Construction Of The Reshaping Anti-CD28 VH Single-Domain Antibody By Means Of Directed Molecular Evolution & Construction And Expression Of A Trispecific Antibody Directed Against Human Ovarian Carcinoma

The first aim of this study was to demonstrate a new and practical method for constructing reshaping antibodies. Different from other methods, it was not necessary to carry out modeling the sequences for determining the human acceptor and which amino acid residues in human acceptor FRs should be substituted, and reshaping and enhancing the antigen binding affinity shared one procedure at the same time. The reshaping anti-CD28 single domain antibodies were constructed by using this method. According to the amino acid sequence of a mouse anti-human CD28 monoclonal antibody VH, two most homologous sequences of human antibodies were searched from GenBank and one of them was used as a main framework residues for constructing the reshaping antibody FRs. The mouse anti-CD28 VH CDRs were still kept, but some amino acid residues in human FRs were substituted according to the comparison of searched human antibody sequences, original mouse antibody sequence, and their consensus sequences. In order to increase diversity of the gene, the amino acid residues planned to substitute were reserved in synthesizing the gene sequences by code degeneracy. Also, when the synthesized nucleotide fragments in different length were assembled and amplified to intact genes by overlap PCR, Taq DNA polymerase and high Mg2+ concentration were used for inducing more mutations randomly. A phage display library was constructed by using the PCR products. The genes of reshaping anti-CD28 VH that had high antigen binding activity were selected after three round panning selections. Two genes were fused with c-myc tag and the hinge region of human IgGS'CL gene, and expressed in E. coli BL21(DE3). The antigen binding affinity and specificity of renatured fusion protein were determined. The results suggested that the procedure we used was feasible and efficient, with which it could be gotten an ideal reshaping antibody in one step and so save the time and labor.The second aim of this study was to construct and express a novel trispecific antibody fragment: anti-human Ovarian Carcinoma scFvX reshaping anti-CD3 scFvX reshaping anti-CD28VH. Two types of the trispecific antibodies were constructed: one was constructed in a cycle formation; another was in a broken cycle formation. Different interlinkers were used in the construction, and some interlinkers could induce some biological properties beside the connection function. The molecular masses of the trispecific antibodies were about 84 kDa, which were suitable for deliver and tumor targeting. The genes of the two type trispecific antibodies were expressed in E.coli BL21(DE3), and the CD28 binding affinity of the expression proteins were determined.