抗自身免疫疾病靶分子CD147和FKBP52的新型双特异人源抗体的构建及功能研究

In recent years, several varieties of monoclonal antibodies (Mabs) created to inhibit proinflammatory cytokines in the pathologic inflammation process have become an active area of drug discovery and development investigation in China and the rest of the world. Many examples now exist which show that humanized and/or fully-human Mabs are effective for clinical therapy for autoimmune diseases including Rheumatoid Arthritis, Psoriasis, and Crohn's Disease. Our previous studies have shown that the intracellular immunophilin FKBP52 (iFKBP52) is elevated upon CD3+ T cell activation and, similar to cyclophilin A, FKBP52 also exists as a soluble mediator (sFKBP52). We have recently shown that the recombinant human form (rhFKBP52) also appears to be a potent Mφ activation factor (MAF). This transmembrane form of FKBP52 (tmFKBP52) forms a binding complex with Extracellular Matrix Metalloproteinase Inducer (CD147; EMMPRIN) and facilitates T cell-mediated Mφ proinflammatory cytokine production in a cell-cell contact manner. siRNA knockdown of either tmFKBP52 or CD147 did not appreciably affect T cell-driven Mφ-cytokine production in vitro, however, T cell-driven Mφ-cytokine levels were decreased by ~95% when expression was simultaneously inhibited by dual siRNA knockdown for both T cell proteins. These data indicate that soluble FKBP52 is an important new therapeutic target of autoimmune disease. This study will use a large, highly-diversified humanized phage display library to produce ScFv's against rhFKBP52 and EMMPRIN. Subsequently, we will construct anti-EMMPRIN and anti-FKBP52 double-specific humanized antibodies to validate their biological function in animal models of autoimmune disease. Our proposal will provide new ideas to help us to discovery more effective and more safer therapeutic drugs for long-term chronic inflammatory diseases.

近年来，针对病理性炎症过程中多种因子及细胞的单抗成为治疗自身免疫性疾病热点。已有多种单抗作为治疗性药物进入临床。前期研究工作发现：免疫抑制剂FK506结合蛋白(FKBP52P)在CD3+T细胞激活时表达量升高，并同环胞霉素A结合蛋白（CyclophilinsA）作用靶点相同，与基质金属蛋白酶诱导因子（CD147）相互作用，参与下游巨噬细胞的激活。siRNA方法单独抑制FKBP52或者CD147不能抑制T细胞介导的巨噬细胞炎症相关细胞因子的分泌，但同时敲除或抑制这两个基因后，T细胞介导的巨噬细胞分泌的炎症相关细胞因子会被抑制。显示其成为治疗自身免疫疾病的新靶点的潜质。本研究将运用人源大容量噬菌体抗体库筛选针对其的人源抗体，并在此基础上组合构建抗CD147和抗FKBP52双特异人源抗体，通过体内外实验探讨其在自身免疫疾病中的作用，为开发出比现有治疗手段更有效，更安全的治疗药物提供新思路。

近年来，针对病理性炎症过程中多种因子及细胞的单抗成为治疗自身免疫性疾病热点。已有多种单抗作为治疗性药物进入临床。双特异性抗体( bispecific antibody) ，又称为双功能抗体或双价抗体，可以同时特异性结合2个不同的抗原，由于其特异性和双功能性，现已成为抗体工程领域的研究热点，目前，至少有30 多种双特异性抗体处于临床研究阶段。在肿瘤、自身免疫病等领域中具有广阔的应用前景。.为此。本课题针对两个潜在的自身免疫疾病靶分子CD147 和FKBP52 开展了新型双特异人源抗体的构建及功能研究，为开发新型治疗自身免疫疾病的抗体药物提供新思路。在本研究中，首先进行了CD147、FKBP52 蛋白的原核表达与纯化，获得纯化的重组蛋白，为使CD147蛋白更符合天然结构，同时构建了CD147真核表达载体，转染293F细胞，获得表达，在此基础上运用Ni-HTA纯化浓缩获得CD147, SDS-PAGE检测纯度达95%以上。对CD147、FKBP52 蛋白在T 细胞激活中的功能进行了测定；其次将原始噬菌体抗体库感染含有cre蛋白的菌株BS1365，通过细胞内定位重组系统，完成了大容量噬菌体抗体库工作库的优化，获得了滴度为１×1013CFU/ml的工作库；随后，通过噬菌体抗体库筛选过程，结合ELISA鉴定、DNA序列测定及分析，分别获得3株抗-CD147和7株抗FKBP52人源单链抗体，并选择相对活性较高的单链抗体进行了初步亲和力等测定；进一步采用同源重组的方法完成单链抗体由库筛选的小分子抗体至完整抗体的转化真核表达载体的构建、DNA测序验证并在成功获得表达；同时完成了抗-CD147及抗-FKBP52完整抗体的分离、纯化及几株抗体的性能评价；在此基础上建立了两种适用于真核表达的双特异抗体（DVD及Cross-mab）表达载体，并成功在真核系统表达获得多种针对不同靶点的双特异抗体，利用亲和层析法对其进行分离纯化的基础上采用ELISA、流式细胞、MTT 等方法对获得双特异抗体初步进行包括细胞增殖、加入抗体前后的促炎症细胞因子(IL32γ, TNFα, IL1β)的变化情况等体内外生物功能评价，结果显示获得的抗体能够在一定程度上抑制炎性细胞因子的产生，具有一定的生物活性及功能，研究结果为探讨新型的治疗性抗体提供新思路。