Effect of Fc core fucosylation and light chain isotype on IgG1 flexibility

Abstract

N-glycosylation plays a key role in modulating the bioactivity of monoclonal antibodies (mAbs), as well as the light chain (LC) isotype can influence their physicochemical properties. However, investigating the impact of such features on mAbs conformational behavior is a big challenge, due to the very high flexibility of these biomolecules. In this work we investigate, by accelerated molecular dynamics (aMD), the conformational behavior of two commercial immunoglobulins G1 (IgG1), representative of κ and λ LCs antibodies, in both their fucosylated and afucosylated forms. Our results show, through the identification of a stable conformation, how the combination of fucosylation and LC isotype modulates the hinge behavior, the Fc conformation and the position of the glycan chains, all factors potentially affecting the binding to the FcγRs. This work also represents a technological enhancement in the conformational exploration of mAbs, making aMD a suitable approach to clarify experimental results.

Fig. 1. Analysis of cMD trajectories.

a Bar plot showing the percentage of frames in which θ angles of both Fab domains present values ≥ or <85° simultaneously. b Bar plot showing the percentage of frames in which θ angle of each Fab domain is ≥85°. c Medoids of the most populated cluster identified for each mAb with the corresponding θ values. d Box plots of CH2 domains distance distribution for each mAb. Plots shown in (a, b and d) were generated by merging three replicas for each system with n = 21,000 frames for each condition.

Fig. 2. Free energy profile along Theta1 and Theta2.

The free energy profile (after reweighting) of the four antibodies along Theta1 and Theta2 angles with the molecular surface of medoid structures isolated from the minimum energy region (in dark red) by cluster analysis. The color bar represents the PMF value in kcal/mol. Black dashed circles show the conformational space explored with cMD simulations, that for avelumab is limited with respect to that sampled via the aMD.

Fig. 3. Δϕ distribution along the minimum energy frames and along the first two eigenvectors.

Fab1 and Fab2 Δϕ distribution in κ (a) and λ (b) mAbs computed for the frames in the energy minimum and along the direction of the first two eigenvectors isolated from the entire trajectories. Density plots show how in each antibody there is at least one eigenvector that can explain the rotations of Fab domains.

Fig. 4. Distribution of CH2 domains distance and of the minimum distance between glycans.

Density plot of CH2 distance distribution (a) and of the minimum distance between glycan chains (b) for each mAb. c Structural superposition of representative Fc structures isolated from medoids, showing the different position of G0 (internal) and G0F glycans (external).

Fig. 5. Analysis of the number of contacts between C-terminal LC residues and hinge region.

Box plots showing the distribution of the number of contacts between C-terminal LC residues and hinge in adalimumab (a) and avelumab (b). The number of frames considered in these analyses is: n = 2649 for adalimumab G0, n = 3440 for adalimumab G0F, n = 3170 for avelumab G0, n = 4110 for avelumab G0F. Structural representation of G0 adalimumab (c) and avelumab (d) to highlight the structural proximity between C-terminal LC residues and the hinge. The secondary structure of Fab domains is shown as lines, the LC residues are shown as sticks, and the molecular surface of the hinge is shown in gray. For clarity, Fc is not displayed.

Fig. 6. Analysis of H-bonds between glycans and adalimumab.

a Dot plot showing the specific interactions made by G0 and G0F chains with adalimumab with a specific color code for HC1, HC2, and LC1. Structural representation of the most relevant H-bonds identified between sugars and G0 (b) and G0F (c) adalimumab. The secondary structure is shown as ribbons, main residues as yellow sticks and the molecular surface of sugars is shown in gray and dark red (fucose).

Fig. 7. Analysis of H-bonds between glycans and avelumab.

a Dot plot showing the specific interactions made by G0 and G0F chains with avelumab with a specific color code for HC1, HC2, and LC2. Structural representation of the most relevant H-bonds identified between sugars and G0 (b) and G0F (c) avelumab. The secondary structure is shown as ribbons, main residues as yellow sticks and the molecular surface of sugars is shown in gray and dark red (fucose).