Engineering Cell Type Specific Toxins

在体外和体内，单抗选择性地结合肿瘤细胞分化抗原。我们已经设计出将剧毒蛋白与抗体连接起来的方法，通过克隆毒素并突变它们来选择性地杀死肿瘤细胞，以降低非靶细胞毒性。一些使用白喉毒素点突变的设计允许对靶肿瘤细胞的毒性是非靶细胞的20万倍。转铁蛋白受体作为脑肿瘤的靶点特别吸引人，因为它在神经元和神经胶质细胞上很低或不存在，而且在许多不同类型的脑肿瘤上都有高表达。在与突变白喉毒素有关的转铁蛋白的I期和II期临床试验中，许多患者显示出治疗后脑瘤大小显着缩小。患者的剂量限制性毒性似乎是血管损伤。在动物模型中，我们探索了一种新的策略来预防这种血管毒性，方法是全身注射氯喹以保护脑血管，而不是保护脑瘤细胞免受免疫毒素的影响。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。这些系统治疗方法的一个局限性是，患者可以对毒素产生免疫反应，这一点可能也适用于脑癌。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。人类细胞毒蛋白，如嗜酸性粒细胞来源的神经毒素，已经被改造并与抗体相连，并被发现能特异性地杀死肿瘤细胞。Bcl2家族中的有毒蛋白Bax和Bad可以诱导细胞凋亡，它们也被设计成可以结合细胞并特异性地杀死它们。由于Bax和Bad是Bcl-2蛋白家族的成员，该家族还包括抗凋亡成员，我们试图开发一种新的策略，通过靶向细胞来防止神经元丢失。最近的研究结果表明，在体外和体内，BclxL可以被传递到细胞内，以防止神经元的凋亡，并有可能在脊髓损伤和中风时抑制神经元的丢失。

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.