Single-chain antibody countermeasures for the Radiation GI Syndrome

描述（由申请人提供）：遗传或药物抑制胃肠道（GI）血管内皮内神经酰胺介导的细胞死亡，保护小鼠免于辐射GI综合征（RGS）的死亡。预防性使用抗神经酰胺抗体可减弱内皮细胞凋亡，从而在致死性辐射暴露后显著保护肠道干细胞和动物存活。辐射暴露24小时后给药同样可以保护隐窝干细胞并显著提高存活率，表明抗神经酰胺抗体是致死性RGS小分子缓解剂的第一种有效抗体。为了提高抗神经酰胺抗体的有效性，我们提出选择含有已知抗神经酰胺抗体的神经酰胺结合区域的重组单链抗体融合蛋白作为一种新的医疗辐射对策。单链抗体融合蛋白是全长抗体的更小的衍生物，因此与全长抗体相比，具有快速进入血液和增加对组织渗透的优势。此外，单链抗体融合蛋白可以通过一般克隆技术进行修饰，以显示首选的靶标结合，从产品开发的角度来看，这些融合蛋白可以很容易地以最低的成本生产。虽然在某些情况下，单链抗体融合蛋白可能无法保留亲本抗体的全部活性，但我们的初步数据表明，酶切人源化抗神经酰胺抗体产生的类似抗体片段，即使在高单剂量暴露24小时后，也能显著保护和减轻辐射诱导的隐窝干细胞的致死率。这些数据表明，单链抗体融合蛋白足以诱导神经酰胺的治疗性中和。因此，一种中和性抗神经酰胺单链抗体融合蛋白代表了一种有希望的候选者，可以完成生物盾计划的任务，在核灾难发生后的头24小时内开发缓解急性辐射综合征的对策。

DESCRIPTION (provided by applicant): Genetic or pharmacologic inhibition of ceramide-mediated cell death within the vascular endothelium of the gastrointestinal tract (GI) protects mice from death from the Radiation GI Syndrome (RGS). Prophylactic administration of anti-ceramide antibody attenuates endothelial apoptosis, resulting in significant protection of intestinal stem cells and animal survival following lethal radiation exposure. Administration 24 h post radiation exposure similarly protects crypt stem cells and dramatically improves survival, indicating that anti-ceramide antibody represents the first effective antibody of small molecule mitigator of lethal RGS. To improve upon the efficacy of anti-ceramide antibody, we propose to select a recombinant single-chain antibody fusion protein containing the known ceramide-binding regions of anti-ceramide antibody as a novel Medical Radiation Countermeasure. Single-chain antibody fusion proteins are smaller derivatives of full length antibodies, and thus offer the advantage of rapid entering into the bloodstream and increased penetration into tissue compared to full-length antibodies. Additionally, single-chain antibody fusion proteins can be modified by general cloning techniques to display preferred target binding, and from a product development standpoint these fusion proteins can be produced easily and at minimal cost. While in some cases single-chain antibody fusion proteins may not retain full activity of the parent antibody, our preliminary data indicates that similar antibody fragments derived by enzymatic digestion of humanized anti-ceramide antibody results in significant protection and mitigation of radiation-induced crypt stem cell lethality, even 24 h after high single-dose exposure. These data indicate that a single-chain antibody fusion protein can be sufficient to induce therapeutic neutralization of ceramide. As such, a neutralizing anti-ceramide single-chain antibody fusion protein represents a promising candidate to fulfill the Project BioShield mandate for development of countermeasures to mitigate acute radiation syndromes within the first 24 h after a nuclear disaster.