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

采用ICOS方法，用人肾癌细胞系Caki-1（2例）、CCF-RC 1（2例）和ACHN（1例）筛选AIMS 5文库。使用临床样品通过免疫染色选择RCC特异性抗体。我们选择了两种不同的抗体（059-152和062-130），其在临床RCC样品上表现出癌细胞特异性染色而不染色正常细胞。这些抗体还与所选人RCC细胞的细胞表面抗原反应。用CCF-RC 1免疫沉淀法分离出两种抗体反应的抗原，证明两种抗体识别的抗原均为表皮生长因子受体（EGFR）。用Caki-1检测了两种抗EGFR抗体对细胞增殖的影响。在低浓度（0.1μg/ml）下，059-152和062-130的细胞增殖率约为70%。在高浓度（10μg/ml）下，我们的两种抗EGFR抗体（059-152，062-130）引起高达40%至60%的细胞增殖。 ...更多信息 行动。在ADCC测定中，059-152具有约35%的ADCC测定。062-130具有约40%ADCC测定。考察了Ab对磷酸化反应的影响。059-152在任何细胞系中都不抑制磷酸化。062-130和西妥昔单抗使用CCF-RC 1和ACHN抑制磷酸化。为了确定这些抗体的作用是否可以转化为体内抑制肿瘤生长，将抗体给予移植有人RCC细胞的小鼠。与对照组相比，用两种抗体治疗对人RCC肿瘤生长产生显著抑制。然而，生长抑制的程度取决于抗体、给药方案和剂量。虽然与化疗药物联合治疗时观察到协同生长抑制作用，但也注意到宿主小鼠体重减轻，这意味着有必要研究联合治疗的不良反应，我们预期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具有足够的效果。由于我们的抗体来源于人源噬菌体展示系统，与动物发生交叉反应的可能性被认为是有限的。预期该抗体将成为临床上治疗肾细胞癌的抗体。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