Examination of the contributions of size and avidity to the neutralization mechanisms of the anti-HIV antibodies b12 and 4E10

Abstract

Monoclonal antibodies b12 and 4E10 are broadly neutralizing against a variety of strains of the human immunodeficiency virus type 1 (HIV-1). The epitope for b12 maps to the CD4-binding site in the gp120 subunit of HIV-1's trimeric gp120-gp41 envelope spike, whereas 4E10 recognizes the membrane-proximal external region (MPER) of gp41. Here, we constructed and compared a series of architectures for the b12 and 4E10 combining sites that differed in size, valency, and flexibility. In a comparative analysis of the ability of the b12 and 4E10 constructs to neutralize a panel of clade B HIV-1 strains, we observed that the ability of bivalent constructs to cross-link envelope spikes on the virion surface made a greater contribution to neutralization by b12 than by 4E10. Increased distance and flexibility between antibody combining sites correlated with enhanced neutralization for both antibodies, suggesting restricted mobility for the trimeric spikes embedded in the virion surface. The size of a construct did not appear to be correlated with neutralization potency for b12, but larger 4E10 constructs exhibited a steric occlusion effect, which we interpret as evidence for restricted access to its gp41 epitope. The combination of limited avidity and steric occlusion suggests a mechanism for evading neutralization by antibodies that target epitopes in the highly conserved MPER of gp41.

Fig. 1.

Structures of antibody constructs. Space-filling models are presented above a description of the domain organization for each construct (V_L, variable light; V_H, variable heavy; (G₄S), Gly-Ser linker; H₆, 6×-His tag). Models were constructed by using coordinates for the heavy (blue) and light (yellow) chains of Fab 4E10 and its peptide epitope (red) (PDB ID code 1TZG) (34). For the diabody model, 2 4E10 V_H–V_L pairs were aligned to the structure of diabody L5MK16 (PDB ID code 1LMK) (30). For the IgG model, 2 4E10 Fabs were used to replace the b12 Fabs in the structure of intact IgG1 b12 (PDB ID code 1HZH) (55). Solid lines indicate approximate dimensions for the scFv, diabody, and Fab. Dotted lines indicate approximate maximal distances between combining sites for the scBvFv and IgG. Curved black arrows indicate axes of rotation.

Fig. 2.

Biophysical characterization of the antibody constructs. (A) Reduced SDS/PAGE. (B) Gel filtration profiles. (C) Molecular weight determinations and binding experiments. Observed results from static light scattering experiments (Obs) are presented beside molecular weights calculated from the relevant sequence (Calc) in column 2 (n.d., not done). Kinetic and equilibrium constants are presented in columns 3–5.

Fig. 3.

Bar graph of ratios of average molar IC₅₀ values (arithmetic means) for b12 constructs (blue) and 4E10 constructs (orange). Reagent pairs with an average ratio of 1.0 (black line) are equal in average potencies. Ratios >1.0 indicate that reagent b is more potent than reagent a. Ratios <1.0 indicate that reagent a is more potent than reagent b. Error bars represent the standard errors calculated from the variability in strain-specific ratios for each pair of reagents.