抗人CD3改形单链抗体的构建、表达及活性测定

ＣＤ３单抗通过多种途径有效地同体的免疫状态，在临床应用中具有极大的潜力。为克服鼠源单抗用于临床的局限性，拟采用抗体工程技术研制抗人ＣＤ３改形单链抗体。首先，将鼠源ＣＤ３单抗ＯＫＴ３轻重链ＣＤＲｓ分别移植到人源抗体ＬＳ１轻链和Ｎｄ重链的框架中，经计算机模拟其空间构象，进行残基替换，确定ＣＤ３改形ＶＬ、ＶＨ氨基酸序列，化学合成改形ＶＬ、ＶＨ基因，将其分别插入至载体ｐＲＯＨ８０中，构建成抗人ＣＤ３改形单

Construction, Expression and Activity Test of a Reshaping Single-chain Antibody Against Human CD3

Monoclonal_antibody_(McAb)_against human CD3 can adjust human body's immune statement in various ways, so that its clinical potential is highly regarded. In order to overcome the immunogenecity related to the murine McAb, this research effort was focused on constructing a reshaping single-chain antibody(scFv) against human CD3 employing antibody engineering. First, the CDRs of the murine McAb against human CD3 OKT3 was transplanted into the light-chain framework regions (FRs) of human McAb LS1 and the heavy-chain FRs of human McAb Nd respectively, spatial conformation was predicted by computer analysis. Then some particular residues were replaced in FRs basing on the result of conformational prediction to draw out the amino acid sequences of the reshaped VL and VH_. The genes were chemically synthesize and inserted into an expression vector pROH80 to construct the reshaping scFv. Inducing the expression of reshaping scFv, the products are mainly as inclusion bodies. The reshaping scFv was expressed in another vector pALM. The inclusion bodies were denatured and then renatured by gel filtration. The renatured products were purified by immobilized metal affinity chromatograph (IMAC). Finally, the antigen-binding activily of the reshaping scFv against human CD3 was testified by the Compelitire in hibilory fluorescenceactivated cell sorting (FACS). The competitive inhibition rate is 18%.