Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

Abstract

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.

Keywords: ADCC, antibody-dependent cell-mediated cytotoxicity; AUC, area-under-the-curve; CD3; EGFR, epidermal growth factor receptor; FITC-CD3ϵγ, fluorescein isothiocyanate-labeled CD3ϵγ; DVD-IgTM, dual variable domain immunoglobulin; FITC-sEGFR, FITC-labeled sEGFR; Fv, variable fragment; ICR, imprinting control region; IgG-like bispecific antibody; MTS, 3-(4, 5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SPR, surface plasmon resonance; SUV, standardized uptake value; T-LAK cells, lymphokine-activated killer cells with the T-cell phenotype; VH, variable heavy domain; VL, variable light domain; antibody engineering; bispecific diabody; bsAb, bispecific antibody; bsDb, bispecific diabody; cancer immunotherapy; effective domain order; epidermal growth factor receptor; sEGFR, soluble EGFR; scDb, single-chain diabody; scFv, single-chain Fv; taFv, tandem scFv.

Figure 1.

Preparation of hEx3-scDb-Fcs with different domain orders. (A) Schematic diagrams of four types of hEx3-scDb-Fc. (B) Schematic diagrams of the expression vectors for hEx3-scDb-Fcs. (C) Gel filtration of hEx3-scDb-Fcs purified through protein A. AU, absorbance unit. (D) SDS-PAGE of each purification step in the preparation of hEx3-scDb-Fc-LH under reducing conditions (lane 1, 3) and non-reducing conditions (lane 2). Lanes 1, 2, after protein A purification; lane 3, peak fraction of gel filtration indicated by the arrow.

Figure 2.

Comparison of growth inhibitory effects of hEx3-scDb-Fcs. hEx3s and T-LAK cells were added to TFK-1 cells (A, D), A431 cells (B), or MCF-7 cells (C) at a ratio of 3:1 (A, D) or 2:1 (B, C). The indicated hEx3s and PBMCs were added to TFK-1 cells at a ratio of 10:1 (E). Data are presented as the mean ± 1 s.d. and are representative of at least two independent experiments.

Figure 3.

Comparison of growth inhibitory effects of hEx3-scDbs prepared from their Fc-fusion formats. (A) Schematic illustration of the preparation of hEx3-scDbs from hEx3-scDb-Fcs. (B) Reducing SDS-PAGE of each purification step in the preparation of hEx3-scDb-LH. Lane 1, protein A chromatography–purified hEx3-scDb-Fc-LH; lane 2, after HRV3C protease digestion; lane 3, after removal of HRV3C protease by Glutathione Sepharose 4B chromatography; lane 4, purified hEx3-scDb-LH after removal of the Fc region by protein A chromatography. (C) hEx3-scDbs and T-LAK cells were added to TFK-1 cells at a ratio of 3:1. Data are presented as the mean ± 1 s.d. and are representative of at least two independent experiments.

Figure 4.

Confirmation of the cross-linking ability of hEx3-scDb-Fcs. A431 and T-LAK cells were incubated with PBS as a negative control (open area) or with each hEx3-scDb-Fc (shaded area); this incubation was followed by staining with FITC-CD3εγ for A431 cells (upper panels) or with FITC-sEGFR for T-LAK cells (lower panels).

Figure 5.

hEx3-scDb-Fcs-mediated cytokine production by T-LAK cells. IFN-γ concentration was evaluated by using an ELISA (A). Time courses of IL-2 (B), IFN-γ (C), GM-CSF (D), and TNF (E) production were evaluated by using an ELISA after 3–15 h of co-culturing 10 nM antibodies with T-LAK cells (1 × 10⁵) in the presence of overnight-adhered TFK-1 cells (5 × 10³).

Figure 6.

Stability assessment of hEx3-scDb-Fcs with different domain orders. (A) Gel filtration of hEx3-scDb-Fcs to assess their stability after storage. Fractionated hEx3-scDb-Fcs were stored for 3 wk at 4°C and then applied to a Hiload Superdex 200-pg column (10/300). (B) Reducing SDS-PAGE of each gel filtration fraction of hEx3-scDb-Fc-HL, indicated by the arrows in A. (C) Blood clearance of bsAbs. Imprinting control region (ICR) mice (n = 5) were injected with each of the ¹²⁵I-labeled bsAbs, and blood samples were collected from tail veins at the indicated time points.

Figure 7.

Comparison of bsAbs with hEx3-scDb-Fc-LH-IgG2. (A) Schematic diagram of hEx3-scDb-Fc-LH-IgG2. Proliferative effects of bsAbs on T-LAK cells (B) and PBMCs (C). Freshly isolated PBMCs or T-LAK cells were incubated for 72 h with the indicated doses of bsAbs. RPMI 1640 medium and phytohemagglutinin (PHA) served as the negative control (N.C.) and positive control (P.C.), respectively. To compare the growth inhibitory effects of bsAbs with that of hEx3-scDb-Fc-LH-IgG2, hEx3s and T-LAK cells (D) or PBMCs (E) were added to TFK-1 cells at ratios of 2:1 and 15:1, respectively. Data are presented as the mean ± 1 s.d. and are representative of at least two independent experiments. A time course of IFN-γ production was evaluated by using an ELISA after 3–15 h of co-culturing 10 nM bsAbs with T-LAK cells (1 × 10⁵) in the presence of overnight-adhered TFK-1 cells (5 × 10³) (F).

Figure 8.

In vivo antitumor effect of hEx3-scDb-Fc-LH. Points indicate the mean tumor volumes from each treatment group; bar, ± 1 s.d.; *, Significant (p < 0.05) difference between hEx3-scDb-Fc-LH and hEx3-LH (A) or hEx3-scDb-Fc-LH-IgG2 (B).