Engineering Cell Type Specific Toxins

工程细胞类型特异性毒素

• 批准号: 6671361
• 负责人: Richard James Youle
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6671361
• 关键词: BCL2 gene /protein; apoptosis; brain neoplasms; clinical research; clinical trial phase I; cytotoxicity; diphtheria toxin; dystonia; endoribonucleases; genetic manipulation; human subject; human therapy evaluation; immunoconjugates; immunotoxicity; molecular cloning; monoclonal antibody; neoplasm /cancer chemotherapy; neoplasm /cancer immunotherapy; neurons; neurotoxins; pancreatic ribonuclease; protein engineering; transferrin; tumor antigens

Monoclonal antibodies selectively bind tumor cell differentiation antigens in vitro and in vivo. We have devised methods of linking extremely toxic proteins to the antibodies to selectively kill tumor cells by cloning the toxins and mutating them to decrease non-target cell toxicity. Some designs using a point mutant of diphtheria toxin allow 200,000 times more toxicity to target tumor cells than nontarget cells. The transferrin receptor is particularly attractive as a target on brain tumors because it is very low or absent on neurons and glial cells and because it is highly expressed on many different types of brain tumor. In phase I and phase II clinical trials of transferrin linked to a mutant diphtheria toxin many patients showed a significant decrease in brain tumor size in response to the treatment. The dose limiting toxicity in patients appeared to be vascular damage. In animal models we have explored a new strategy to prevent this vascular toxicity by systemic delivery of chloroquine to protect the brain vasculature but not protect the brain tumor cells for the immunotoxin. We have also developed immunotoxins for treatment of dystonia and other muscle spasm diseases by targeting ricin to muscle cells. One limitation of these approaches for systemic therapy and perhaps also for brain cancer is that patients can develop an immune response to the toxin. Therefore we have explored ways to use human proteins that may be nonimmunogenic to initiate tumor cell death. Human cytotoxic proteins such as the eosinophil derived neurotoxin have been engineered and linked to antibodies and found to specifically kill tumor cells. Toxic proteins in the Bcl-2 family, Bax and Bad, that induce apoptosis, have also been engineered to bind cells and specifically kill them. As Bax and Bad are members of the Bcl-2 family of proteins that also includes anti-apoptotic members we sought to develop a new strategy to prevent neuron loss by targeting Bcl-xl to cells. Recent results show that Bcl-xl can be delivered to cells in vitro and in vivo to prevent neuron apoptosis and may have potential for inhibiting neuron loss during spinal cord injury and stroke.

单克隆抗体在体外和体内选择性结合肿瘤细胞分化抗原。我们设计了将毒性极强的蛋白质与抗体连接起来的方法，通过克隆毒素并使其突变来降低非靶细胞毒性，从而选择性地杀死肿瘤细胞。一些使用白喉毒素点突变体的设计对靶肿瘤细胞的毒性比非靶细胞高 200,000 倍。转铁蛋白受体作为脑肿瘤的靶标特别有吸引力，因为它在神经元和神经胶质细胞上的含量非常低或不存在，并且因为它在许多不同类型的脑肿瘤中高度表达。在与突变白喉毒素相关的转铁蛋白的 I 期和 II 期临床试验中，许多患者显示出治疗后脑肿瘤大小显着减小。患者的剂量限制性毒性似乎是血管损伤。在动物模型中，我们探索了一种新策略，通过全身递送氯喹来保护脑血管系统，但不保护脑肿瘤细胞免受免疫毒素的影响，从而预防这种血管毒性。我们还开发了免疫毒素，通过将蓖麻毒素靶向肌肉细胞来治疗肌张力障碍和其他肌肉痉挛疾病。这些方法对于全身治疗（或许也适用于脑癌）的一个局限性是患者可能会对毒素产生免疫反应。因此，我们探索了使用可能非免疫原性的人类蛋白质来启动肿瘤细胞死亡的方法。人类细胞毒性蛋白（例如嗜酸性粒细胞衍生的神经毒素）已被设计并与抗体连接，并被发现可以特异性杀死肿瘤细胞。 Bcl-2 家族中的有毒蛋白 Bax 和 Bad 可以诱导细胞凋亡，也被设计用来结合细胞并特异性杀死它们。由于 Bax 和 Bad 是 Bcl-2 蛋白家族的成员，该蛋白家族还包括抗凋亡成员，因此我们试图开发一种新策略，通过将 Bcl-xl 靶向细胞来防止神经元丢失。最近的结果表明，Bcl-xl 可以在体外和体内递送至细胞以防止神经元凋亡，并且可能具有抑制脊髓损伤和中风期间神经元损失的潜力。