Anti-hepatitis b virus antibodies and use thereof
a technology of hepatitis b virus and antibody, applied in the field of molecular virology and immunology, can solve the problems of less sources of high-potency plasma, no definitively significant and effective active immunotherapy drug/vaccine that can be used to treat chronic hbv infection, and high price, so as to effectively eliminate hbv and hbv-infected cells, neutralize the virulence of hbv, and reduce the serum level of hbv
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example 1
eening of pH-Dependent Anti-HBsAg Antibodies
1.1 Determination of Mutation Sites for pH-Dependent Antibody Modification
[0191]The anti-HBV humanized antibody 162 (detailed in Chinese patent application 201610879693.5) developed in the laboratory was used as the parent antibody, and its variable region were modified for pH-dependent antigen binding. As shown in FIG. 1, the modified 162 could maintain the antigen-binding activity under neutral conditions, but its antigen-binding activity under acidic conditions was greatly reduced. The dissociated modified 162 could bind to intracellular FcRn so as to return to the plasma and bind to the antigen again, so that one molecule of the modified 162 with pH-dependent antigen binding ability could repeatedly bind and neutralize a plurality of molecules of antigen. Histidine was protonated under acidic conditions and was a key amino acid to bring the pH-dependent antigen binding properties. The 162 Fab had an analyzed crystal structure, the anal...
example 2
on of pH-Dependent Anti-HBsAg Antibodies
2.1 Construction of Recombinant Vector for Eukaryotic Expression
[0195]In the present invention, a large amount of antibody recombination needed to be carried out, so it was necessary to construct a set of light and heavy chain vectors that can efficiently recombine antibodies. In the present invention, the existing eukaryotic expression vector pTT5 in the laboratory was specially modified to construct a set of light and heavy chain recombinant vectors for double plasmid co-transfection. MGWSCIILFLVATATGVHS (SEQ ID NO: 54) was used as the signal peptide for the light and heavy chains. The sequences encoding the constant regions of the human antibody light and heavy chains were separately ligated to the downstream of signal peptide to construct a set of eukaryotic expression vectors pTT5-CH, pTT5-Cκ and pTT5-Cλ that facilitated antibody recombination.
[0196]The six scFv antibodies obtained in 1.3 were used to amplify the light and heavy chain var...
example 3
Analysis and Functional Evaluation of pH-Dependent Anti-HB sAg Antibodies
[0200]Through the method of Example 1, six strains of pH-dependent phage antibodies that bound to HBsAg were obtained by preliminary screening, named C26, C27, C32, D3, D4 and D5, respectively. Furthermore, the small-scale eukaryotic expression and purification of the 6 strains of phage antibodies were carried out by the method of Example 2. The VH and VL amino acid sequences of the 6 antibodies were shown in Table 4 below. In addition, the CDR sequences of the 6 antibodies were determined, and the CDR amino acid sequences of the heavy chain variable regions and the light chain variable regions thereof were shown in Table 5. The mutation sites that endowed C26, C27, C32, D3, D4 and D5 with pH-dependent antigen binding properties to HBsAg were summarized in FIG. 5.
TABLE 4Amino acid sequences of C26 / C27 / C32 / D3 / D4 / D5 light and heavy chain variable regionsSequence SEQ nameID NOAmino acid sequence C26 VH3EVQLQESGPGL...
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