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系统，应用于不能切除的胆管癌的免疫治疗，构建了来源于原始杂交瘤细胞株的抗MUC-1 IgG、抗CD 3 IgG和抗CD 28 IgG可变区的细菌表达系统。克隆编码免疫球蛋白相应可变区的每个cDNA，然后用柔性肽接头（GlyGlyGlyGlySer）3连接这些区域。编码单链抗体片段的基因以VH-接头-VL或VL-接头-VH排列。表达的不溶性蛋白用盐酸胍溶解，复性，并通过金属螯合层析纯化。制备的MUSE-11、抗CD 28和抗CD 3单链抗体对各原始IgG具有相同的特异性。近年来，一种与肿瘤相关抗原MUC 1和T细胞表面CD 3结合的双特异性双抗体的研究受到关注。也就是说，一条链由MUC 1特异性VH和CD 3特异性VL通过短多肽接头（GGGGS）连接组成。第二种是由MUC 1特异性的VL与CD 3特异性的VH连接组成。这两个异源sc Fv分别从大肠杆菌的胞内不溶性组分中获得。大肠杆菌，纯化和化学计量混合，并通过改进的透析法复性。重折叠的双异源scFv具有异源二聚体结构，对两种靶细胞具有不同的特异性。通过癌细胞的生长抑制测定，用双抗体评价T-LAK的体外功效，证明在效应物：靶比为10时，癌细胞的最大生长抑制达到约98%，几乎与用抗MUC 1 x抗CD 3化学合成的BsAb的相同。双特异性双抗体可能是新的过继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.