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Published September 27, 2018 | Submitted
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Quantifying the Entropic and Energetic Effects of Linker Length and Rigidity within Synthetic HIV-1 Antibodies designed to Bind Bivalently to Env Spikes


Due to the low density of envelope (Env) spikes on the surface of HIV-1, neutralizing IgG antibodies rarely bind bivalently using both antigen-binding arms (Fabs) to crosslink between spikes, instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Synthetic antibodies capable of bivalently binding within single Env trimers (intra-spike crosslinking) were previously shown to exhibit increased neutralization potencies. In the initial work, diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA) were considered. Here we investigated whether linkers with different rigidities could enhance diFab potency by synthesizing DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and created a model that predicts their neutralization potencies. Model predictions, verified by experimental data, show that although a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom during bivalent binding. In contrast, the strongest neutralization potencies require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer. These results inform the design of bivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. We thank Anthony Bartolotta, Justin Bois, Jim Eisenstein, Vahe Galstyan, Peng He, Willem Hegel, David Hsieh, Giacomo Koszegi, Pankaj Mehta, Jiseon Min, Olexei Motrunich, Noah Olsman, Vahe Singh, and Richard Zhu for useful discussions, Christopher Barnes for measuring modeled 3BNC60-Env complexes, Aaron Coey for discussions about triFabs IC_(50)s, and affinities, and Marta Murphy for help with preparing figures. This research was supported by National Institute of Allergy and Infectious Diseases of the National Institutes of Health grants 1R01AI129784 and HIVRAD P01 AI100148 (P.J.B.), the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery Grant 1040753 (P.J.B.), La Fondation Pierre-Gilles de Gennes (R.P.), the Rosen Center at Caltech (R.P.), the National Institutes of Health DP1 OD000217 (Director's Pioneer Award), R01 GM085286, and 1R35 GM118043-01 (MIRA) (R.P.), and a Caltech-COH Biomedical Research Initiative (to P.J.B.). We are grateful to the Burroughs-Wellcome Fund for its support of the Physiology Course at the Marine Biological Laboratory, where part of the work on this work was done, and for a post-course research grant (S.Y.). Author Contributions: R.P.G., T.E., A.P.W., R.P. and P.J.B. conceived the project and interpreted data; R.P.G. designed, constructed, and characterized diFabs; T.E., S.Y., and R.P developed the model and performed analyses; P.N.P.G. and A.M.L. performed in vitro neutralization assays; D.S.J, and A.M.L. assisted with diFab construction; T.E., R.P., and P.J.B. wrote the paper with input from other authors.

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