Preparation And Evaluation Of A Human Anti-Toll Like Receptor 4 Antibody Fab Fragment

TLR4 receptor is a kind of pattern recognition receptors. In the study found that TLR4 plays a key regulatory role in LPS‐mediated severe reactions. TLR4 adjust the intracellular signal transduction of LPS through My D88‐dependent pathway or no My D88‐dependent, to control endotoxin biological effect. Blocking TLR4 mediated signal transduction of endotoxin into the cells, is an effective way to reduce the biological effects of endotoxin. In our previous studies on murine anti TLR4 antibody, a mouse anti‐human TLR4 monoclonal antibody has been successfully prepared, which showed that it can significantly inhibit the generation of TNF‐α stimulated by LPS. However, it is worth to note the immunogenicity of m Abs when they were used in human body as a foreign protein. The approach of development of human antibody is cumbersome and difficult to achieve. So it is necessary to develop new human and chimeric antibodies to reduce immunogenicity to eliminate adverse reactions of exogenous protein from murine and maintain high affinity to antigen. In this study, we prepared a chimeric Fab antibody using variable regions of anti‐TLR4 m Ab combined with human constant regions by splicing by overlap extension. Then, the chimeric Fab was expressed and evaluated.Methods:(1) Preparation of human anti‐chimeric Fab antibody fragments TLR4. The variable regions of the heavy chain(VH) and the light chain(VL) of positive clones of phage which were screened In the previous studies were amplified by PCR. Human constant regions of the heavy chain domain 1(CH1) and the light chain(CL) were also amplified by PCR. Chimeric heavy chain(Fd) and light chain were prepared by overlap extension(SOE). Recombinant plasmid was constructed and transformed into competent E. coli BL21. After expression and purification, chimeric Fab antibody was obtained.(2)Identification of Fab antibody and antigen interaction, determination of antibody affinity constant. The ability of antibody Fab fragments specific recognized TLR4 antigen was detected by flow cytometry. The antibody affinity constant was detected by anti‐TLR4 Biacore X100.(3) Evaluation of the neutralization effects of Fab against TNF‐α in vitro.Results:(1) Recombinant plasmid p ETDuet TM‐1‐Fab was constructed. High purity of TLR4 chimeric antibody Fab fragments was obtained.(2)The ELISA and flow cytometry showed that the antibodies produced in this study can be combined with h TLR4 specificity.(3) TLR4 chimeric anti‐Fab antibody affinity constants in this study prepared was 1.619 * 10‐7.(4) The chimeric Fab antibody could effectively protect cells and neutralize TNF‐α in vitro.Conclusion:We have developed a human/murine chimeric Fab against TLR4 to reduce immunogenicity of murine m Ab. The chimeric Fab could interact with TLR4 with high affinity,and it also could neutralize TNF‐α in vitro. Thus we believe that the chimeric Fab may provide a new biologic agent in prevention and therapy of the diseases where TLR4‐mediated signal transduction plays a pathophysiological role.