Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

Abstract

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.

Figure 1

Target residues for library construction. Amino acid sequence of the variable regions of D5 heavy chain and light chain are shown with the CDR regions underlined. The target residues that were randomized in the maturation libraries are shown in red bold type. Two libraries were designed for each of the CDR3 regions, with six overlapping codons in each. These libraries are indicated as VH CDR3A&B (DNPTLL, LLGSDY) and VL CDR3A&B (QQYSNY, SNYPLT).

Figure 2

Reproducibility of single-cycle HIV-1 infectivity assay in functional screening. Two sets of scFv were made and tested in separate infectivity assays 2A and 2B. Results of 11 clones are shown from each of the two runs. S1 to S4 indicate the dilution of each sample. The columns indicate the amount of HIV-1 that enters the P4/R5 cells. Clones D5 and D7 show inhibition at all 4 dilutions. Clone D9 shows the least amount of viral inhibition.

Figure 3

Structural rationale for D5 scFv affinity maturation clones. (A) D5 CDR loops (colored and labeled) contacting the gp41 N-peptide trimeric inner core (shades of yellow and labeled). (B) D5 HC CDR2 loop entering gp41 hydrophobic pocket. Core D5 residues are labeled in blue and gp41 residues are labeled in black. (C) D5-derived ‘Trp 571’ pocket. Several residues from five D5 CDR loops surround the gp41 N-peptide. Loops and specific contact residues are colored and labeled as follows: H1 (green), H2 (purple), H3 (cyan), L1 (orange), L3 (peach). Solvent molecules are labeled in the interface and hydrogen bonds are shown as dotted lines. (D) Model of D5 mutant with alanine at position HC 33 substituted for glutamine. In this model, the water molecules w1 and w2 are replaced by the glutamine side chain such that new hydrogen bonds with gp41 Gln575, D5 HC Asp95 and D5 LC Tyr94 may exist. (E) The region of D5 HC CDR1 behind HC CDR2 contains conserved aromatic side chains (Phe29 and Trp36). In D5, the 34 position is an isoleucine, however several affinity matured clones contain a tyrosine at this residue.

Figure 4

Correlations between neutralization potency (IC₅₀) and in vitro binding affinity (K_d) for scFvs vs. IgGs of selected D5 variants. IC₅₀ for the single-cycle HIV-1 infectivity assay vs. in vitro binding affinity determined by surface plasmon resonance for (A) scFv variants and (B) corresponding IgG variants. Inhibition of infection and binding affinity correlate for scFvs (R² = 0.50) but not for IgGs (R² = 0.07). Note that X and Y-axis scales are different between panels A and B to show correlations.

Figure 5

Schematic model of designed peptide antigen (Biotin-CCIZN23)₃. The IZN helices forming a homotrimeric coiled-coil are represented by cylinders. The N peptide consists of an N-terminal designed trimeric coiled-coil (IZ, light gray) fused to a portion of the sequence from the NHR of gp41 (dark gray), namely N23 (residues 559–581 of HIV-HXB2). The (CCIZN23)₃ helices are covalently stabilized at the N-termini by three interchain disulfides between pair of cysteines of each peptide chain. Only one of the possible combinations of the three disulfides is drawn. The Biotin-Gly residues (not shown in the model) are assembled at the N-terminus of the CC-IZN23 sequence.

Table 3

Clones from first screen tested in single-cycle infectivity assay

Table 4

scFv screening results from duplicate runs in the single-cycle HIV-1 infectivity assay

Table 5

D5 scFv variants with undetectable functional activity