Kipriyanov S M, Moldenhauer G, Strauss G, Little M
Recombinant Antibody Research Group, Diagnostics and Experimental Therapy Program, German Cancer Research Center (DKFZ), Heidelberg.
Int J Cancer. 1998 Aug 31;77(5):763-72. doi: 10.1002/(sici)1097-0215(19980831)77:5<763::aid-ijc16>3.0.co;2-2.
For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector containing a dicistronic operon for co-secretion of V(H)3-V(L)19 and V(H)19-V(L)3 single-chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatography (IMAC) from the periplasmic extract and culture medium. Flow cytometry experiments revealed specific interactions of the diabody with both CD3 and CD19 positive cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti-CD3 scFv but more stable than anti-CD19 scFv when incubated in human serum at 37 degrees C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepared by the quadroma technique.
为了治疗白血病和恶性淋巴瘤患者的微小残留病，我们构建了一种异源二聚体双特异性抗体，它对B细胞上的人CD19和T细胞受体复合物的CD3ε链具有特异性。使用含有双顺反子操纵子的载体在大肠杆菌中表达双特异性双抗体，用于共分泌V(H)3-V(L)19和V(H)19-V(L)3单链Fv片段（scFv）。通过固定化金属亲和色谱（IMAC）从周质提取物和培养基中一步纯化。流式细胞术实验揭示了双抗体与CD3和CD19阳性细胞的特异性相互作用，其结合亲和力接近亲本scFv。当在37℃的人血清中孵育时，它比抗CD3 scFv稳定性差，但比抗CD19 scFv稳定性好。在细胞毒性试验中，双抗体被证明是一种有效的试剂，可将外周血淋巴细胞重新靶向以裂解表达CD19抗原的肿瘤细胞。细胞裂解效率与通过四瘤技术制备的具有相同双特异性的双特异性抗体（BsAb）相当。
