Preparation of human recombinant Fab against tumor-associated antigen, CD98 and its application for diagnosis and therapy of tumor diseases

本研究的目的是制备抗肿瘤相关抗原CD98的人源性重组Fab（rFab），并将其应用于肿瘤疾病的诊断和治疗。我从一名MOLT淋巴瘤患者的骨髓细胞中构建了人IgG 1，κ组合文库（1.3 x 10^7 cfu）。尽管针对抗体捕获的CD98抗原对该文库进行了五轮淘选，但未获得CD98反应性rFab。因此，我们将抗原来源从CD98蛋白改变为活的CD98阳性细胞（HeLa细胞悬浮液：sHeLa）。经过5轮淘选，观察到文库富集了240倍。用间接免疫荧光法从40个阳性克隆中筛选出39个阳性克隆。发现阳性克隆与它们的BstNI指纹图谱相同。我们接下来对克隆的rFab（称为AHSA抗HeLa表面抗原rFab）进行分子结构和功能表征。发现AHSA rFab的可变重链和轻链序列与衍生的生殖系基因序列高度同源。AHSA rFab以浓度依赖性方式与sHeLa反应，尽管它不与粘附的HeLaS3细胞以及与人或小鼠来源的其他细胞系反应。AHSA rFab对sHeLa的反应性降低，与长期培养引起的簇形成效率的损失平行。抗Fab交联的AHSA rFab体外处理培养的sHeLa细胞可抑制sHeLa的簇形成。这些结果表明，AHSA rFab识别sHeLa细胞表面上的簇形成相关抗原。

The goal of this research project is to prepare human recombinant Fab (rFab) against tumor-associated antigen, CD98 and its application to diagnosis and therapy of tumor diseases. I constructed human IgG1, kappa combinatorial library (1.3 x 10^7 cfu) from bone marrow cells of a MOLT lymphoma patient. Although five-rounds of panning was performed on this library against antibody-captured CD98 antigen, no CD98-reactive rFab was obtained. We therefore changed the antigen source from CD98 protein to live CD98-positive cells (suspension form HeLa cells : sHeLa). After five-rounds of panning, 240-fold enrichment of library was observed.. We obtained 39 positives from 40 tested clones by indirect immunofluorescence assay. Positive clones were found to be identical from their BstNI fingerprinting patterns. We next performed the molecular structural and functional characterization of cloned rFab, termed AHSA anti-HeLa surface antigen) rFab. Variable heavy-and light-chain sequences of AHSA rFab were found to be highly homologous with the derived germ line gene sequences. AHSA rFab reacted with sHeLa in a concentration dependent manner, though it did not react with adherent HeLaS3 cells, and with other cell lines of human or mouse origin. Reactivity of AHSA rFab against sHeLa was decreased in parallel with the loss of cluster formation efficiency caused by a long-term culture. Cluster formation of sHeLa was inhibited by treatment of anti-Fab-cross-linked AHSA rFab against cultured sHeLa cells in vitro. These results suggest that AHSA rFab recognizes the cluster formation-associated antigen on the surface of sHeLa cells.