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

生腱蛋白-C和细胞纤连蛋白是已知的在胚胎组织和癌间质中表达的癌胚细胞外基质（ECM）蛋白。这些分子由间充质细胞和上皮细胞在这样的微环境下产生，并以比正常成人组织更高的水平沉积在组织中。此外，存在这些分子的各种剪接变体，其似乎仅在癌组织中表达。本研究旨在探索肿瘤间质的特异性，制备肿瘤间质特异性单克隆抗体，并开发适用于肿瘤治疗的方法。1)首先，我们研究了已知ECM蛋白在癌间质中的特异性表达。2)我们试图通过免疫从基质中提取的蛋白质来产生对癌症基质特异的单克隆抗体。3)我们还测试了在将荧光标记的抗体注射到荷瘤小鼠中后，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在癌间质中的沉积是相对特异的，这可能在癌症进展中起重要作用。2)我们产生了新的抗癌基质蛋白，可能是FN和蛋白聚糖的单克隆抗体。3)我们通过免疫TNC-缺失小鼠产生了抗TNC的高亲和力单克隆抗体。注射后，抗体仅在小鼠乳腺肿瘤间质中积累。此外，产生了针对TNC剪接位点的抗体，所述TNC剪接位点包括在癌间质中，但不包括在正常组织中。抗体对癌间质呈阳性反应，而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.