Efficacy Evaluation of Combination Treatment Using Gemcitabine and Radioimmunotherapy with 90Y-Labeled Fully Human Anti-CD147 Monoclonal Antibody 059-053 in a BxPC-3 Xenograft Mouse Model of Refractory Pancreatic Cancer

Abstract

The poor prognosis of pancreatic cancer requires the development of more effective therapy. CD147 expresses in pancreatic cancer with high incidence and has a crucial role in invasion and metastasis. We developed a fully human monoclonal antibody (059-053) with high affinity for CD147. Here we evaluated the efficacy of combined treatment using radioimmunotherapy (RIT) with ⁹⁰Y-labeled 059-053 and gemcitabine in a BxPC-3 xenograft mouse model. Expression of CD147 and matrix metalloproteinase-2 (MMP2) in BxPC-3 tumors was evaluated. In vitro and in vivo properties of 059-053 were evaluated using ¹¹¹In-labeled 059-053 and a pancreatic cancer model BxPC-3. Tumor volume and body weight were periodically measured in mice receiving gemcitabine, RIT, and both RIT and gemcitabine (one cycle and two cycles). High expression of CD147 and MMP2 was observed in BxPC-3 tumors and suppressed by 059-053 injection. Radiolabeled 059-053 bound specifically to BxPC-3 cells and accumulated highly in BxPC-3 tumors but low in major organs. Combined treatment using RIT with gemcitabine (one cycle) significantly suppressed tumor growth and prolonged survival with tolerable toxicity. The two-cycle regimen had the highest anti-tumor effect, but was not tolerable. Combined treatment with ⁹⁰Y-labeled 059-053 and gemcitabine is a promising therapeutic option for pancreatic cancer.

Keywords: combination therapy; extracellular matrix metalloproteinase inducer; gemcitabine; pancreatic cancer; radioimmunotherapy.

Figure 1

Immunohistochemical staining of CD147 and MMP2 in BxPC-3 xenografted tumors at days 1, 3, and 7 after i.v. injection of intact anti-CD147 antibody 059-053 (25 μg). Sections were immunostained with a goat anti-EMMPRIN/CD147 antibody (diluted 1:500) or an anti-MMP2 polyclonal antibody (diluted 1:200) as the primary antibody.

Figure 2

Cell binding and competitive inhibition assays of anti-CD147 antibody 059-053 using BxPC-3 cells. (A) Cell binding of ¹¹¹In-labeled 059-053 to BxPC-3 cells. The cell suspensions were incubated with ¹¹¹In-labeled 059-053 on ice for 60 min, and the radioactivity bound to cells after washing was measured. (B) Competitive inhibition assay for intact 059-053 (black circles and solid line) and CHX-A″-DTPA-conjugated 059-053 (white circle and dashed line) using BxPC-3 cells. The ¹¹¹In-059-053 was incubated with BxPC-3 cells in the presence of varying concentrations of intact or CHX-A″-DTPA-conjugated 059-053 on ice for 60 min, and the radioactivity bound to the cells was counted after washing.

Figure 3

Growth curves of BxPC-3 tumors in mice injected with ⁹⁰Y-hs8001 alone. The injected doses were 0 (antibody only), 0.925, 1.85 and 3.7 MBq of ⁹⁰Y-hs8001. Tumor size was measured at least twice a week. Data are presented as mean ± SD. Individual animal tumor growth curves (A) are shown as well as a survival curve (B) based on the endpoint of 150% tumor volume.

Figure 4

Individual body weight curves of mice bearing BxPC-3 tumors injected with ⁹⁰Y-labeled anti-CD147 antibody 059-053. The injected doses were 0 (intact antibody only), 0.925 MBq, 1.85 MBq and 3.7 MBq of ⁹⁰Y-059-053. Body weight was measured at least twice a week.

Figure 5

Individual tumor growth curves of BxPC-3 tumors in mice treated with single and combination regimens. The mice were injected with gemcitabine (gem, 240 mg/kg body weight) alone, 3.7 MBq of ⁹⁰Y-labeled anti-CD147 antibody 059-053 (RIT) alone, and the combination of gem plus RIT (one and two cycles) shown in the (A). Tumor size was measured at least twice a week. Survival curves were plotted based on the endpoint of 150% tumor volume (B).

Figure 6

Individual body weight curves of mice bearing BxPC-3 tumors treated with single and combination regimens. The mice were injected with gemcitabine (gem, 240 mg/kg body weight) alone, 3.7 MBq of ⁹⁰Y-labeled anti-CD147 antibody 059-053 (RIT) alone, and the combination of gem plus RIT (one and two cycles). Body weight was measured at least twice a week.

Figure 7

H&E-stained BxPC-3-tumor sections at days 1, 3, and 7 after i.v. injection of intact anti-CD147 antibody 059-053 (0 MBq), gemcitabine (gem, 240 mg/kg body weight) alone, 3.7 MBq of ⁹⁰Y-labeled 059-053 (RIT) alone, and the combination of gem plus RIT (one-cycle regimen).

Figure 8

Ki-67-stained BxPC-3-tumor sections at days 1, 3, and 7 after i.v. injection of intact anti-CD147 antibody 059-053 (0 MBq), gemcitabine (gem, 240 mg/kg body weight) alone, 3.7 MBq of ⁹⁰Y-labeled 059-053 (RIT) alone, and the combination of gem plus RIT (one-cycle regimen). The Ki-67 antigen was detected using an anti-human Ki-67 polyclonal antibody as the primary antibody (diluted 1:100).

Figure 9

Individual tumor growth curves of BxPC-3 tumors treated with X-ray radiation with or without gemcitabine (gem). 0 Gy, gem (240 mg/kg body weight) alone, 5 Gy, gem plus 5 Gy, 15 Gy, gem plus 15 Gy, 30 Gy, and gem plus 30 Gy shown in the (A). Tumor size was measured at least twice a week. Survival curves were plotted based on the endpoint of 150% tumor volume (B).

Figure 10

Individual body weight curves of mice bearing BxPC-3 tumors treated with X-ray radiation with or without gemcitabine (gem). 0 Gy, gem (240 mg/kg body weight) alone, 5 Gy, gem plus 5 Gy, 15 Gy, gem plus 15 Gy, 30 Gy, and gem plus 30 Gy. Body weight was measured at least twice a week.