Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units

Abstract

During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the "mispairing problem." Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce "bsAb by external pairing and excision" (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells.

Keywords: Bispecific antibody; Cancer; Chain pairing; SnoopCatcher; SpyCatcher.

Fig. 1

Overview of the BAPE (bsAb by external pairing and excision) procedure. Two different antibody clones are shown in black and white. Two external pairing units and protease cleavage sites are also indicated

Fig. 2

(A) Construction of CD3-Her2(Spy-Snoop) for BAPE. (B) SDS-PAGE of purified CD3-Her2(Spy-Snoop) with a reduced condition (Lane 1) and CD3-Her2 bsAb molecules following cleavage and purification (Lane 2). M: molecular weight marker. Arrows indicate labeled proteins. (C) Analytical size-exclusion chromatography after the removal of the pairing tags (black line). Red dashed line indicates the IgG elution profile under the same experimental settings. (D) Differential scanning fluorometry. (E) Flow cytometry results for Her2-positive SK-BR-3 cells (top) and CD3-positive HPB-ALL cells (bottom). Black: without antibody, red: constructed bsAb, blue: positive control antibody. (F) In vitro cytotoxicity assay. Tumor cell: SK-BR-3, Effector cell: Activated T-cell (T-LAK), E/T = 5, incubation time 48 h. Error bars indicate standard deviation

Fig. 3

(A) Construction of CD3-EGFR(Spy-Snoop) via BAPE. (B) SDS-PAGE of the purified precursor bsAb with a reduced condition (Lane 1) and the final bsAb molecule after cleavage and purification (Lane 2). M: molecular weight marker. Arrows indicate labeled proteins. (C) Analytical size exclusion chromatography. The elution profile for CD3-EGFR(Spy-Snoop) is shown with a blue line. The Red dashed line indicates the IgG elution profile in the same experimental settings. (D) Differential scanning fluorometry. (E) Flow cytometry results for EGFR-positive A431 cells (top) and CD3-positive HPB-ALL cells (bottom). Black: without antibody, green: constructed bsAb, red: positive control antibody. (F) In vitro cytotoxicity assay. Tumor cell: TFK-1, Effector cell: Activated T-cells (T-LAK). E/T = 2, incubation time = 20 h. Error bars indicate standard deviation