Human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive HIV-1 neutralizers

Abstract

The antibody access to some conserved structures on the HIV-1 envelope glycoprotein (Env) is sterically restricted. We have hypothesized that the smallest independently folded antibody fragments (domains) could exhibit exceptionally potent and broadly cross-reactive neutralizing activity by targeting hidden conserved epitopes that are not accessible by larger antibodies. To test this hypothesis, we constructed a large (size 2.5 x 10(10)), highly diversified library of human antibody variable domains (domain antibodies) and used it for selection of binders to conserved Env structures by panning sequentially against Envs from different isolates. The highest affinity binder, m36, neutralized all tested HIV-1 isolates from clades A- D with an activity on average higher than that of C34, a peptide similar to the fusion inhibitor T20, which is in clinical use, and that of m9, which exhibits a neutralizing activity superior to known potent cross-reactive antibodies. Large-size fusion proteins of m36 exhibited diminished neutralizing activity but preincubation of virions with soluble CD4 restored it, suggesting that m36 epitope is sterically restricted and induced by CD4 (CD4i). M36 bound to gp120-CD4 complexes better than to gp120 alone and competed with CD4i antibodies. M36 is the only reported representative of a promising class of potent, broadly cross-reactive HIV-1 inhibitors based on human domain antibodies. It has potential for prevention and therapy and as an agent for exploration of the closely guarded conserved Env structures with implications for design of small molecule inhibitors and elucidation of mechanisms of virus entry and evasion of immune responses.

Fig. 1.

Construction of a human antibody VH library. A stable VH (m0) was used as a scaffold for grafting CDR2s and CDR3s from five human antibody Fab libraries. CDR1 residues 27, 29, 31, and 32 (ImMunoGeneTics numbering system) were randomized to A, D, S, or Y. The numbers denote the positions of the amino acid residues corresponding to the respective regions of the antibody VH gene where the CDRs were grafted; the # denotes the positions of the CDR1 randomization. The SfiI denotes the restriction enzyme sites used for cloning.

Fig. 2.

Potent m36-mediated neutralization of viruses pseodotyped with Envs of HIV-1 primary isolates. (A) Dose-dependent inhibition of Bal by m36, scFv m9, and C34, respectively. (B) Percentage inhibition of a panel of viruses by m36, scFv m9, and C34 at 667 nM, respectively.

Fig. 3.

Binding of m36 to gp120 is induced by the gp120 interaction with CD4. (A) Specific binding of m36 to gp120_Bal-CD4 but not to gp120_Bal alone. (B) Competition of m36 with a CD4i antibody (IgG m16) for binding to gp120_Bal-CD4. The CD4bs antibody m14 was used as a negative control.

Fig. 4.

Design of m36 fusion proteins. Schematic representation of m36 fused with SAbp, human IgG1 CH3 domain, or Fc. The names of the constructs and their molecular weights are also shown. The sequences of the SAbp and the linkers used to join m36 with Fc by human IgG1 and IgG3 hinge, and camel IgG2 hinge are QRHPEDICLPRWGCLWGDDD, DKTHTCPPCP, EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP, and ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCP, respectively.

Fig. 5.

Comparable binding activity of m36 and its fusion proteins. (A) Binding of m36, m36SAbp, m36CH3, and m36b0Fc to gp120_Bal-CD4 and gp120_Bal, respectively. (B) Binding of m36h1Fc, m36c2Fc, and m36h3Fc to gp120_Bal-CD4 and gp120_Bal, respectively.

Fig. 6.

Pretriggering (sensitization) of virus by sCD4 dramatically increases neutralization by large molecules fused with m36. Viruses were preinoculated with different concentrations of antibodies and/or sCD4 at 8 nM for 1 h at 37°C, and then the mixture was added to 1.5 × 10⁴ HOS-CD4-CCR5 cells grown in each well of 96-well plates. Luminesence was measured 48-h postinfection and percentage inhibition was calculated as described in Materials and Methods.