Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction

Abstract

Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

Fig 1. SDS-PAGE analysis of cell-free synthesized 059-152-Fv and 059-152-Fab.

(A) 059-152-Fv was synthesized under a series of different concentrations (0, 0.2, and 0.4 mg/ml) of DsbC, as indicated. Total (T) and soluble (S) fractions of the internal solution were analyzed by reducing SDS-PAGE. (B) Purified 059-152-Fv was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fv are indicated under each lane of the non-reducing SDS polyacrylamide gel image. (C) 059-152-Fab was synthesized in the presence of 0, 0.2, 0.4, and 0.8 mg/ml of DsbC, as indicated. (D) Purified 059-152-Fab was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fab are indicated under each lane of the non-reducing SDS polyacrylamide gel image. BG: cell-free synthesis without template DNA. VH: cell-free synthesis of VH without DsbC. VL: cell-free synthesis of VL without DsbC. VHCH1: cell-free synthesis of VHCH1 without DsbC. L chain: cell-free synthesis of light chain without DsbC. Gels were stained with CBB.

Fig 2. SDS-PAGE analysis of site-specific fluorescent-labeled 059-152-Fv and 059-152-Fab.

Reducing and non-reducing SDS-PAGE analysis of 059-152-Fv (A) and 059-152-Fab (B). (lane 1) 059-152-Fv, (lane 2) AzF-incorporated 059-152-Fv, (lane 3) Alexa-488 conjugated 059-152-Fv, (lane 4) 059-152-Fab, (lane 5) AzF-incorporated 059-152-Fab, and (lane 6) Alexa-488 conjugated 059-152-Fab. Fluorescent images and CBB-stained images were acquired from the same gels.

Fig 3. Crystal structure of the 059-152-Fv•EGFR-ECD complex.

(A) Ribbon representation of the 059-152-Fv•EGFR-ECD complex. VH and VL domains of 059-152-Fv are respectively colored cyan and green. External region of the EGFR is shown with domain I in yellow, domain II in orange, domain III in red, and domain IV in purple. (B, C) Close up-view of the interactions between CDR loops of 059–152 and domain III. For clarity, interactions of CDR-H and CDR-L are separately shown. CDR-H loops, CDR-L loops, and domain III are colored cyan, green, and gray, respectively. Residues that make key interactions are shown in the stick models. Hydrogen bonds are indicated by gray dotted lines.

Fig 4. Contacting region of 059-152-Fv, cetuximab, and EGF on domain III.

(A) Ribbon representations of domain III in complex with 059-152-Fv, EGF, and cetuximab. VH, VL, EGF, and domain III are respectively shown in cyan, green, yellow, and gray. (B) Surface representations of domain III with the contact region. Surface of domain III (gray) contacting (within 4 Å) 059–152, EGF, and cetuximab are shown in purple, red, and orange, respectively. These are viewed from approximately the same orientations onto the domain III binding site. Residues on domain III that form hydrogen bonds with EGF are shown in white.

Fig 5. Electron density map of intradomain disulfide bonds of 059-152-Fv.

Close-up view of the electron density maps (blue mesh) and stick models of 059-152-VH (A) and 059-152-VL (B) are shown. The stick models are shown with carbons colored yellow; nitrogens, blue; oxygens, red; and sulfurs, green.