We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.
Submitter: Marie Ary
Submission Date: Nov. 19, 2018, 10:53 a.m.
No info on whether antigen (EGFR-C-lambda fusion protein) had linker, so just added human IgG1-lambda sequence directly to end of EGFR extracellular domain sequence for ER data. For SPR data, specified EGFR extracellular domain alone as antigen (no fusion). ER data reported is the average of values given for the synonymous codons. Did not include data in Table 1 (cetuximab and chimeric 225).
|Number of data points||1207|
|Proteins||Anti-EGFR antibody (hu225)|
|Assays/Quantities/Protocols||Experimental Assay: k_a ; Experimental Assay: Enrichment ratio (ER) ; Experimental Assay: Kd ; Experimental Assay: k_d ; Experimental Assay: FACS affinity (xWT) ; Derived Quantity: SPR affinity (WT/var)|
|Libraries||Binding kinetics by SPR (Table 4) ; FACS affinity relative to WT for 67 high-affinity variants (Table S6) ; ER for all CDR point mutations (Table S3)|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|90.4||H||Anti-EGFR antibody (hu225)||P0DOX5||IGG1_HUMAN|
|99.7||H||Anti-EGFR antibody (hu225)||P01857||IGHG1_HUMAN|
|90.6||H||Anti-EGFR antibody (hu225)||P01859||IGHG2_HUMAN|
|90.6||H||Anti-EGFR antibody (hu225)||P01861||IGHG4_HUMAN|
|99.1||L||Anti-EGFR antibody (hu225)||P01834||IGKC_HUMAN|