Highly protective antimalarial antibodies via precision library generation and yeast display screening

Abstract

The monoclonal antibody CIS43 targets the Plasmodium falciparum circumsporozoite protein (PfCSP) and prevents malaria infection in humans for up to 9 mo following a single intravenous administration. To enhance the potency and clinical utility of CIS43, we used iterative site-saturation mutagenesis and DNA shuffling to screen precise gene-variant yeast display libraries for improved PfCSP antigen recognition. We identified several mutations that improved recognition, predominately in framework regions, and combined these to produce a panel of antibody variants. The most improved antibody, CIS43_Var10, had three mutations and showed approximately sixfold enhanced protective potency in vivo compared to CIS43. Co-crystal and cryo-electron microscopy structures of CIS43_Var10 with the peptide epitope or with PfCSP, respectively, revealed functional roles for each of these mutations. The unbiased site-directed mutagenesis and screening pipeline described here represent a powerful approach to enhance protective potency and to enable broader clinical use of antimalarial antibodies.

Challenges in antibody structure prediction

“…These structural inaccuracies affect the results of structure-based biophysical property predictions. 36 , 37 In addition to the Jain et al dataset, 34 we predicted the structure of the CIS43 antibody, where the experimental X-ray structure (PDB accession code: 7SG5) 38 was released after the structure prediction tools were published, to have an experimental reference structure outside of the used training set. Figure 1 shows an overlay of all obtained structure models and reveals an overall high structural similarity, reflected in low overall RMSD values (~1 Å).…”

Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model

“…Our study focused on the use of a cytokine as a model immunogen to compare genotypic and phenotypic responses across different lymphoid organs, and thus we did not examine additional functional features of the selected mAbs and the in vivo efficacy in animal models. We conducted affinity titrations with multiple antigen concentrations ( 77 , 84 ) rather than simpler affinity gating as in some previous studies ( 57 , 58 , 70 , 85 ) to more accurately predict the relative binding affinity of antibodies in our yeast surface display platform. We used affinity titrations because the different titration groups can analyze a broader range of antibody affinities than affinity gating, which uses only a single (albeit carefully selected) antigen concentration.…”

An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries

“…After fluorophore labeling, the cells were pelleted and washed with 1 mL of ice-cold PBSF, and pellets were left covered on ice until loading onto Sony SH800 cell sorter, at which time each pellet was resuspended in 5 mL of ice-cold PBSF. Cells were first gated for yeast cells and single cells (drawn according to Banach et al 66 to avoid collection of clumped yeast of irregular large yeast aggregates), and then a gate for positive Fab expression was drawn and 200,000 cells were collected as the library reference population ( Extended Data Figure 6 ). Sorting bins for the Top 25% and Next 25% of binding based on PE signal were gated from the display positive population and 200,000 cells were collected in each bin ( Extended Data Figure 6 ).…”