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1.
Commun Biol ; 6(1): 997, 2023 09 29.
Article in English | MEDLINE | ID: mdl-37773269

ABSTRACT

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1ß antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1ß complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.


Subject(s)
Antibodies , Interleukin-17 , Epitopes/chemistry , Antigens , Binding Sites, Antibody
2.
Sci Rep ; 8(1): 3538, 2018 02 23.
Article in English | MEDLINE | ID: mdl-29476161

ABSTRACT

Mammalian SIRT6 is a well-studied histone deacetylase that was recently shown to exhibit high protein deacylation activity enabling the removal of long chain fatty acyl groups from proteins. SIRT6 was shown to play key roles in cellular homeostasis by regulating a variety of cellular processes including DNA repair and glucose metabolism. However, the link between SIRT6 enzymatic activities and its cellular functions is not clear. Here, we utilized a directed enzyme evolution approach to generate SIRT6 mutants with improved deacylation activity. We found that while two mutants show increased deacylation activity at high substrate concentration and improved glucose metabolism they exhibit no improvement and even abolished deacetylation activity on H3K9Ac and H3K56Ac in cells. Our results demonstrate the separation of function between SIRT6 catalytic activities and suggest that SIRT6 deacylation activity in cells is important for glucose metabolism and can be mediated by still unknown acylated cellular proteins.


Subject(s)
Directed Molecular Evolution/methods , Glucose/metabolism , Histones/chemistry , Protein Engineering/methods , Sirtuins/chemistry , Tumor Necrosis Factor-alpha/chemistry , Acylation , Animals , Binding Sites , Biocatalysis , Gene Library , HEK293 Cells , Histones/genetics , Histones/metabolism , Homeostasis/genetics , Humans , Hydrolysis , Kinetics , Models, Molecular , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Sirtuins/deficiency , Sirtuins/genetics , Substrate Specificity , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
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