A human IgG-like bispecific antibody co-targeting epidermal growth factor receptor and the vascular endothelial growth factor receptor 2 for enhanced antitumor activity

Figures & data

Figure 1. The structure, stability and bispecificity of Bi-Ab. Structure of the anti-EGFR/VEGFR2 bispecific antibody (A) V_LE, variable light region of cetuximab; V_HE, variable heavy region of cetuximab; V_LK, variable light region of mAb-04; V_HK, variable heavy region of mAb-04. SDS-PAGE analysis of the purified Bi-Ab (B) Lane M, marker; lane 1, cetuximab; lane 2, mAb-04; lane 3, Bi-Ab. SDS-PAGE was used to analyze the thermostability of Bi-Ab (C) Lane M, marker; lane 1–6, sample incubated for 0, 3, 6, 9, 12 and 15 d at 37℃. The surface plasmon resonance spectroscopy analysis (D) VEGFR2 was injected after Bi-Ab were flowed over the EGFR-immobilized sensor chip. RU, resonance units.

Table 1 SPR analysis of the binding affinities of antibodies to EGFR or VEGFR2.

Binding	ka (M^-1s^-1)	kd (s^-1)	KD (M)	Chi^2
Bi-Ab-EGFR	2.22×10^6	3.21×10^-3	1.45×10^-9	0.856
Bi-Ab-VEGFR2	3.51×10^6	8.32×10^-3	2.37×10^-9	1.721
cetuximab-EGFR	1.83×10^6	1.35×10^-3	7.38×10^-10	0.538
mAb-04-VEGFR2	1.91×10^6	2.71×10^-3	1.42×10^-9	1.204

Chi², correlation fit between the calculated and experimental data. The data on sensorgrams of mAb-04-VEGFR2 and cetuximab-EGFR are not shown.

Figure 2. The binding of Bi-Ab to recombinant EGFR/VEGFR2 ectodomains and membrane-associated EGFR/VEGFR2. Surface plasmon resonance spectroscopy was used to analysis the binding kinetics of Bi-Ab to recombinant EGFR and VEGFR2 ectodomains ((A) and B). The equilibrium dissociation rate constant (K_D) of Bi-Ab to EGFR or VEGFR2 were 1.45×10 ⁻⁹M and 2.37×10 ⁻⁹M respectively. Flow cytometry was used to investigate the binding of Bi-Ab to membrane-associated EGFR and VEGFR2 ((C)- F). The histogram overlay showed that Bi-Ab binds to HT-29 and SKOV-3 cells line with a relatively high binding levels.

Figure 3. Bi-Ab inhibits phosphorylation of EGFR and VEGFR2 and down-regulates AKT and MAPK signaling. (A) Western blot to show the inhibition on phosphorylation of EGFR, VEGFR2, AKT and Erk1/2 in HT-29 and SKOV-3 cells after antibodies treatment. ((B) and C) Phosphorylation level of AKT and Erk1/2 in HT-29 and SKOV-3 cells. The data presented as the mean ± SD, are from a representative experiment, n = 3. *P < 0.05; **P < 0.01.

Figure 4. Bi-Ab showed the most effective inhibition of proliferation on HT-29 and SKOV-3 cells compared to mAb-04, cetuximab or Combi with EGF and VEGF stimulated ((A)and B). Three independent experiments were performed in triplicate, the means ± SD of triplicate experiment are shown, *P < 0.05; **P < 0.01 versus treatment with Bi-Ab treatment. Photomicrographs of transwell invasion assay indicated that Bi-Ab could effectively inhibit the invasion of HT-29 and SKOV-3 cells induced by EGF and VEGF ((C)and D). Quantitative analysis of the transwell invasion assay showing that Bi-Ab treatment significantly increased the inhibition of HT-29 and SKOV-3 cells invasion when compared to mAb-04 and cetuximab. The data presented as the mean ± SD, are from a representative experiment, 5 independent experiments were performed in triplicate, *P < 0.05; **P < 0.01. Percent ADCC of the antibodies on HT-29 and SKOV-3 (E). The data presented as the mean ± SD, each antibody was tested in triplicate, the assays were repeated once, n = 3, *P < 0.05.

Figure 5. The apoptosis was analyzed by flow cytometry, endothelial tube formation was performed using HUVECs tube formation assay. ((A)and C) Bi-Ab treatment increased apoptosis in SKOV-3 cells than cetuximab and mAb-04 alone or Combi, but not HT-29. (B) HUVEC tube-like photomicrographs showing the significant effects of Bi-Ab on HUVECs tube formation. (D) Similar to the Combi, Bi-Ab demonstrated relatively more potent restraining effect on tube formation by HUVEC cells compared to mAb-04 or cetuximab. Three independent experiments were performed in triplicate, the means ± SD of triplicate experiment are shown (*P <0 .05; **P <0 .01 vs. Bi-Ab treatment).

Table 2 Median survival and 100-day survival rate (%).

Drug	Median survival (d) (HT-29)	Survival at 100d (%) (HT-29)	Median survival (d) (SKOV-3)	Survival at 100d (%) (SKOV-3)
PBS	61**	0	54**	0
mAb-04	73.5*	0	59.5**	0
cetuximab	76.5*	0	67*	0
Combi	85.5	16.7	73.5*	0
Bi-Ab	92	33.3	84	16.7

*P < 0.05

**P < 0.02, versus Bi-Ab treatment, analyzed by log rank tests.

Figure 6. The Bi-Ab shows potent antitumor effect on HT-29 and SKOV-3 tumor xenografts in nude mice. ((A) and B) Bi-Ab suppressed tumor growth, tumor diameter was measured with a vernier caliper (*P < 0.05; **P < 0.01; ***P <0 .005 versus treatment with Bi-Ab). The survival rates of HT-29 and SKOV-3 tumor-bearing mice ((C)and D). The median survival and terminal survival rate were shown in Table 2.

Figure 7. Immunohistochemical analysis was used to measure the effect of Bi-Ab treatment on proliferation, apoptosis and angiogenesis in vivo. In HT-29 (A) and SKOV-3 (B) tumors, proliferation and apoptosis were evaluated with Ki-67 and cleaved caspase-3 staining methods respectively, VEGF and CD31 were used to evaluate tumor angiogenesis. (C) Proliferative cells (Ki-67 positive cells) decreased in Bi-Ab-treated HT-29 and SKOV-3 tumors, and it was more effective compared to mAb-04- and cetuximab-treated tumors. (D) Apoptotic cells (cleaved caspase-3 positive cells) increased in Bi-Ab-treated HT-29 and SKOV-3 tumors. (E) The density of CD31-positive blood vessels decreased in Bi-Ab-treated HT-29 and SKOV-3 tumors. The data presented as the mean ± SD, are from a representative experiment, n = 5, *P < 0.05; **P < 0.005; ***P < 0.0005 vs. Bi-Ab treatment.