Establishment of the adoptive anticancer immunotherapy for the unresectable cholangiocarcinoma using the bispecific antibody - Construction of bacterial expression system of bispecific antibodies

为了构建稳定的供应体系，应用于不可切除胆管癌的免疫治疗，构建了原杂交瘤细胞系抗muc1 IgG、抗cd3 IgG和抗cd28 IgG变区细菌表达体系。克隆每个编码免疫球蛋白相应可变区域的cDNA，然后用柔性肽连接体（glyglyglyser）3连接这些区域。编码单链Fv片段的基因排列顺序为vh - link_vl或vl - link_vh。表达的不溶性蛋白用胍-盐酸溶解，再折叠，用金属螯合化学法纯化。制备的MUSE-11、抗cd28和抗cd3 scFv对每种原始IgG具有相同的特异性。在t细胞上，一种与腺癌相关抗原MUC1和CD3结合的双特异性糖尿病已被关注。也就是说，一条链由MUC1特异性的VH与CD3特异性的VL通过短多肽连接物（GGGGS）连接而成。其次，它们由MUC1特异性的VL与CD3特异性的VH连接而成。从大肠杆菌的细胞内不溶性组分中独立获得两种异质sc Fvs，经过化学计量学纯化和混合，并通过改进的透析方法进行折叠。重新折叠的两个异源单链单链病毒具有异二聚体结构，对两个靶细胞都具有明显的特异性。通过对癌细胞的生长抑制实验评价T-LAK与机体的体外疗效，结果表明，当效应靶比为10时，T-LAK对癌细胞的生长抑制最大可达98%左右，与化学合成抗muc1 x抗cd3的bsab几乎相同。双特异性糖尿病可能是新的过继性靶向免疫治疗的良好候选者。

In order to construct the stabel supply system and apply to the immunotherapy for unresectable cholangiocarcinoma, the bacterial expression system of variable regions of anti-MUC-1 IgG, anti-CD3 IgG and anti-CD28 IgG derived from original hybridoma cell line was constructed. Each cDNA encoding corresponding variable region of immunoglobulin was cloned, followed by linking the regions with flexible peptide linker (GlyGlyGlyGlySer)3. The genes encoding single chain Fv fragment were arranged with either VH-linker-VL or VL-linker-VH. The expressed insoluble proteins were solubillized by Guanidine-HCl, refolded, and purified by metal-chelating chematography. Prepared MUSE-11, anti-CD28 and anti-CD3 scFv had same specificity for each original IgG. And a bispecific diabody binding to adenocarcinoma associated antigen MUC1 and to CD3 on Tcells has been focused. Namely, one chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). They second was composed of the VL specific for MUC1 linked to the VH specific for CD3. The two hetero sc Fvs were independently obtained from intracellular insoluble fractions of E. coli, purified and mixed stoichiometrically and refolded by the modified dialysis method. The refolded two hetero scFv has hetero-dimeric structure, with distinct specificity for both target cells. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach about 98% at the effector:target ratio of 10, almost identical to that with anti-MUC1 x anti-CD3 chemically synthesized BsAbs. Bispecific diabody may be good candidates for new adoptive targeted immunotherapy.