Precise directed evolution techniques were used to optimize the mAb CIS43 for enhanced binding to PfCSP epitopes . (a) SSM libraries were generated separately for the VH and VL of the antimalarial antibody CIS43 and cloned into yeast Fab display vectors for affinity-based FACS. Plasmid DNA was extracted from high-affinity sorted libraries, and additional combinatorial mutagenesis and screening was performed to select high-affinity multi-mutation variants by FACS. Sorted libraries were analyzed using NGS to quantitatively track each variant across sorting rounds. NGS data were mined to identify improved CIS43 antibody gene variants. After Illumina 2 × 300 bp sequencing, raw fastq sequence files were merged and quality-filtered to obtain high-quality in-frame antibody amino acid sequences. Unique amino acid sequences were compiled into library prevalence values, which were followed to track changes in variant populations across each round of sorting. (b) Five CSP-derived antigen probes were used for yeast display screening. Probes include fl_PfCSP, an N-terminal domain stabilized truncated-CSP format (NTDS_5/3_CSP), and three peptides derived from PfCSP (Pep21, Pep25, and Pep29.). (c) VH-SSM and VL-SSM single-mutation libraries were sorted for high affinity against fl_PfCSP and Pep21 (red) compared to wild-type CIS43 (cyan), which revealed minimal enhancements in library-scale affinity in single-mutation libraries. (d) Flow cytometric analysis of combinatorial multi-mutation Libraries 1, 2, and 3 after three rounds of sorting showed substantially enhanced affinity against fl_PfCSP, NTDS_5/3_CSP, Pep21, Pep25, and Pep29 (red) compared to wild-type CIS43 (cyan).