Development of a monoclonal antibody specific to cancer stroma and a basic study for the medical application

Tenascin-C和细胞纤维连接蛋白被称为癌胎细胞外基质（ECM）蛋白，在胚胎组织和癌间质中表达。这些分子是由间充质细胞和上皮细胞在这种微环境下产生的，并且在组织中的沉积水平高于正常成人组织。此外，这些分子有多种剪接变体，似乎只在癌组织中表达。在本研究中，我们将探索肿瘤间质的特异性，制备针对肿瘤间质的单克隆抗体，并开发适用于肿瘤治疗的方法。1)首先，我们研究了已知ECM蛋白在肿瘤基质中的特异性表达。2)我们尝试用从肿瘤基质中提取的蛋白免疫制备肿瘤基质特异性单克隆抗体。3)检测荷瘤小鼠注射荧光标记抗体后，抗体是否在癌间质中积累。对于1，我们发现癌细胞自身产生的TN-C和细胞FN沉积在癌间质中具有相对特异性，可能在癌症进展中发挥重要作用。2)我们制备了新的针对肿瘤基质蛋白的单克隆抗体，可能是FN和蛋白多糖。3)通过免疫tnc缺失小鼠，制备了高亲和的tnc单克隆抗体。注射后抗体在小鼠乳腺肿瘤间质中独占积累。此外，还产生了针对TNC剪接位点的抗体，该位点包含在癌症基质中，但不存在于正常组织中。该抗体对癌间质呈阳性反应，对正常组织呈阴性反应。

Tenascin-C and cellular fibronectin are known as onco-fetal extracellular matrix (ECM) proteins expressed in embryonic tissues and cancer stroma. These molecules are produced by mesenchymal and epithelial cells under such microenvironments, and deposits in the tissues in higher levels than in normal adult tissues. Furthermore, there are various splicing variants of these molecules, which seem to be exclusively expressed in cacer tissues. In this study, we explore specificities of cancer stroma, produce a monoclonal antibody specific to cancer stroma, and develop the applicable methods for cancer treatments. 1) First we studied specific expression of known ECM proteins in cancer stroma. 2) we attempted to produce monoclonal antibodies specific to cancer stroma by immunization of extracted proteins from the stroma. 3) we also tested whether, after injection of fluorescence-lableled antibody in tumor-bearing mice, the antibody was accumulated in cancer stroma. For 1, we found that deposition of TN-C and cellular FN produced by cancer cells themselves are relatively specific in cancer stroma, which may play important roles in cancer progression. 2) we produced new monoclonal antibodies against cancer stromal proteins, possibly FN and proteoglycan. 3) we produced high affinity-monoclonal antibodies against TN-C by immunization to TNC-null mice. After the injection, the antibody was exclusively accumulated in stroma of mouse mammary tumor. Furthermore, antibodies against a spliced site of TNC which is included in cancer stromal one but not in normal tissue, were produced. The antibody showed positive reaction to cancer stroma, but negative in normal tissues.