Immunotoxins in bone marrow transplantation

描述：（申请人的摘要）移植物抗宿主病（GVHD）是一种主要的 骨髓（BM）移植并发症，尽管尝试 限制它，GVHD仍然发生在超过60%的匹配移植中， 死亡率。在过去的15年里，我们已经构建了攻击性小鼠 GVHD模型基于这一赠款，并发现最好的方法之一， 靶向正在进行的GVHD是与有效的免疫毒素（IT），抗体连接， 催化毒素我们研究了传统的生化连接剂， 可能的，但研究显示毒性，使我们得出结论， IT的局限性必须通过可以在基因上 修改.因此，我们组装了一个可修饰的载体， 单链Fv（抗原识别的最小单位）剪接到截短的 白喉毒素DT390抗mCD3sFv蛋白具有较强的抗GVHD作用 即使在MHC完全不同的受体小鼠中也有效果，但其剂量受到器官的限制 毒性在上一轮融资中，我们筛选了几个潜在的 修改，并使用了一个积极的小鼠GVHD模型，以发现一个 明显降低器官毒性的修饰，增加耐受性 剂量，创造一个治疗窗口（TW），其中没有以前存在。这是 通过在sFv部分的下游插入半胱氨酸残基来实现。 C-末端。这种修饰产生了分子间二硫键桥连 从而产生称为MuSS2的独特的二价IT。在这轮融资中，我们计划 为了确定MuSS 2是否在更具攻击性的动物模型中成功， GVHD更接近我们使用它的方式 临床上这些包括正在进行的已建立的GVHD的治疗模型， 以确定MuSS 2是否对扩增的引起GVHD的T细胞有效。 第二个模型将采用高剂量辐射调节，因为我们的第一个模型 在临床情况下， 条件反射攻击性。同种异体骨髓移植的一个优点是T 细胞介导的移植物抗白血病（GVL）反应。第三种小鼠模型 我们小组建立的机制将用于解决一个重要问题， MuSS 2诱导的抗T细胞GVHD效应将干扰有利的T细胞增殖。 细胞GVL应答。我们的数据表明，它是不稳定的单体 通过过滤进入肾脏杀死老鼠因此，稳定IT 防止其崩溃可能会增加其效力，甚至进一步扩大 TW。因此，我们将测试2个额外的修改，旨在进一步 增强IT在体内的稳定性。其中一项修改将针对 通过引入稳定的分子间二硫键改进MuSS 2 专门设计用于减少这些键的体内谷胱甘肽还原。 第二种不同的方法将涉及插入Fc结构域 稳定序列已显示调节抗体催化剂。如果 如果成功，这些方法将应用于人类的最终设计。 目前正在开发中。

DESCRIPTION: (Applicant's Abstract) Graft-versus-host-disease (GVHD) is a major complication in bone marrow (BM) transplantation and despite attempts at limiting it, GVHD still occurs in over 60% of matched transplants with high mortality rates. Over the past 15 years, we have constructed aggressive murine GVHD models based on this very grant and found that one of the best ways to target ongoing GVHD is with potent immunotoxins (IT), antibodies linked to catalytic toxins. We pursued conventional biochemically linked agents as far as possible, but studies revealed toxicity and caused us to conclude that the limitations of IT must be addressed with agents that can be genetically modified. Thus, we assembled a modifiable vector consisting of the anti-CD3 single chain Fv (smallest unit of antigen recognition) spliced to truncated diphtheria toxin. DT390anti-mCD3sFv protein was capable of potent anti-GVHD effects even in MHC disparate recipient mice, but its dose was limited by organ toxicity. In the last round of funding, we screened several potential modifications and used an aggressive mouse GVHD model to discover a modification that markedly decreased organ toxicity, increased tolerated dosage, creating a therapeutic window (TW) where none existed before. This was achieved by inserting a cysteine residue downstream of the sFv moiety at the c-terminus. This modification produced intermolecular disulfide bridging resulting in unique bivalent IT called MuSS2. In this round of funding, we plan to determine whether MuSS2 is successful in more aggressive models of animal GVHD that more closely approximate the manner in which we will be using it clinically. These include a therapy model of ongoing established GVHD designed to determine whether MuSS2 is effective against expanding GVHD-causing T cells. A second model will employ high dose irradiation conditioning since our first usage will be likely in clinical situations where patients have been conditioned aggressively. One advantage to allogeneic BM transplantation is a T cell-mediated graft-versus-leukemia (GVL) response. A third murine model established by our group will be used to address the important issue of whether anti-T cell GVHD effects induced by MuSS2 will interfere with advantageous T cell GVL responses. Our data indicate that it is the non-stabilized monomer that kills mice due to its filtration into kidneys. Therefore, stabilizing IT and preventing its breakdown may increase its efficacy even further widening the TW. Therefore, we will test 2 additional modifications designed to further enhance IT stability in vivo. One of these modifications will be directed at improving MuSS2 by introducing a stabilized intermolecular disulfide linkage specifically designed to curtail in vivo glutathione reduction of these bonds. A second, different approach will involve the insertion of Fc domain stabilizing sequences that have been shown to regulate antibody catabolism. If successful, these approaches would be applied to the final design of a human homologue currently under development.