Developing affinity reagents recognizing and modulating GPCR function by traditional animal immunization or in vitro screening methods is challenging. Some anti-GPCR antibodies exist on the market, but the success rate of development is still poor compared with antibodies targeting soluble or peripherally anchored proteins. More importantly, the majority of these antibodies do not modulate GPCR function. The current pipeline for antibody development primarily screens for affinity rather than epitope recognition. We propose to develop a new strategy utilizing natural ligand affinity to generate a library of antibody variants with inherent bias toward the active site of the GPCR. Instead of using phage libraries displaying antibodies with random CDR sequences, we will generate focused antibody libraries with a natural ligand encoded within or cross-linked to one of the CDRs or the N-terminus. We will limit randomization of the antibody to amino acids in regions around the ligand while leaving the ligand carrying part unaltered to tailor binding to the active site. A second round of randomization of the ligand carrying part will then be performed to eliminate the bias of the ligand. If successful, the proposed pipeline will enable the rapid generation of functional antibodies (both agonists and antagonists) against high value targets with poor epitope exposures including GPCR and other integral membrane proteins.

Public Health Relevance

Developing affinity reagents recognizing and modulating GPCR function by traditional animal immunization or in vitro screening methods is challenging. We hereby propose a general method to develop scFvs and IgGs that specifically target the functional epitope of GPCRs and other similar integral membrane proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21EB023445-02
Application #
9530655
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Rampulla, David
Project Start
2017-08-01
Project End
2019-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Abcam, Inc.
Department
Type
DUNS #
139406602
City
Cambridge
State
MA
Country
United States
Zip Code
Zhao, Qi; Buhr, Diane; Gunter, Courtney et al. (2018) Rational library design by functional CDR resampling. N Biotechnol 45:89-97