Rybak S M, Hoogenboom H R, Newton D L, Raus J C, Youle R J
Surgical Neurology Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892.
Cell Biophys. 1992 Aug-Dec;21(1-3):121-38. doi: 10.1007/BF02789483.
Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M, whereas > 10(-4) M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.
胰腺核糖核酸酶（RNase）家族成员对RNA具有多种活性，这可能使它们在宿主防御和生理性细胞死亡途径中发挥作用。通过将核糖核酸酶与细胞结合配体偶联，可利用这种活性将它们设计成细胞类型特异性细胞毒素。因此，通过二硫键将牛胰腺核糖核酸酶A与人转铁蛋白或转铁蛋白受体抗体相连，探索了核糖核酸酶的细胞毒性潜力。这些核糖核酸酶杂合蛋白在体外对K562人红白血病细胞具有细胞毒性，IC50约为10^(-7) M，而抑制蛋白质合成则需要>10^(-4) M的天然核糖核酸酶。细胞毒性需要偶联物的两个组分，因为向培养基中添加过量的转铁蛋白或核糖核酸酶抑制剂可保护细胞免受转铁蛋白 - 核糖核酸酶毒性的影响。重要的是，发现核糖核酸酶偶联物在体内具有强大的抗肿瘤作用。还开发了嵌合核糖核酸酶融合蛋白。通过将人核糖核酸酶基因与嵌合抗转铁蛋白受体重链基因相连，制备了F(ab')2样抗体 - 酶融合物。将抗体酶融合基因导入分泌相同抗体嵌合轻链的转染瘤中，并克隆出能合成和分泌预期大小的抗体 - 酶融合蛋白、浓度为1 - 5 ng/mL的细胞系。分泌融合蛋白的克隆的培养上清液对表达人转铁蛋白受体的K562细胞具有生长抑制和蛋白质合成抑制作用，但对非人类来源的细胞系则无此作用。由于与抗体偶联的人核糖核酸酶也表现出受体介导的毒性，因此提供了一种选择性细胞杀伤的新方法。这可能有助于开发新的癌症治疗药物制剂，与目前使用的配体 - 毒素偶联物相比全身毒性更小，重要的是免疫原性更低。
