工業技術研究院

技術簡介

人類表皮生長因子受體(EGFR)已被視為是有效的目標物用於發展抗腫瘤治療。可表現EGFR之32D(32D-EGFR)細胞株已被報導可用於EGFR相關訊息傳遞路徑的抑制劑篩選。利用32D-EGFR細胞與重組之EGFR細胞外區域(EGFR-ECD)自單域抗體庫中針對EGFR進行噬菌體展示篩選。利用此合併篩選策略成功篩選出一個新穎抗人類EGFR抗體。利用人類抗體Fc片段融合單域抗體使其成為二價抗體形態並利用EGFR過量表現的A431細胞測定出其結合強度為~52 nM。同時也利用A431細胞進行細胞內噬作用與抑制細胞生長能力分析。綜合上述結果，利用合併32D-EGFR細胞與重組之EGFR細胞外區域(EGFR-ECD)針對EGFR進行噬菌體展示篩選為有效率策略，因為不需要額外免疫動物用以產生抗體庫。

Abstract

Epidermal growth factor receptor (ErbB1, EGFR) has been considered as a valid target for development anti-cancer targeted therapies. The EGFR-transfected 32D (non-tumorigenic murine hematopoetic cells) (32D-EGFR) cell line has been reported as a suitable EGFR pathway inhibitor screening system. Domain antibodies (dAbs) specific for EGFR have been directly isolated via panning a phage display human domain antibody library on 32D-EGFR and recombinant extracellular domain of EGFR (EGFR-ECD). One novel human EGFR specific domain antibody was isolated from this combinative screening strategy. A recombinant dimeric form of dAb, dAb-hFc, has been constructed by fusing dAb to the fragment crystallizable region (Fc) of human IgG1. The KD value of dAb-hFc was ~52 nM as determined on EGFR-overexpressing A431 cells. Internalized and growth inhibition activity of dAb -hFc were also confirmed as determined on A431 cells. Taken together, phage display screening of EGFR specific antibody based on combination of 32D-EGFR cell and EGFR-ECD is an efficient strategy without additional animal immunization to construct antibody library.

技術規格

1.為全人源抗體序列。 2.該抗體具有與人類表皮生長因子受體專一性結合的能力。 3.抗體可藉由一受體媒介內噬作用進入細胞中。 4.兩價單域抗體與人類表皮生長因子受體結合強度落於52 nM左右。

Technical Specification

1.Fully human antibody sequence. 2.The anti-human EGFR antibody is capable of specifically binding to a human EGFR. 3.The anti-human EGFR antibody is capable of entering a cell through receptor-mediated endocytosis. 4.The equilibrium constant (KD) values of dimeric form antibody to human EGFR is ~ 52 nM.

技術特色

利用小鼠32D(有無EGFR表現)細胞與嗜菌體展示(phage display)技術在人類單域抗體庫(domain antibody library)內進行針對人類表皮生長因子受體(EGFR)抗體篩選。經過序列分析比對證實dAb-D2單域抗體其變異區(variable region)為未曾被報導過的新穎序列。利用EGFR過量表現之人類A431細胞進行抗體相關之活性分析，dAb-D2-hFc重組抗體與A431細胞具有高度親和性(結合強度落於nanomolar range)並同時具有細胞內噬作用之能力(internalized activity)，細胞生長抑制實驗也證實dAb-D2-hFc重組抗體可抑制A431細胞生長。dAb-D2-hFc重組抗體對於EGFR具有高度親和性與抑制細胞生長能力可應用於標靶傳遞藥物(targeted drug delivery)或抑制腫瘤細胞生長。

應用範圍

可應用於生物醫學相關市場，如診斷試劑開發、治療用抗體開發等領域。

接受技術者具備基礎建議（設備）

1.哺乳類細胞培養設備。 2.蛋白質純化設備。

接受技術者具備基礎建議（專業）

1.分子生物學。 2.哺乳類細胞培養技術。 3.蛋白質純化技術。

聯絡資訊