Using function-based structure models and alanine scanning, we have identified a potentially general mode for phospho-amino acid- and context sequence-binding by IgG and scFv Abs. We propose to exploit this phenomenon to design a phospho-focused scFv library in which the phospho-amino acid binding hypervariable complementarity determining region (CDR) H2 of the Ab is held constant while sequences along the protein- and peptide-binding groove composed of the other 5 CDRs are mutated to accommodate various context sequences. This phospho- focused library will be used to both bias- and direct the binding of Abs to a specific appropriately modified amino acid on a peptide based on surface loops near enzyme active site. The Abs will be affinity matured against non-phosphorylated peptide and/or full length protein. The matured Abs will be tested for their ability to act as steric inhibitors to the enzyme to which they were designed. If successful, our system will enable the rapid generation of reversible inhibitors with utility in biochemistry and pharmacology.

Public Health Relevance

We propose to develop an approach toward making anti-enzyme Ab steric inhibitors. We will use a phospho-focused library based on an Ab framework with a pre-existing propensity to bind phosphorylated amino acids and a general mode to bind context sequences. This phospho- specific library will be used to direct Ab-binding to phospho-modified peptides composed of the amino acid sequence of surface-exposed loops surrounding the active sites of enzymes. The Abs will be affinity matured against non-phosphorylated peptide and/or full length protein. The matured Abs will be tested for their ability to act as steric inhibitors to the enzyme to which they were designed. If successful, our system will enable the rapid generation of reversible inhibitors with utility in biochemistry and pharmacology.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI135900-01
Application #
9431361
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Ferguson, Stacy E
Project Start
2018-01-01
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Abcam, Inc.
Department
Type
DUNS #
139406602
City
Cambridge
State
MA
Country
United States
Zip Code