title: Affinity and cross-reactivity engineering of CTLA4-Ig to modulate T cell costimulation.
abstract: 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.
authors.affiliation: Abbott Biotherapeutics Corp., Redwood City, CA 94063, USA.
authors.forename: Zhenghai
authors.initials: Z
authors.lastname: Xu
authors.forename: Veronica
authors.initials: V
authors.lastname: Juan
authors.forename: Alexander
authors.initials: A
authors.lastname: Ivanov
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Ma
authors.forename: Dixie
authors.initials: D
authors.lastname: Polakoff
authors.forename: David B
authors.initials: DB
authors.lastname: Powers
authors.forename: Robert B
authors.initials: RB
authors.lastname: Dubridge
authors.forename: Keith
authors.initials: K
authors.lastname: Wilson
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Akamatsu
mesh_terms: D000069594:Abatacept; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000473:pathology / Q000628:therapy; D018122:B7-1 Antigen / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D051940:B7-2 Antigen / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003429:Cross Reactions / Q000235:genetics / Q000276:immunology; D005813:Genes, Synthetic / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D018796:Immunoconjugates / Q000235:genetics* / Q000378:metabolism* / Q000627:therapeutic use; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000235:genetics* / Q000276:immunology*; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism*
chemical_list: D018122:B7-1 Antigen; D051940:B7-2 Antigen; D018796:Immunoconjugates; D019151:Peptide Library; D000069594:Abatacept