Recombinant Human Anti-HIV-1 gp120 Antibody (PGT123)

Competitors (IgG) are listed at the left and analytes (biotinylated IgG) at the top. Some bNAbs could not be biotinylated. In those cases (indicated by *), the competitors were Fabs and the analytes were IgG. The bNAb clusters were arranged according to their location on the Env spike from top to bottom (membrane-distal to membrane-proximal). Within clusters, the bNAbs were arranged in such a way as to obtain an optimal inhibitory effect along the diagonal axis. Color-coding depicts the extent of competition or enhancement: red and orange indicate strong and intermediate inhibition, yellow indicates weak inhibition and green indicates no effect. Weak and strong enhancement effects are indicated in turquoise and blue. The numbers inside each box are derived from 6–9 independent values and indicate the percentage of residual binding of the analyte relative to the control (100%). The standard errors for all data points are plotted in the accompanying matrix in S1B Fig. The 8ANC195 Fab was unable to self-compete, and the 1NC9 and 35O22 Fabs were only weakly self-competitive. The bold numbers for 8ANC195 and 35O22 represent competition values obtained by SPR, not ELISA, and are means of two replicates.

Derking, R., Ozorowski, G., Sliepen, K., Yasmeen, A., Cupo, A., Torres, J. L., ... & Connors, M. (2015). Comprehensive antigenic map of a cleaved soluble HIV-1 envelope trimer. PLoS pathogens, 11(3), e1004767.

The order from left to right is as on the inset legend from top to bottom. Negative values indicate a decrease in binding affinity of sCD4 or PGV04 in the presence of PGT121 antibodies, positive values represent an increase in binding affinity and a value of ~1 represents no effective change in binding affinity. Sequential ITC binding experiments show that sCD4 has over 100 to 650 fold decrease in binding affinity to gp120 when antibodies of the PGT121 family are pre-complexed with gp120. On the other hand, pre-complexing PGT121 antibodies with gp120 does not lead to a change in binding affinity for PGV04 to gp120. These results suggest that antibodies of the PGT121 family do not sterically block access to the CD4 binding site (Fig. S8A), but disturb CD4 binding, possibly by interfering with gp120 conformational changes associated with CD4 binding. No sCD4 competition by PGT121 antibodies is observed for a core-miniV3 gp120 construct, indicating that modulatory elements of gp120 such as C1, V1/V2 and fully length V3 are important in modulating sCD4 competition.

Julien, J. P., Sok, D., Khayat, R., Lee, J. H., Doores, K. J., Walker, L. M., ... & Katpally, U. (2013). Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. PLoS pathogens, 9(5), e1003342.

17b+gp120 (black) and 2G12+gp120 (orange) complexes bound well to CD4+ TZM-bl cells. On the other hand, a PGT123+gp120 complex (red) was not able to engage CD4+ TZM-bl cells. Lack of binding of the PGT123+gp120 complex to CD4+ TZM-bl cells is comparable to similar lack of binding of gp120 in complex with the CD4-binding site antibody PGV04 (blue). A similar inability to bind CD4+ TZM-bl cells was observed for gp120 in complex with PGT121 and PGT122 antibodies (Fig. S9). PE-A represents the relative intensity of detected Fab on the surface of CD4+ TZM-bl cells. Curves with filled areas indicate Fab alone (negative control), whereas curves with hollow areas are for the Fab-gp120 complexes.

Julien, J. P., Sok, D., Khayat, R., Lee, J. H., Doores, K. J., Walker, L. M., ... & Katpally, U. (2013). Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. PLoS pathogens, 9(5), e1003342.

PGT123 Fab (pink), b12 Fab (yellow) and 17b Fab (green) were pre-incubated with the cells in titrating amounts at 37uC before being exposed to a constant amount of either C) sCD4 or D) PGV04. As expected, b12 Fab that targets the CD4 binding site directly competes with CD4 binding, and 17b Fab, which binds to the co-receptor binding site, enhances CD4 binding. PGT123 Fab competes with sCD4 binding to the same extent as b12 Fab. A similar level of sCD4 competition was observed for PGT121 and PGT122 antibodies (Fig. S9). On the other hand, PGT123 Fab does not compete with PGV04, a CD4 binding site targeted antibody that does not induce conformational changes upon binding. Binding curves are represented by plotting the dimensionless mean fluorescence intensity (MFI) of C) sCD4 and D) PGV04 binding as a function of Fab concentration.

Julien, J. P., Sok, D., Khayat, R., Lee, J. H., Doores, K. J., Walker, L. M., ... & Katpally, U. (2013). Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. PLoS pathogens, 9(5), e1003342.

A) SEC-purified complexes of gp120 with various Fabs were tested for their ability to bind CD4⁺ TZM-bl cells by FACS. Whereas 17b+gp120 (black) and 2G12+gp120 (orange) complexes bound well to CD4⁺ TZM-bl cells, PGT121+gp120, PGT122+gp120 and PGT123+gp120 complexes (red) were not able to engage CD4⁺ TZM-bl cells. Lack of binding of the gp120 complexes with PGT121, PGT122 and PGT123 is comparable to that of the CD4-binding site antibody PGV04 in complex with gp120 (blue). On the x-axis, PE-A represents the relative intensity of detected Fab on the surface of CD4⁺ TZM-bl cells. Filled areas indicate Fab alone (negative control), whereas hollow areas are for the Fab-gp120 complexes. B) Binding curves of elements used in the competition assays to cell-surface HIV-1 JRFL Env. C) sCD4 binding to cells expressing JRFL Env on their surface as observed by flow cytometry. PGT121 (brown), 122 (green) and 123 (blue) Fab were pre-incubated with the cells in titrating amounts at 37°C before being exposed to a constant amount of sCD4. Antibodies of the PGT121 family compete with sCD4 to the same extent. D) VRC01 binding to cells expressing JRFL Env on their surface as observed by flow cytometry. PGT123 Fab (pink), b12 Fab (yellow) and 17b Fab (green) were pre-incubated with the cells in titrating amounts at 37°C before being exposed to a constant amount of VRC01. As expected, b12 Fab that targets the CD4 binding site directly competes with VRC01 binding, and 17b Fab, which binds to the co-receptor binding site, shows no competition with VRC01 binding. PGT123 Fab does not significantly compete with VRC01, a CD4 binding site targeted antibody that does not induce conformational changes upon binding. Binding curves are represented by plotting the dimensionless mean fluorescence intensity (MFI) of VRC01 binding as a function of Fab concentration.

Julien, J. P., Sok, D., Khayat, R., Lee, J. H., Doores, K. J., Walker, L. M., ... & Katpally, U. (2013). Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. PLoS pathogens, 9(5), e1003342.