Analysis of biological function and efficacy of anti EGFR antibodies isolated from screening of human antibody phage display library specific to human renal cell carcinoma

用ICOS方法筛选人肾癌细胞系Caki-1(2例)、CCF-RC1(2例)和ACHN(1例)的AIMS5文库。用临床标本进行免疫染色，筛选肾细胞癌特异性抗体。我们选择了两种不同的抗体(059-152和062-130)，它们在临床肾细胞癌标本上显示出癌细胞特异性染色，而不染色正常细胞。这些抗体还与选定的人肾细胞癌细胞表面抗原发生反应。这两种抗体反应的抗原是从CCF-Rc1免疫沉淀中分离出来的，从而证明这两种抗体识别的抗原是表皮生长因子受体(EGFR)。用CAKI-1对分离的两种不同的抗表皮生长因子受体抗体的细胞增殖活性进行了评价。在低浓度(0.1μg/ml)时，059-152和062-130对细胞的增殖活性约为70%。在高浓度(10μg/ml)下，我们的两种抗EGFR抗体(059-152、062-130)诱导了高达40-60%的细胞增殖…。更多的时间。在ADCC检测中，059-152有约35%的ADCC检测。062-130有约40%的ADCC检测。考察了抗体对磷酸化反应的影响。059-152不抑制任何细胞系的磷酸化。062-130和西妥昔单抗通过CCF-RC1和ACCN抑制磷酸化。为了确定这些抗体的作用是否可以转化为抑制体内肿瘤生长，将抗体给予移植了人RCC细胞的小鼠。与对照组相比，两种抗体对人肾细胞癌生长均有明显的抑制作用。然而，生长抑制的程度取决于抗体、给药程序和剂量。虽然与化疗药物联合使用可观察到协同生长抑制作用，但宿主小鼠体重减轻也被注意到，这意味着有必要研究联合治疗的不良反应，我们认为这些抗体是候选治疗抗体。我们发现，我们的抗EGFR抗体，特别是062-130有足够的效果。由于我们的抗体来源于人噬菌体展示系统，因此与动物发生交叉反应的可能性被认为是有限的。我们期待该抗体能成为临床上用于治疗肾癌的抗体。较少

Using the ICOS method, AIMS5 library was screened with human RCC cell lines, Caki-1(2 cases), CCF-RC1(2 cases) and ACHN(1 case). RCC-specific antibodies were chosen by immunostaining using clinical samples. We chose two different antibodies(059-152 and 062-130) that exhibited cancer cell-specific staining without staining normal cells on clinical RCC sample. These antibodies also reacted to the cell surface antigen of the selected human RCC cells. Antigens these two antibodies reacted were isolated from immunoprecipitation with CCF-RC1.Consequently, the recognized antigen by these two antibodies was proved to be epidermal growth factor receptor(EGFR). Isolated two different anti-EGFR antibodies were assessed for their functional activity on cell proliferation using Caki-1.At the low concentration(0.1μg/ml), 059-152 and 062-130 exhibited about 70% cell proliferation. At the high concentration(10μg/ml), our two anti-EGFR antibodies(059-152, 062-130) elicited up to 40 to 60% cell prolifer … More ation. In ADCC assay, 059-152 had about 35% ADCC assay. 062-130 had about 40% ADCC assay. The effects of the Ab on the phosphorylation reaction were examined. 059-152 did not inhibit phosphorylation in any cell lines. 062-130 and Cetuximab inhibited phosphorylation using CCF-RC1 and ACHN.To determine if the effects of these antibodies could be translated to inhibition of tumor growth in vivo, the antibody was given to mice transplanted with human RCC cells. Treatment with both antibodies developed significant inhibition on human RCC tumor growth compared with control group. The degree of growth inhibition, however, were dependent on the antibody, administration schedule and doses. Although, synergistic growth inhibitions were observed in combination with chemotherapeutic agents, reduction of host mice body weights reduction were also noted, which meant the necessity of investigation of adverse effects in combination therapy.We expected these antibodies were candidate therapeutic antibodies. We showed, our anti-EGFR antibodies, especially 062-130 had sufficient effect. Because our antibodies were originated from human phage display system, possibility of cross reaction over animals were thought to be limited. We expect that this antibody will become a therapeutic antibody for RCC in clinical use. Less