以表達CD158d或T細胞受體-CD3複合物的細胞株進行噬菌體呈現庫的生物淘選

Abstract

以噬菌體呈現庫（phage displayed libraries）進行生物淘選（bio-panning）被運用在許多種蛋白質交互作用的研究上。在藥物學中，許多針對潛在治療標靶的配體被發現並且應用在藥物發展。我們選擇了兩種抗原作為標靶：TCR（T細胞受體）／CD3複合物中的CD3和KIR家族（殺手細胞免疫球蛋白樣受體）中的CD158d。CD3是在T細胞淋巴癌中的一種譜系專一抗原，而CD158和其他KIR家族蛋白有不同特性，存在於大部分的NK細胞淋巴癌。兩者都有做為潛在治療標靶來研究，如CD3為雙特異性抗體pasotuxizumab的標靶。 我們首先利用許多組重組質體將CD158d或CD3分子表現在293T及CHO-K1細胞株的表面上。接下來我們測試了兩種條件，透過反覆數輪的淘選去篩選M13噬菌體庫。每一輪包含一次負向淘選然後一次正向淘選。對於負向淘選，噬菌體庫被結合到空載體轉染的細胞內；而正向淘選中，則是結合到CD158d或CD3轉染細胞或未轉染的CD158+或CD3+細胞株。在正向淘選後，細胞分選的技術和anti-M13抗體被使用於分離出標靶專一性的噬菌體。在三輪和四輪的淘選後，我們挑出一個主要的殖株，植株2，並利用數種細胞株來確認結合專一性。這個殖株結合到T細胞來源的細胞株及293T細胞比B細胞或NK細胞來源的細胞株還要好。 由於穿膜結構、多次單元的複合物構造和較低的原始表現量，要找出CD3和CD158d專一的多肽可能很困難的。透過噬菌體庫結合到不同細胞株，我們嘗試降低其背景值並分離出最具專一性的殖株。未來我們將會嘗試ZAP和Notch報導蛋白作為替代方式去找出更高CD158d或CD3專一性的多肽。

Bio-panning with phage displayed libraries is used in a wide range of studies on protein-protein interactions. In pharmacologic researches, many ligands against potential therapeutic targets were found and used in drug development. We chose two antigens as targets: CD3 of the TCR (T cell receptor)/CD3 complex and CD158d of the KIR (killer cell immunoglobulin-like receptor) family. CD3 is a lineage-specific antigen in T cell lymphomas and CD158d, with distinct characteristics from other KIRs, is present in most NK-cell lymphoma. Both have been studied as potential therapeutic targets, for example, CD3 as the target of a bi-specific antibody, pasotuxizumab. We first expressed CD158d or CD3 on the surface of 293T and CHO-K1 cells with various construct sets. Next, we tested two conditions for screening of a M13 phage library through repetitive rounds of selection. Each round included a negative selection followed by a positive selection. For the negative selection, the library was bound onto empty vector-transfected cells, and, for the positive selection, on CD158d- or CD3- transfected cells, or non-transfected CD158+ or CD3+ cell lines. After positive selections, cell sorting with anti-M13 antibody was used to isolate target-specific phages. At the end of three and four rounds of selection, we picked a major clone, clone 2, to check binding specificity with several cell lines. The clone bound cell lines of T cell origin and 293T cells better than cell lines of B or NK cell origin. Due to the trans-membrane structure, a multi-subunit complex formation, and lower native expression, it’s probably difficult to identify CD158d and CD3-specific peptides. Through binding of the library with various cell lines, we tried to reduce the background and to isolate the most specific clones. In the future, we will try ZAP70 and Notch reporter as an alternative approach to identify peptides with higher CD158d or CD3-specificity.