Study On The Construction And Activity Of Monovalent Form Of HIV-1 ENV Specific Antibodies

Neutralizing antibody is an important protective immune response in the human body, so finding broadly neutralizing antibodies against HIV-1 is very important for AIDS prevention and therapy. As a consequence of its high variability, HIV rapidly develops resistance to a neutralizing antibody. Therefore, combination of different broadly neutralizing antibodies can significantly increase the efficiency of HIV-1control. However, the effect of antibodies combination may be damped due to the steric hindrance of several antibodies simultaneously binding to viral particles.Monovalent antibody with smaller molecular weight may be a good substitution for IgG when several antibodies be used simultaneously. There are two kinds of monovalent antibody, the N-terminally truncated human IgG(IgG-tH) with one Fab and one Fc, and the half molecule of IgG(IgG-HM) which consists of a heavy chain and a light chain moiety. In this study, the known HIV-1 neutralizing antibody 2G12 was uesed to construct IgG-tH and IgG-HM firstly, and their binding and neutralizing activity were compared. Then, the better monovalent antibody form which characterized with good binding and neutralizing activity and easy to expression and purificaton was chosed to construct other monovalent antibodies and applied to antibodies combination against HIV-1.To obtain the monovalent form HIV-1 specific antibody 2G12 with 1 Fab and1Fc(2G12-tH), an expression vector containing DNA encoding the IgG1 hinge,CH2 and CH3 domain exons was constructed and co-transfected with the plasmids expressing 2G12 heavy and light chain. And to obtain the half molecule form 2G12(2G12-HM), the disulfide bond at hinge region and the stable non-covalent bonds in CH2, CH3 regions was removed by site-directed mutation on 2G12 heavy chain gene. The mutated heavy chain gene was co-transfected with light chain expressing plasmid. Purified 2G12-tH and 2G12-HM were analyzed by non-reducing SDS-PAGE. The antigen specific bindingand neutralizing activity against HIV-1was compared with parent IgG by ELISA and micro-neutralizing assay. Results showed that two forms of monovalent 2G12 antibody, 2G12-tH and 2G12-HM,were constructed and expressed correctly. Both 2G12-tH and 2G12-HM effectively combined with HIV-1 Env. The results of neutralizing assay based on pseudoviruses of HIV-1 subtype B indicated that monovalent form antibodies had similarneutralizing activity with parent IgG. But 2G12-HM was easier to be expressed and purified.To construct other monovalent antibodies, the 2G12-HM antibody variable region of heavy chain and light chain were substituted by corresponding part of different antibodies which were identified by our lab and other’s. 11 HM type of monovalent antibodies were successfully obtained. The neutralizing activity of these IgG-HMs were evaluated by micro-neutralization assay against HIV-1 pseudovirus in TZM-bl cells at the concentration of 25μg/mL. It was found that the neutralizing activities of most IgG-HMs were similar with their parent Ig G, but there were two IgG-HMs(DF07-2A5-HM and DF07-2G5-HM) neutralized pseudovirus TRO.11 which could not be neutralized by their parent IgGs. The HMs of broadly neutralizing antibodies VRC01, NIH45-46 and PG9 displayed the most potent and broadly neutralizing activity. Thereafter, we focused on comparing these there HMs with their parent IgGs on binding activity, affinity, and neutralizing activity when used alone or combined with other antibodies.The results showed that, compared with the parent IgG, the binding activity and affinity of monovalent antibodies were no significant difference. But different antibodies showed varient changing in the micro-neutralizing assay based on a panel of 9 subtype B pseudoviruses. When used alone, VRC01-HM antibody showed more potent neutralizing activity than VRC01-IgG against 5 of 9 pseudoviruses and no significant diffefence against others. NIH45-46-HM and PG9-HM displayed enhancement, decrease and similar neutralizing activity compared with IgG dependent on different pseudoviruses. When two antibodies used together, all the combinations of IgG-HMs showed lower IC50 than that of parent IgGs and more additive or synergistic effect were observed in IgG-HM group than IgG group.Especially in the combination of NIH45-46-HM and PG9-HM, the additive or synergistic effect were observed against 6 of 9 pseudoviruses, whereas in the combination of IgG was 2 of 9.In summary, half molecular form of monovalent antibodies were successfully constructed by modification of the constant region of IgG heavy chain. Compared with parent IgG, monovalent antibodies showed similar binding activity and affinity;When used alone, different IgG-HM showed different changing trend in neutralizing activity dependent on pseudovirus used for assay. However, the IgG-HM groupdisplayed more potent neutralizing activity than IgG group when two antibodies used in combination, which indicated the advantage of IgG-HMs when two or more antibodies used simutaneously.