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的人重组Fab(RFab)，并将其应用于肿瘤疾病的诊断和治疗。我从一例恶性淋巴瘤患者的骨髓细胞中构建了人IgG1kappa组合文库(1.3x10^7CFU)。尽管对抗体捕获的CD98抗原对该文库进行了五轮淘洗，但没有获得CD98反应的rFab。因此，我们将抗原来源从CD98蛋白改为活的CD98阳性细胞(悬浮形式为HeLa细胞：SHELA)。经过五轮淘洗，观察到文库丰富了240倍。用间接免疫荧光法从40个供试克隆中获得39个阳性克隆。阳性克隆与其BstNI指纹图谱一致。接下来，我们对克隆的rFab进行了分子结构和功能鉴定，命名为AHSA抗HeLa表面抗原rFab。AHSA rFab的重链和轻链可变序列与所获得的生殖系基因序列高度同源。Ahsa rFab以浓度依赖的方式与Shela反应，但不与贴壁的HeLaS3细胞以及人或小鼠来源的其他细胞系反应。AHSA rFab对SHELA的反应性降低，与长期培养导致的簇形成效率的丧失平行。体外培养的SHELA细胞经抗Fab-交联型AHSA rFab处理后，可抑制SHELA细胞簇的形成。这些结果表明，AHSA rFab识别SHELA细胞表面的簇形成相关抗原。

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.