Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system

Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

“…For these reasons, EGFR is considered an important target for cancer therapeutic reagents, including therapeutic antibodies. Two anti-EGFR therapeutic IgGs, Cetuximab and Panitumumab, are currently approved by the US Food and Drug Administration, and others are undergoing clinical trials [4]. However, such intact IgG has several limitations including high production costs, because of the requirement for a mammalian expression system, and poor penetration into tumor tissues [5,6].…”

Isolation of human Fab antibodies specific for the low-affinity IgE receptor (CD23) by selecting a hierarchical antibody library system against B lymphoblastic IM-9 cells

“…Enrichment of phage antibodies bound to IM-9 cells by BRASIL cell panning and identification of the human Fab clones specific for the CD23 antigen Recombinant phage antibodies were prepared from the primary combinatorial Fab antibody library HuDVFab-8L which had been was created by combining the HuNH-D2 sublibrary (4.5 × 10 9 diversity of human naïve heavy chains) with eight discrete human naïve kappa light chains (HuL1-HuL8) with undefined antigenbinding specificity [19]. After depletion of phage antibodies with human promonocytic U937 cells to remove non-specific binders, four rounds of cell panning via the 'BRASIL procedure' were carried out to isolate phage antibodies that bound to the human B lymphoblastic IM-9 cells.…”

“…Briefly, recombinant phage antibodies were prepared from the primary antibody library HuDVFab-8L (IG Therapy Co., South Korea) using Ex12 helper phage (IG Therapy Co.) [19]. A total of 10 12 cfu of the phage antibodies were pre-incubated with 1 × 10 6 U937 cells in 1 ml of ice-cold RPMI containing 1% bovine serum albumin (BSA) (Sigma-Aldrich, USA).…”

“…Soluble Fab molecules were prepared by subcloning the Fd (V H + C H1 ) genes into the pFdEx plasmid (IG Therapy Co.), followed by cultivation of E. coli TG1 cells in 2× YT/AT supplemented with 0.02% arabinose and 0.1 mM IPTG and purification using CNBr-activated sepharose (Amersham-Pharmacia, Sweden) conjugated with anti-human IgG (Fab ) 2 antibody (Pierce, USA) [19].…”

“…We previously reported the DVS-III vector system for L chain shuffling and the human naïve kappa light chain sublibrary HuNL-D3 which possesses 1.4 × 10 8 diversity of human naïve kappa L repertoire [19,20]. To generate improved derivatives of IM-L6-E and IM-L8-G Fab clones through L chain shuffling two secondary antibody libraries were constructed by combining the H chain of IM-L6-E with HuNL-D3 and the H chain of IM-L8-G with HuNL-D3, respectively, and two rounds of biopanning were carried out against the recombinant CD23 protein immobilized on microtiter plates.…”