Common framework mutations impact antibody interfacial dynamics and flexibility

Abstract

IntroductionWith the flood of engineered antibodies, there is a heightened need to elucidate the structural features of antibodies that contribute to specificity, stability, and breadth. While antibody flexibility and interface angle have begun to be explored, design rules have yet to emerge, as their impact on the metrics above remains unclear. Furthermore, the purpose of framework mutations in mature antibodies is highly convoluted.MethodsTo this end, a case study utilizing molecular dynamics simulations was undertaken to determine the impact framework mutations have on the VH-VL interface. We further sought to elucidate the governing mechanisms by which changes in the VH-VL interface angle impact structural elements of mature antibodies by looking at root mean squared deviations, root mean squared fluctuations, and solvent accessible surface area.Results and discussionOverall, our results suggest framework mutations can significantly shift the distribution of VH-VL interface angles, which leads to local changes in antibody flexibility through local changes in the solvent accessible surface area. The data presented herein highlights the need to reject the dogma of static antibody crystal structures and exemplifies the dynamic nature of these proteins in solution. Findings from this work further demonstrate the importance of framework mutations on antibody structure and lay the foundation for establishing design principles to create antibodies with increased specificity, stability, and breadth.

Humanization of Pan-HLA-DR mAb 44H10 Hinges on Critical Residues in the Antibody Framework

“…Framework regions can also indirectly modulate antibody paratopes by providing support critical for shaping CDRs. Such framework residues constitute the “Vernier Zone” (VZ), and have been shown to impact antigen binding, antibody energetics, and antibody activity [ 10 , 11 , 12 , 13 ]. Notably, one of the most recognized examples of a VZ residue is position 71 in the antibody heavy chain (H71), which was also identified here as critical for enabling humanized 44H10 binding to HLA-DR.…”

“…Here, approaches that maximize either sequence or structural homology to the parental framework have been proposed to increase the likelihood of positive outcomes since homologous frameworks provide the same scaffold for the CDRs implicated in antigen binding [ 8 ]. In addition to the selection of appropriate CDRs and framework regions, back-mutations of select residues in the framework are often necessary, as they are known to play a role in shaping antibody paratopes and may also directly contact the antigen [ 10 , 11 , 12 , 13 , 14 ]. Thus, striking a balance between maximizing human content whilst retaining the parental antibody’s specificity and affinity for the antigen of interest can be a tricky endeavor.…”

Humanization of pan-HLA-DR mAb 44H10 Hinges on Critical Residues in the Antibody Framework

“…2 framework have been proposed to increase the likelihood of positive outcomes since homologous frameworks provide the same scaffold for the CDRs implicated in antigen binding [8]. In addition to the selection of appropriate CDRs and framework regions, back-mutations of select residues in the framework are often necessary, as they are known to play a role in shaping antibody paratopes and may also directly contact the antigen [10][11][12][13][14]. Thus, striking a balance between maximizing human content whilst retaining the parental antibody's specificity and affinity for the antigen of interest can be a tricky endeavor.…”