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.
Subject(s)
Amino Acid Substitution , Antibodies, Monoclonal, Humanized , Antineoplastic Agents , Complementarity Determining Regions , ErbB Receptors/immunology , Mutation, Missense , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/genetics , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacology , Cetuximab , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , ErbB Receptors/genetics , HEK293 Cells , HumansABSTRACT
CTLA4-Ig is an Fc fusion protein containing the extracellular domain of CTLA-4, a receptor known to deliver a negative signal to T cells. CTLA4-Ig modulates T cell costimulatory signals by blocking the CD80 and CD86 ligands from binding to CD28, which delivers a positive T cell costimulatory signal. To engineer CTLA4-Ig variants with altered binding affinity to CD80 and CD86, we employed a high-throughput protein engineering method to map the ligand binding surface of CTLA-4. The resulting mutagenesis map identified positions critical for the recognition of each ligand on the three CDR-like loops of CTLA-4, consistent with the published site-directed mutagenesis and x-ray crystal structures of the CTLA-4/CD80 and CTLA-4/CD86 complexes. A number of single amino acid substitutions were identified that equally affected the binding affinity of CTLA4-Ig for both ligands as well as those that differentially affected binding. All of the high-affinity variants showed improved off-rates, with the best one being a 17.5-fold improved off-rate over parental CTLA4-Ig binding to CD86. Allostimulation of human CD4(+) T cells showed that improvement of CD80 and CD86 binding activity augmented inhibition of naive and primed T cell activation. In general, increased affinity for CD86 resulted in more potent inhibition of T cell response than did increased affinity for CD80. Optimization of the affinity balance to CD80 and CD86 to particular disease settings may lead to development of a CTLA4-Ig molecule with improved efficacy and safety profiles.
