以基因工程產生 YKL-40 單鏈變異區片段抗體 (scFv) 用於犬癌症之研究

Abstract

YKL-40是一種分泌型的醣蛋白，因其結構類似幾丁酶，具有與幾丁質結合之能力，但不具有酵素的功能，又名為CHI3L1 (chitinase-3-like-1)，在近期研究中被認為可能是人類癌症的一個biomarker；在人醫許多癌症研究中發現，當患者血清中YKL-40 含量較高時，其預後及存活率較差。我們先前在犬隻相關的研究中也有相似的發現 ，因此研究室已經製備了三株抗犬YKL-40之單株抗體，這些單株抗體除了可以在不同試驗中標定YKL40外，在細胞學試驗也證實他們具有抑制因YKL40所促發的移行 (migration)、侵潤 (invasion) 以及管腺生成 (tubulogenesis） 等作用；然而傳統的融合瘤製備及保存方式較為繁瑣，且較不穩定，因此，本研究擬選殖出上述三株抗犬YKL-40單株抗體之單鏈變異區片段抗體 (single-chain variable fragment antibody, scFv Ab)，並以大腸桿菌表現系統進行大量表現，以應用於犬癌症的相關研究。首先，以基因工程技術選殖出上述單株抗體變異區的重鏈及輕鏈基因序列，透過重疊延伸聚合酶鏈鎖反應 (SOE-PCR) 將兩段序列連接，之後架構於載體中，再由大腸桿菌表現系統進行表現及純化。接著以酵素免疫分析法 (ELISA)、西方墨點法、免疫螢光染色確認產製的單鏈變異區片段抗體能夠辨識犬隻重組YKL-40 (rcYKL-40)及天然型態YKL-40 (nYKL-40)。另外在以犬黑色素瘤為標的細胞學試驗中發現，本研究中製備的scFv/M2及scFv/M3抗體則能有效抑制因YKL-40所促發的細胞移行及侵潤能力；在細胞存活試驗中，添加scFv/M2抗體後能夠降低細胞存活率，且能幫助化療藥物抑制腫瘤增生。在細胞訊息傳遞路徑上，發現YKL-40具有活化MAPK的能力，而添加scFv/M2抗體能夠抑制AKT及MAPK的磷酸化。最後，以scFv/M2抗體建立的酵素免疫分析法檢測犬血清中YKL-40的含量，癌症犬血清中的YKL-40量顯著地較非癌症犬為高。綜合以上的結果，本研究成功以基因工程方式產出犬YKL-40的scFv Abs，這些scFv Abs除了可以標定犬YKL-40外，也可以抑制犬黑色素瘤細胞的一些癌化特性，同時也用來建立ELISA，確認癌症犬血中有較高量的YKL-40，因此這些scFv Abs未來有許多有潛力開發作為犬腫瘤治療及診斷之用。

YKL-40, also known as CHI3L1 (chitinase-3-like-1), is a secretory glycoprotein with chitinase like structure and chitin-binding affinity, but has no chitinase activity. In recent study, YKL-40 is considered a cancer biomarker in human cancers. Patients with elevated serum level of YKL-40 is correlated with poor survival and bad prognosis in a variety of human cancers. Our study in dog cancer cases has also revealed this similar finding. Inspired by this, previous studies in our lab have developed three mouse mAbs (monoclonal antibodies) against canine YKL-40 through hybridoma technology. Except recognized YKL-40 in several assay, the developed mouse mAbs also has presented the ability to inhibit YKL-40 induced cellular functions including migration, invasion and tubulogenesis, etc. However, through the hybridoma technology to produce antibody is complex and unstable. Thus, to preserve these mAbs forever and expand their applications, the aims of my study are to generate their scFv (single-chain variable fragment) Abs, which can be expressed through the prokaryotic expression system to facilitate mass production, and characterize them for future applications on canine cancer research. To these ends, the DNA fragments of variable region of heavy chain and light chain were respectively amplified from the hybridoma cells and were connected become scFv DNA fragments through splicing by SOE-PCR (overlap extension polymerase chain reaction). The scFv DNA fragments were ligated to the vector and expressed by E.coli expression system. Next, various assays including ELISA, Western blot, IP and immunofluorescence assay were used to verify the binding specificity of purified scFv Abs to the rcYKL-40 (recombinant canine) and nature form YKL-40. In cellular assays of canine melanoma cells, the migration and invasion ability elicited by rcYKL-40 was blocked by two of scFv Abs, scFv/M2 and scFv/M3. Next, we III found application of scFv Abs, scFv/M2, decreased the cell viability of cancer cells and promoted the chemotherapy to inhibit the cell proliferation. In signal transduction analysis, MAPK activity could be activated by rcYKL-40, and the M2/scFv could effectively reduce activation of Akt and MAPK. Finally, an indirect ELISA was established using scFv/M2 to detect YKL-40 levels in canine serum. The results showed serum YKL-40 levels of cancer dog were significantly elevated compared to that in non-cancer dog. In conclusions, canine YKL-40 scFv Abs were successfully cloned and expressed through the genetic engineering. These scFv Abs could recognize canine YKL-40 and inhibit the various cellular functions elicited by rcYKL-40 on canine melanoma cells. Also, an established ELISA using our scFv mAbs reconfirmed our previous finding which elevated YKL-40 levels existed in dogs with cancer. Thus, these scFv Abs we generated might have the potential to develop as diagnostic tools and therapeutic agents for canine cancer in the future.