用于促进脊髓损伤后神经轴突再生的人源化抗NB-3单克隆抗体的研发及转化医学研究

After spinal cord injury (SCI), the structure of spinal motor circuit (SMC) is permeant damaged because traumatic axons can’t regrowth in CNS. The patients lose locomotion ability partly or entirely with paralysis. SCI induced paralysis currently is one of incurable symptoms in spinal cord injury. In our previous study, we demonstrated that NB-3 trans-homophilic interactions mediated the cross-talk between post-traumatic axons and scar-forming cells and impaired the intrinsic growth ability of injured axons. The regrowth of corticospinal axons was significantly enhanced in vivo with interruption of NB-3 transhomophilic interactions. In vivo evidence also demonstrated NB-3 deficiency promoted locomotion, electrophysiological transmission, and synapse reformation. Based on our previous studies, we plan to identify the function domain that contains the binding site and mediates the NB-3 trans-homophilic interactions in this proposal. Moreover, we will develop the specific monoclonal antibody against NB-3 that targets at the trans-homophilic binding site to interrupt the NB-3 trans-homophilic interactions. The anti-NB-3 monoclonal antibody will be screened and verified in vivo to obtain the effective monoclonal antibody against NB-3 trans-homophilic interactions. The efficiency and reliability of its application leading to axonal regeneration, neural circuit reconstruction and locomotion recovery will be evaluated in SCI mice. Moreover the humanized anti-NB-3 monoclonal antibody will be developed by genetic engineering technology, and its treatment effect on SCI will be evaluated in the SCI animal model. All these studies will provide the strong support to carry out further investigations for translation medical research in the future.

脊髓损伤后由于中枢神经轴突不能再生、脊髓运动神经环路被破坏，导致运动功能丧失，出现截瘫，是难以治愈的重大神经系统疾病。目前在临床上尚无有效促进脊髓损伤后中枢神经再生和环路重建的药物。我们研究发现：脊髓损伤后神经识别分子NB-3通过反式同源性结合介导瘢痕形成细胞和损伤皮质脊髓束神经轴突之间的分子对话，抑制神经元内在的轴突再生能力；而阻断NB-3反式同源性结合，可以显著提高损伤神经轴突的再生能力，促进神经轴突再生、运动功能改善和突触重建。基于此发现，本课题拟定位NB-3反式同源性结合的结构区域，以此为靶点研发特异性阻断NB-3反式同源性结合的抗NB-3单克隆抗体，并在动物模型上验证其对于促进脊髓损伤后神经轴突再生、神经环路重建和运动功能恢复的有效性和可靠性。进一步发展人源化抗NB-3单克隆抗体, 并在动物模型上评价其治疗脊髓损伤的作用，为将来的临床转化医学研究打下坚实的基础。

脊髓损伤后由于中枢神经轴突不能再生、脊髓运动神经环路被破坏，导致运动功能丧失，出现截瘫，是难以治愈的重大神经系统疾病。目前在临床上尚无有效促进脊髓损伤后中枢神经再生和环路重建的药物。本项目拟在前期研究的基础上解析NB-3反式同源性结合所依赖的功能结构区域，以此为靶点研发特异性阻断NB-3反式同源性结合的抗NB-3单克隆抗体，并在动物模型上验证其对于促进脊髓损伤后神经轴突再生、神经环路重建和运动功能恢复的有效性和可靠性。本项目开展的研究工作明确了NB-3反式同源性结合所依赖的胞外段蛋白质功能结构域及关键氨基酸位点，获得了促进神经轴突再生的潜在药物靶点；应用小鼠杂交瘤技术获得抗NB-3单克隆抗体细胞株，通过对其特异性、功能有效性等的筛选，获得了具有能够识别NB-3特异性功能结构域、有效阻断其反式同源性作用的抗NB-3单克隆抗体；通过体外和体内实验验证，筛选到的抗NB-3单克隆抗体能够有效促进脊髓损伤后神经轴突再生和运动功能改善; 通过对抗体进行基因工程替换制备了人源化抗NB-3单克隆抗体。这些工作为使用抗NB-3单克隆抗体治疗脊髓损伤提供了重要依据，为将来开展临床转化医学研究打下了坚实的基础。