Anti-Malaria Antibody Engineering Broadens Recognition Motifs and Reveals New Homotypic Interactions that Enhance Protective Breadth

The monoclonal antibody L9 mediates high-level protection against malaria in children for up to 6 months in Africa. L9 preferentially binds with high affinity to the NVDP minor repeat on the P. falciparum circumsporozoite protein (PfCSP). Structure-function studies show that closely spaced minor repeats are important for multi-epitope avidity interactions and optimal protection in pre-clinical models. Here, we assessed monoclonal antibody variants of L9 for protection against chimeric transgenic Plasmodium CSP parasite strains that had altered the number and spacing of minor repeats within PfCSP. We used site-saturation mutagenesis and DNA shuffling to generate libraries for yeast display-affinity screening. Sequencing of enriched yeast display libraries identified a panel of L9 variants with improved affinity to Peptide 22, the canonical motif containing the sequential minor and major repeats, with maintained binding to PfCSP. In vivo challenge showed that the affinity-improved variant L9_yd19 was modestly more potent than L9 against a chimeric transgenic parasite similar to the PfCSP sequence of an East African Pf strain with two widely spaced minor repeats, and comparable potency to L9 against the benchmark 3D7 strain. L9_yd19 also had high affinity against NANP major repeats and was highly protective against transgenic Plasmodium CSP containing only NANP major repeats (NANP12). Cryo-EM analyses revealed L9_yd19 recognition of PfCSP to comprise two distinct homotypic interfaces: a side-to-side interface with an asymmetric antibody trimer, and a head-to-head interface between the two antibody trimers. These combined to yield a higher-order complex consisting of two trimeric layers of antibodies related by 2-fold symmetry; one layer recognizing the minor repeat epitope (if available) and the second recognizing major repeats. These structure-function data thus reveal a new antibody mechanism that incorporates dual homotypic interfaces for potentially improved malaria prevention with monoclonal antibodies.