| The highly pathogenic avian influenza (HPAI) is a severe infectious disease caused by H5N1 virus. As the virus spread worldwide in recent years, the poultry industry has suffered large economic loss and the virus has broken the restriction of interspecies transmission to infect human. The current problem of H5N1 is the high mortality and the lacking of effective drugs. The pandemic virus strains with different antigenicity prevalence at the same period due to the persistent mutation. It is necessary to develop drugs of wide range to H5N1 to meet the need of long-term strategic reserve.Neutralizing antibodies is an effective way to control viral diseases. However, the neutralizing antibody to H5N1 has limit spectrum to a few strains because of the highly frequent mutation of virus genes, especially the hemagglutinin (HA) genes, which restricts the application of neutralizing antibodies.We has constructed a H5-specific antibody panel with hemagglutinin inhibition(HI) to 41 strains of virus belonging to 10 clades and some of them had broad-spectrum protection to mouse in vivo. In the paper, four strains of mouse antibodies designated as 4D1, 10F7, 8H5 and 13D4 with high activity in neutralizing assay are humanized to reduce the human anti-mouse antibody (HAMA) in therapy, which is fundamental to the clinical application. It is also expected to construct a platform for therapeutic antibody humanization including gene modification, construction of expressing vector, stable cell strain of highly expression, scale-up culture with serum-free and antibody purification.In our research, the variable genes were obtained from murine hybridoma cells by RT-PCR and combined with the constant region of human IgG1. The genes were then inserted into pcDNA3.1 to transiently expressing chimeric antibody. The activity was tested by HI, ELISA and Immunofluorescence. To accumulate the appropriate amount of antibody for further research, we used Flp-In expression system which could stably express antibody with high efficiency by FRT targeted integration. After the screening procedure, we obtained several stable cell lines and the production of antibody increased 3-4 times.To fulfill the purification and scale-up production, we developed the CHO culture with serum-free system, including nutrient content in medium, roller bottle culture. We found that the addition of BSA could increase the production significantly, especially 3% BSA addition which could increase by 8 times but influenced the purification. The expression strategy could be optimized considering the purity and yield of antibody. The affinity chromatography by protein A was a specific and effective way for antibody purification. We explored eluting buffer with different pH and obtained the appropriate one with final purity of 95% and yield of 90%.To confirm the humanization and expression procedure had no influence on the activity of variable genes, the purified antibody from serum-free culture was tested by HI and neutralizing assay to several strains of H5N1 virus to test the broad-spectrum neutralization. The protection assay in mouse was performed to the 13D4cAb with broad-spectrum neutralization. It showed that the 13D4cAb had 100% therapeutic activity to 4 clades of virus.In summary, we successfully constructed four strains of chimeric antibodies to HPAI H5N1. They maintained the broad-spectrum neutralizing activity as murine antibodies and reduced the HAMA in therapy, which laid a foundation to antibody therapy for H5N1 infection.
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