以小鼠模式研發對抗VEGFR2之人類抗體於癌症治療之研究

Abstract

腫瘤血管新生 (angiogenesis) 是癌症的主要特徵之一。根據以往研究顯示，血管內皮生長因子A（VEGF-A）及其受體（VEGFR-2）在腫瘤生長過程中扮演調節血管生成的重要因子，血管內皮細胞藉由 VEGF/VEGFR-2訊息傳遞路徑促進細胞之增生、存活力、遷移及轉移。因此抗血管新生策略在癌症治療中是具有前景的治療方式。為了開發抗體標靶治療，實驗室先前透過噬菌體顯現法 (phage display) 和親和力成熟技術 (affinity maturation)，篩選出專一性結合VEGFR-2的人類抗體 (anti-VEGFR2-AF antibody)。本篇研究中，我們想測試anti-VEGFR2-AF抗體對於腫瘤血管新生及抑制腫瘤生長的能力，首先，我們透過即時聚合酶鏈鎖反應(real time-PCR) 觀察到在人臍靜脈內皮細胞（HUVEC）、前列腺癌細胞株（PC-3）和白血病細胞株 (HL-60）有VEGFR-2的表現，進一步利用流式細胞儀，確認anti-VEGFR2-AF antibody專一性結合到內皮細胞表面VEGFR-2比IMC- 1121B (ramucirumab) 結合力更強，IMC- 1121B是對抗VEGFR-2單株抗體，於2014年4月經由FDA通過用於胃癌晚期治療。隨後，我們發現anti-VEGFR2-AF 抗體可與VEGF-A競爭結合到內皮細胞的VEGFR-2，進而抑制VEGFR-2磷酸化和VEGFR-2 下游訊號因子Akt / MAPK / FAK 的活化。此外，anti-VEGFR2-AF抗體不只能夠引發補體依賴性細胞毒性（complement dependent cytotoxicity）和抗體依賴性細胞毒性作用（antibody-dependent cell-mediated cytotoxicity），我們發現，IMC- 1121B及 anti-VEGFR2-AF 抗體同時處理內皮細胞會導致內皮細胞中VEGFR-2蛋白降解。進一步表明，anti-VEGFR2-AF抗體明顯抑制攝護腺癌細胞 (PC-3) 的遷移，侵襲，集落形成和增生能力。在人類前列腺癌細胞異種移植的免疫缺陷型小鼠中，我們發現anti-VEGFR2-AF抗體不論單一治療或者結合化療藥物 (Docetaxel)，治療後明顯抑制腫瘤的生長和腫瘤血管新生，其抑制能力更甚於IMC-1121B。另一方面，在人類急性前骨髓性白血病癌細胞異種移植的免疫缺陷型小鼠中，anti-VEGFR2-AF抗體比IMC- 1121B有更顯著性的治療效果，不僅延長存活率，甚至降低癌細胞浸潤卵巢及淋巴。綜合以上之結果， anti-VEGFR2-AF抗體透過直接標靶VEGFR-2表達的腫瘤細胞進而抑制腫瘤血管新生進及延遲腫瘤生長，未來，anti-VEGFR2-AF抗體在腫瘤治療中是具有極大的潛力。

Angiogenesis is one of the key hallmarks of cancer. Vascular endothelial growth factor A (VEGF-A) and its receptor (VEGFR-2) play the major roles to modulate angiogenesis, which is essential for solid tumor progression. Therefore, anti-angiogenesis treatment has been a promising new form of cancer therapy. To develop antibody-based targeted therapy for cancer, we have previously performed phage display and affinity maturation techniques to generate a fully human antibody against VEGFR-2, anti-VEGFR2-AF antibody. In this study, we have investigated the potential of anti-VEGFR2-AF antibody in anti-angiogenesis and tumor suppression in vitro and in vivo. First, we observed that VEGFR-2 is expressed not only in human umbilical vein endothelial cells (HUVECs), but it is also found in prostate cancer cell lines (PC-3) and leukemia cell lines (HL-60), as shown by quantitative RT-PCR. Using flow cytometry, anti-VEGFR2-AF antibody bound to HUVECs was stronger than IMC-1121B (ramucirumab), which is an anti-VEGFR2 antibody. On April 21, 2014, the U. S. Food and Drug Administration approved ramucirumab for use as a single agent for the treatment of patients with advanced or metastatic gastric cancer. Subsequently, we showed that anti-VEGFR2-AF antibody inhibited VEGF-A-induced phosphorylation of VEGFR-2 and suppressed activation of Akt/MAPK/FAK signal transduction cascades in HUVECs. We also demonstrated that anti-VEGFR2-AF antibody was able to trigger complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. In addition, we found that treatment of HUVECs with IMC-1121B and anti-VEGFR2-AF simultaneously would cause VEGFR-2 protein degradation in HUVECs. Compared to IMC-1121B, anti-VEGFR2-AF exhibited superior ability to inhibit VEGF-A-induced migration, invasion, colony formation and viability of PC-3 cells. In prostate cancer (PC-3) xenograft mouse models, treatment with anti-VEGFR2-AF antibody both monotherapy and combination with docetaxel resulted in significantly higher reduction in tumor growth and tumor angiogenesis than treatment with IMC-1121B. Moreover, anti-VEGFR2-AF antibody possessed more significant efficacy than IMC-1121B in prolonging survival and decreasing myeloma cells infiltration of ovaries and lymph nodes in the treatment of NSG mice bearing human leukemia (HL-60). Taken together, our findings strongly suggest that anti-VEGFR2-AF human antibody may have clinical potential for cancer therapy by exerting anti-angiogenesis or by directly targeting VEGFR-2-expressing tumor cells.