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Instruments / Technical Area	Publication Details	Published	Actions
I04-Macromolecular Crystallography	Fragment-based computational design of antibodies targeting structured epitopes Mauricio Aguilar Rangel , Alice Bedwell , Elisa Costanzi , Ross J. Taylor , Rosaria Russo , Gonçalo J. L. Bernardes , Stefano Ricagno , Judith Frydman , Michele Vendruscolo , Pietro Sormanni Science Advances 8 DOI: 10.1126/sciadv.abp9540 Diamond Proposal Number(s): [20221] Open Access Show Abstract ▼ Abstract: De novo design methods hold the promise of reducing the time and cost of antibody discovery while enabling the facile and precise targeting of predetermined epitopes. Here, we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterization showed that all designs are stable and bind their intended targets with affinities in the nanomolar range without in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as similar predictions are obtained when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to preselected epitopes.	Nov 2022
I03-Macromolecular Crystallography Krios II-Titan Krios II at Diamond Krios IV-Titan Krios IV at Diamond	Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes Halina Mikolajek , Miriam Weckener , Z. Faidon Brotzakis , Jiandong Huo , Evmorfia V. Dalietou , Audrey Le Bas , Pietro Sormanni , Peter J. Harrison , Philip N. Ward , Steven Truong , Lucile Moynie , Daniel K. Clare , Maud Dumoux , Joshua Dormon , Chelsea Norman , Naveed Hussain , Vinod Vogirala , Raymond J. Owens , Michele Vendruscolo , James Naismith Proceedings Of The National Academy Of Sciences 119 DOI: 10.1073/pnas.2205412119 Diamond Proposal Number(s): [27031, 27051, 29666] Open Access Show Abstract ▼ Abstract: Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.	Jul 2022

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