GENETICALLY OPTIMIZED IMMUNOTOXINS FOR LEUKEMIA THERAPY

描述：（申请人摘要）申请人的长期目标是 开发用于治疗人类T细胞癌的新型靶向治疗剂， 恶性肿瘤。免疫毒素（IT）是一类具有高毒性的治疗剂。 特异性和独特的作用机制。IT是一个混合体 由与毒素连接的靶向部分组成的分子。靶向 部分选择性地结合到肿瘤细胞并靶向其通过免疫细胞化学途径死亡。 附着毒素一般来说，IT对癌症细胞具有特异性效力， 体外和人恶性肿瘤的动物模型中。然而，IT是有限的 在临床上通过免疫原性、毒性和/或不稳定性。临床级IT DA 7是在明尼苏达大学通过生物化学方法合成的， 将去糖基化蓖麻毒素A链（dgRTA）连接至单克隆抗体 对T细胞相关抗原CD 7具有特异性。DA 7的I期临床研究 显示其疗效主要受到不稳定性和非特异性的限制， 血管毒性尽管存在这些局限性，DA 7仍实现了客观的临床效果。 最大耐受剂量下的反应。如果不稳定和血管 DA 7的毒性降低，那么申请人认为DA 7可以找到一种 在治疗难治性T细胞疾病中的治疗小生境，或作为 辅助手术或放化疗。本次修订的目标 应用是利用基因工程来扩大“治疗窗口” 通过增加其稳定性和降低其毒性来提高DA 7的活性。第一 具体目标集中在重组IT的构建和测试上 含有与CD 7特异性单克隆抗体连接的白喉毒素（DT）衍生物， 链Fv（sFv）片段。最初将使用DT，因为它是 唯一一种FDA批准的免疫毒素sFv结构的修饰将是 以增强稳定性。高水平表达的新方法 可溶性融合毒素，并用于IT介导的肿瘤的直接可视化 体内细胞杀伤包括在该目的中。第二个具体目标是 通过连接最稳定的sFv来降低毒性（和免疫原性） 在Specific Aim 1中鉴定的结构与人RNA酶的结合。RNA酶将 被工程化以既抵抗RNA酶抑制剂， 细胞毒性一旦内化。具体目标1中提出的所有免疫毒素， 将检测特异性目标2的稳定性、相对亲和力， 药代动力学、毒性和抗肿瘤活性。这些实验将 允许申请人更精确地确定 稳定性、毒性和功效。他的最终目标是重返诊所 更强的DA 7

DESCRIPTION: (Applicant's Abstract) The applicant's long-term goal is to develop novel, targeted therapeutics for the treatment of human T cell malignancies. Immunotoxins (ITs) are a class of therapeutic agents with a high degree of specificity and a unique mechanism of action. An IT is a hybrid molecule consisting of a targeting moiety linked to a toxin. The targeting moiety selectively binds to a tumor cell and targets it for death via the attached toxin. Generally, ITs are specifically potent against cancer cells in vitro and in animal models of human malignancies. However, ITs are limited clinically by immunogenicity, toxicity, and/or instability. A clinical grade IT called DA7 was synthesized at the University of Minnesota by biochemically linking deglycosylated ricin toxin A chain (dgRTA) to a monoclonal antibody specific for the T cell-associated antigen CD7. A Phase I clinical study of DA7 revealed that its efficacy was primarily limited by instability and nonspecific vascular toxicity. Despite these limitations, DA7 achieved objective clinical responses at the maximal tolerated dose. If the instability and vascular toxicity of DA7 were reduced, then the applicant contends that DA7 could find a therapeutic niche in the treatment of refractory T cell disease, or as an adjuvant to surgery or chemoradiotherapy. The objective of this revised application is to use genetic engineering to enlarge the 'therapeutic window' of DA7 by increasing its stability and decreasing its toxicity. The first specific aim focuses on the construction and testing of recombinant ITs containing a derivative of Diphtheria toxin (DT) linked to CD7-specffic single chain Fv (sFv) fragments. DT will be used initially since it is a component of the only FDA-approved immunotoxin. Modifications of the sFv structure will be made to enhance stability. Novel approaches for the high-level expression of soluble fusion toxins, and for the direct visualization of IT-mediated tumor cell killing in vivo, are included in this aim. The second specific aim focuses on decreasing toxicity (and immunogenicity) by linking the most stable sFv structure identified in Specific Aim l to human RNAses. The RNAses will be engineered to be both resistant to RNAse inhibitors and to be optimally cytotoxic once internalized. All immunotoxins proposed in Specific Aim 1 and Specific Aim 2 will be tested for their stability, relative affinity, pharmacokinetics, toxicity, and anti-tumor activity. These experiments will allow the applicant to determine more precisely the relationships between stability, toxicity, and efficacy. His ultimate goal is to return to the clinic with a more potent version of DA7.