外，第二型嵌膜絲胺酸蛋白酶II亦被報導有外泌或脫落(shed)的現象。因此，第二型嵌膜絲胺酸蛋白酶II在攝護腺癌的臨床應用上，具有診斷及作為治療標的的潛力。我的研究致力於建立抗第二型嵌膜絲胺酸蛋白酶II的抗體，並鑑尋出具攝護腺癌診斷及治療潛力的多株或單株抗體。我首先建立了穩定表達外泌型第二型嵌膜絲胺酸蛋白酶II重組蛋白的中國倉鼠卵巢細胞株，從其細胞的培養基中純化出此重組蛋白質並以此純化蛋白質作為免疫動物的抗原。我已從被抗原免疫過的天竺鼠和兔子製作出多株抗體及免疫小鼠製備單株抗體。抗第二型嵌膜絲胺酸蛋白酶II之天竺鼠源多株抗體可成功辨識第二型嵌膜絲胺酸蛋白酶的水解作用區塊。兔源多株抗體(Rabbi anti-TM2 pAb)具有良好的專一性，但其中和抑制第二型嵌膜絲胺酸蛋白酶II重組蛋白活性的能力，較不明顯。我們用兔源多株抗體可在攝護腺癌細胞培養基中以及在攝護腺癌病人的尿液檢體中偵測到分泌型的第二型嵌膜絲胺酸蛋白酶II。未來要利用兔源多株抗體建立ELISA，大量分析攝護腺癌病人尿液中第二型嵌膜絲胺酸蛋白酶II的含量，探討其作為攝護腺癌進程指標的潛力。在建立鼠源單株抗體的方面，經過細胞融合瘤的實驗，我初步得到一株可以辨認第二型嵌膜絲胺酸蛋白酶II重組蛋白的單株抗體融合瘤，並將釐清其應用於臨床的潛力。同時我們正在努力建立更多鼠源單株抗體，期望未來可以挑選到具中和第二型嵌膜絲胺酸蛋白酶II活性的單株抗體，以應用於臨床診斷或治療。

關鍵字

攝護腺癌 ； 細胞外基質水解 ； 第二型嵌膜絲胺酸蛋白酶II ； 偵測性多株抗體 ； 中和性單株抗體

並列摘要

Androgen signaling and pericellular proteolysis play important roles in prostate cancer (PCa) progression and metastasis. Among them, type II transmembrane serine proteases II (TMPRSS2) receives an attention because we found that TMPRSS2 could promote PCa cells invasion, tumor growth and metastasis through activation of its proteolytic cascade and extracellular matrix degradation. TMPRSS2 has been also reported to be shed out from cells after its activation. Thus, TMPRSS2 may serve as a biomarker for PCa diagnosis/prognosis and a therapeutic target in PCa. In this thesis, I aimed to generate anti-TMPRSS2 antibodies with a potential of diagnosis, prognosis or therapeutic usages. I used a mammalian secretory expression system to express recombinant TMPRSS2 proteins in CHO cells and purify those proteins from the conditioned media. I successfully used the purified rTMPRSS2 to be antigens for polyclonal antibody generation from a guinea pig and a rabbit. Guinea pig anti-TM2 antibody could recognize the protease domain of TMPRSS2. Rabbit anti-TM2 polyclonal antibody showed a great specificity against TMPRSS2 in cell lysates and could also recognize the shed TMPRSS2 in the conditioned media of PCa cells and PCa patients’ urine samples. The establishment of neutralizing monoclonal antibodies against TMPRSS2 are still undergoing. Thus, the results together indicate that the anti-TM2 antibodies are successfully generated from the animals of guinea pig and rabbit with a potential of clinical application.

並列關鍵字

prostate cancer ； pericellular proteolysis ； type II transmembrane serine protease II ； polyclonal antibody ； neutralizing antibody

參考文獻

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