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A Single‐Framework Synthetic Antibody Library Containing a Combination of Canonical and Variable Complementarity‐Determining Regions
Author(s) -
Maruthachalam Bharathikumar Vellalore,
ElSayed Ayman,
Liu Jianghai,
Sutherland Ashley R.,
Hill Wayne,
Alam Md Kausar,
Pastushok Landon,
Fonge Humphrey,
Barreto Kris,
Geyer C. Ronald
Publication year - 2017
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700279
Subject(s) - complementarity determining region , antibody , peptide library , phage display , complementarity (molecular biology) , computational biology , immunoglobulin fab fragments , affinity maturation , aptamer , microbiology and biotechnology , chemistry , human epidermal growth factor receptor 2 , fragment crystallizable region , biology , immunoglobulin light chain , genetics , peptide sequence , gene , cancer , breast cancer
Synthetic antibody libraries have been used to generate antibodies with favorable biophysical and pharmacological properties. Here, we describe the design, construction, and validation of a phage‐displayed antigen‐binding fragment (Fab) library built on a modified trastuzumab framework with four fixed and two diversified complementarity‐determining regions (CDRs). CDRs L1, L2, H1, and H2 were fixed to preserve the most commonly observed “canonical” CDR conformation preferred by the modified trastuzumab Fab framework. The library diversity was engineered within CDRs L3 and H3 by use of custom‐designed trinucleotide phosphoramidite mixes and biased towards human antibody CDR sequences. The library contained ≈7.6 billion unique Fabs, and >95 % of the library correctly encoded both diversified CDR sequences. We used this library to conduct selections against the human epidermal growth factor receptor‐3 extracellular domain (HER3‐ECD) and compared the CDR diversity of the naïve library and the anti‐HER3 selection pool by use of next‐generation sequencing. The most commonly observed CDR combination isolated, named Her3‐3, was overexpressed and purified in Fab and immunoglobulin G (IgG) formats. Fab HER3‐3 bound to HER3‐ECD with a K D value of 2.14 n m and recognized cell‐surface HER3. Although HER3‐3 IgG bound to cell‐surface HER3, it did not inhibit the proliferation of HER3‐positive cells. Near‐infrared imaging showed that Fab HER3‐3 selectively accumulated in a murine HER3‐postive xenograft, thus providing a lead for the development of HER3 imaging probes.