GENETICALLY OPTIMIZED IMMUNOTOXINS FOR LEUKEMIA THERAPY

描述：(申请人摘要)申请人的长期目标是 开发治疗人类T细胞的新型靶向疗法 恶性肿瘤。免疫毒素(ITS)是一类高活性的治疗药物。 特异性程度和独特的作用机制。IT是混合体 由与毒素相连的靶向部分组成的分子。目标定位 部分选择性地与肿瘤细胞结合，并通过 附着的毒素。一般说来，它对人体内的癌细胞有特效。 在人类恶性肿瘤的体外和动物模型中。然而，它是有限的。 临床上由免疫原性、毒性和/或不稳定性决定。A临床级别的IT 明尼苏达大学通过生物化学合成了DA7 脱糖化蓖麻毒素A链(DgRTA)与单抗的连接 针对T细胞相关抗原CD7。DA7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。尽管有这些限制，DA7还是达到了客观的临床效果 在最大耐受剂量下的反应。如果不稳定和血管 DA7的毒性降低，那么申请人认为DA7可以找到一种 治疗难治性T细胞疾病的利基，或作为一种 手术或放化疗的辅助剂。这一修订的目标是 应用是利用基因工程扩大“治疗窗口” 通过增加DA7的稳定性和降低其毒性。第一 具体目的在于重组ITS的构建和检测 含有与CD7-SPECFICS单链的白喉毒素(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结构的修改将是 为了增强稳定性而制造的。用于高水平表达的新方法 可溶性融合毒素，用于IT介导的肿瘤的直接可视化 在体内杀死细胞，都包括在这个目标中。第二个具体目标集中在 通过连接最稳定的SFV来降低毒性(和免疫原性) 结构鉴定在特定的目的L对人类核糖核酸。RNase将是 设计成既能抵抗核糖核酸酶抑制剂，又能最优地 一旦内化就会产生细胞毒性。在特定目标1中提出的所有免疫毒素和 将测试特定目标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 允许申请人更准确地确定 稳定性、毒性和有效性。他的最终目标是回到诊所 使用更强大的DA7版本。

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.