The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.

中文翻译：

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使用新型抗 Tn 重组抗体鉴定人类癌症中表达 Tn 抗原 O-GalNAc 的糖蛋白。

Tn 抗原是一种在许多人类癌症中异常表达的新抗原，其表达与转移和较差的存活率相关。为了探索其生物标志物的潜力，需要新的抗体来特异性识别肿瘤中的这种抗原。在这里，我们生成了两种针对 Tn 抗原的重组抗体，Remab6 作为嵌合人 IgG1 抗体和 ReBaGs6 作为鼠 IgM 抗体，并使用多种生化和生物学方法表征了它们的特异性。Remab6 和 ReBaGs6 都能识别成簇的 Tn 结构，但最重要的是不识别包含潜在交叉反应性 Tn 抗原结构的人 IgA1 糖型。在流式细胞术和免疫荧光分析中，Remab6 识别表达 Tn 抗原的人类癌细胞系，但不能识别其 Tn 阴性对应物。在免疫组织化学 (IHC) 中，Remab6 以组织阵列形式对许多人类癌症进行染色，但很少对正常组织染色，然后主要在细胞内染色。我们使用这些抗体在 Tn 阳性 Colo205 细胞中鉴定了几种独特的含 Tn 糖蛋白，表明它们在未来生物标志物研究中对糖蛋白质组学的效用。因此，重组 Remab6 和 ReBaGs6 可用于癌细胞的生化表征和肿瘤的 IHC，并代表独立于 IgA1 识别的 Tn 生物标志物发现的有前途的工具。