Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing.


Abstract

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.

Submission Details

ID: DWx5bcAe

Submitter: Marie Ary

Submission Date: Nov. 19, 2018, 10:53 a.m.

Version: 1

Publication Details
Forsyth CM;Juan V;Akamatsu Y;DuBridge RB;Doan M;Ivanov AV;Ma Z;Polakoff D;Razo J;Wilson K;Powers DB,MAbs (2013) Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing. PMID:23765106
Additional Information

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).

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4HAF 2012-09-26T00:00:00+0000 2.04 Crystal structure of fc-fragment of human IgG2 antibody (primitive crystal form)
4HAG 2012-09-26T00:00:00+0000 3.4 Crystal structure of fc-fragment of human IgG2 antibody (centered crystal form)
4L4J 2013-06-07T00:00:00+0000 1.92 Crystal structure of fc-fragment of human IgG2-Sigma antibody
7LUS 2021-02-23T00:00:00+0000 2.45 IgG2 Fc Charge Pair Mutation version 1 (CPMv1)
7LUS 2021-02-23T00:00:00+0000 2.45 IgG2 Fc Charge Pair Mutation version 1 (CPMv1)
1ADQ 1997-02-18T00:00:00+0000 3.15 CRYSTAL STRUCTURE OF A HUMAN IGM RHEUMATOID FACTOR FAB IN COMPLEX WITH ITS AUTOANTIGEN IGG FC
1BBJ 1992-04-30T00:00:00+0000 3.1 CRYSTAL STRUCTURE OF A CHIMERIC FAB' FRAGMENT OF AN ANTIBODY BINDING TUMOUR CELLS
3EO1 2008-09-26T00:00:00+0000 3.1 Structure of the Fab Fragment of GC-1008 in Complex with Transforming Growth Factor-Beta 3
4B53 2012-08-02T00:00:00+0000 1.8 Crystal structure of the isolated IgG4 CH3 domain
4C54 2013-09-10T00:00:00+0000 1.9 Crystal structure of recombinant human IgG4 Fc

Relevant UniProtKB Entries

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