製備應用於犬癌症研究的犬YKL-40 單株抗體

Abstract

近年來，癌症已成為國內外犬隻的第一大死因，但目前大多數犬癌症的治療仍以傳統的手術切除、化學療法及放射療法為主，療效大多不好；與人類癌症相似的是，早期的診斷能讓癌症患犬得到較好的療效及復原狀況，因此，現階段我們希望找到一個能用於犬癌症早期診斷與治療的生物標記（biomarker）。YKL-40為一種分泌型之醣蛋白，因其結構類似幾丁酶，具有與幾丁質結合之能力而有別名CHI3L1（chitinase-3-like-1），但其酵素活性區域之谷氨酸（glutamic acid）被白胺酸（leucine）所取代，因此不具幾丁酶之活性。而在YKL-40與癌症相關研究中，一般認為血清含有高量YKL-40可能與癌細胞的轉移及病人較短的存活時間有關；除此之外，許多證據顯示YKL-40可能是一種自體抗原，與多種發炎性疾病及發炎所引起的腫瘤有關。然而目前YKL-40的研究都以人類和實驗鼠為主，還沒有犬YKL-40的相關研究，因此，本研究擬純化重組的犬YKL-40及製備抗犬YKL-40之單株抗體，並將其應用於犬癌症醫學之研究。首先，將純化後之犬YKL-40重組蛋白作為免疫抗原，以融合瘤技術製備並經由酵素免疫分析法(ELISA)、免疫墨點法(immunoblot)、免疫螢光染色(immunofluorescence)和免疫組織化學染色(immunohistochemistry)分析後得到專一性的單株抗體，接著以ELISA技術來檢測健康犬與癌症犬血清中的YKL-40和自體抗體(YKL-40 autoantibody, YAA)，並以統計方法分析兩者與犬癌症發展之間的相關性。最後經由生物功能試驗如細胞增生試驗（proliferation）、移行試驗（migration）、侵入能力試驗（invasion）以及管腺生成試驗（tubulogenesis）等來評估犬YKL-40對於癌症細胞的生理功能以及單株抗體的中和效力。由實驗結果得知，犬YKL-40重組蛋白能促進犬淋巴癌細胞的移行，犬腎上皮細胞的增生和管腺生成，而本研究中製備的單株抗體19C12M2則能抑制YKL-40促進的管腺生成。另外，由血清學的檢測和統計分析得知，腫瘤患犬的血清YAA低於健康犬，而YKL-40則高於健康犬；此外，血清YKL-40含量較高的患犬，會有較高的腫瘤復發率及轉移率，一年存活率也顯著較低。綜合以上分析結果，單株抗體19C12M2能阻斷犬YKL-40所促進的管腺生成，血清中高量的YKL-40可能可作為犬癌症預後不良的評估指標。

Cancer is now the number one killer of canine in worldwide. While canine cancer can be treated with surgery, chemotherapy, radiation, and immunotherapy, the prognosis is generally poor. Similar to human cancer, early detection of cancer is most critical for successful treatment and recovery. Therefore, it is urgent to find a biomarker for diagnosis and treatment in early stages of canine cancer. YKL-40 protein, also known as CHI3L1 (chitinase-3-like-1), is a glycoprotein with chitin-binding affinity and lacks chitinase activity due to mutations within the active site. It is considered that high serum level of YKL-40 is correlated with metastasis and poor survival in a variety of human cancer. In addition, YKL-40 may serve as an autoantigen which mediates multiple inflammatory diseases and cancers. However, the role of YKL-40 in dog is still under evaluation. The aim of this study is to develop and characterize monoclonal antibody (mAb) against canine YKL-40 for canine cancer research. Purified recombinant canine YKL-40 (rcYKL-40) was used to develop mouse mAb by hybridoma technology. An ELISA method was then established to detect serum YKL-40 and its autoantibody (YAA) in dogs. Afterward, the bio function of canine YKL-40 were investigated by cell proliferation, migration, invasion assay. Our data show that rcYKL-40 promotes cell migration of canine lymphoma cells, CLC and CLBL-1; mAb 19C12M2 might have potential to block YKL-40 associate cell migration. We also found that patients with higher serum YKL-40 present poor prognosis and worse one-year survival. In conclusion, mAbs we developed could be a useful tool in canine YKL-40 detection, and serum level of YKL-40 might be an independent prognostic factor for canine cancers.