Immunotoxins in bone marrow transplantation

描述：(申请人摘要)移植物抗宿主病(GVHD)是一种 骨髓(BM)移植的并发症和尽管尝试 限制它，GVHD仍然发生在60%以上的配对移植物中 死亡率。在过去的15年里，我们构建了具有攻击性的小鼠 基于这种赠款的GVHD模型发现，最好的方法之一是 目标正在进行的GVHD带有强大的免疫毒素(IT)，抗体与 催化性毒素。我们一直在寻找传统的生物化学连接剂 有可能，但研究显示了毒性，并使我们得出结论 IT的局限性必须通过可遗传的代理来解决 修改过的。因此，我们组装了一个由抗CD3抗体组成的可修饰载体 单链Fv(抗原识别的最小单位)被剪接到截断 白喉毒素。DT390抗mCD3sFv蛋白具有较强的抗GVHD作用 即使在MHC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 并使用攻击性小鼠GVHD模型发现了 显著降低器官毒性、增加耐受性的修饰 剂量，创造了一个以前不存在的治疗窗口(TW)。这是 通过在SFV部分下游插入半胱氨酸残基在 C-末端。这种修饰产生了分子间的二硫键桥联。 从而产生了被称为MuSS2的独特的二价IT。在这一轮融资中，我们计划 确定MuSS2在更具攻击性的动物模型中是否成功 GVHD更接近于我们将使用它的方式 从临床上看。其中包括正在建立的GVHD治疗模型 以确定MuSS2是否有效地对抗导致GVHD的T细胞的扩张。 第二个模型将使用高剂量辐射调节，因为我们的第一个模型 可能会在临床情况下使用，因为患者已经 有侵略性的条件反射。异基因骨髓移植的一个优点是T细胞 细胞介导的移植物抗白血病(GVL)反应。第三种小鼠模型 由我们小组建立的将被用来解决是否 MuSS2诱导的抗T细胞GVHD效应将干扰优势T细胞 细胞GVL反应。我们的数据表明它是不稳定的单体。 这会导致老鼠死亡，因为它会渗入肾脏。因此，稳定IT 而防止它的崩溃可能会增加它的效力，甚至进一步扩大 TW。因此，我们将测试两个额外的修改，旨在进一步 增强IT在体内的稳定性。其中一项修改将针对 引入稳定的分子间二硫键改进MuSS2 专门设计用来减少体内谷胱甘肽对这些键的还原。 第二种不同的方法将涉及插入FC域 已被证明能调节抗体分解代谢的稳定序列。如果 如果成功，这些方法将被应用于人类的最终设计 同源物目前正在开发中。

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.