Therapeutic efficacy evaluation of radioimmunotherapy with 90 Y-labeled anti-podoplanin antibody NZ-12 for mesothelioma

Abstract

Podoplanin is a type I transmembrane sialomucin-like glycoprotein that is highly expressed in malignant mesothelioma. The rat-human chimeric antibody NZ-12 has high affinity for human podoplanin and antibody-dependent cellular cytotoxicity and is applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the present study, we evaluated the in vivo and in vitro properties of radiolabeled NZ-12 and the antitumor effect of RIT with ⁹⁰ Y-labeled NZ-12 in an NCI-H226 (H226) malignant mesothelioma xenograft mouse model. ¹¹¹ In-labeled NZ-12 bound specifically to H226 cells with high affinity, and accumulation was high in H226 tumors but low in major organs. RIT with ⁹⁰ Y-labeled NZ-12 significantly suppressed tumor growth and prolonged survival without body weight loss and obvious adverse effects. Higher podoplanin expression levels were observed in human mesothelioma specimens, suggesting higher tumor accumulation of ⁹⁰ Y-labeled NZ-12 in patients compared with the H226 tumor xenografts. Our findings suggest that ⁹⁰ Y-labeled NZ-12 is a promising RIT agent as a new therapeutic option for malignant mesothelioma that warrants further clinical studies to evaluate the dosimetry and efficacy in patients.

Keywords: antibody; malignant mesothelioma; podoplanin; radiation; radioimmunotherapy.

Figure 1

Immunofluorescence staining of H226 (podoplanin‐positive) and 211H (podoplanin‐negative) cells using anti‐podoplanin antibody LpMab‐17 (red). The nuclei were counterstained with DAPI (blue)

Figure 2

In vitro characterization of radiolabeled antibody NZ‐12. A, Cell binding assay of ¹¹¹In‐labeled NZ‐12 with H226 (black circles) and 211H cells. (white circles). B, Cell binding assay of ¹¹¹In‐labeled control antibody (IgG_1λ) with H226. C, Competitive inhibition assay for intact NZ‐12 (black circles) and CHX‐A′′‐DTPA‐conjugated NZ‐12 (white circles) with H226 cells. D, Competitive inhibition assay for control antibody (IgG_1λ)

Figure 3

Therapeutic efficacy of ⁹⁰Y‐labeled antibodies in nude mice bearing H226 tumors. A, Tumor growth curves after injection with ⁹⁰Y‐labeled NZ‐12 and control antibody. Data indicate mean and SD (n = 10). **P < .01 vs 0 MBq of ⁹⁰Y‐labeled NZ‐12 (intact NZ‐12 only). B, Representative images of mice at day 21 after injection with ⁹⁰Y‐labeled NZ‐12 and control antibody. Dashed circles indicate tumors. C, Kaplan‐Meier survival curves based on the endpoints of tumor volume of 500 mm³. **P < .01, 0 MBq and 1.85 MBq of ⁹⁰Y‐labeled NZ‐12 vs 3.70 MBq of ⁹⁰Y‐labeled NZ‐12; *P < .05, 3.70 MBq of ⁹⁰Y‐labeled control antibody vs 3.70 MBq of ⁹⁰Y‐labeled NZ‐12. D, Body weight changes after injection with ⁹⁰Y‐labeled NZ‐12 and control antibody. Data indicate mean and SD (n = 10). *P < .05 vs 0 MBq of ⁹⁰Y‐labeled NZ‐12

Figure 4

Histological analysis of H226 tumors after injection with 0 MBq (intact NZ‐12 only) and 3.70 MBq of ⁹⁰Y‐labeled NZ‐12. Hematoxylin‐and‐eosin‐stained sections of H226 tumors at days 1, 3, and 7 after injection. White arrowheads indicate necrosis and black arrowheads indicate hemorrhage

Figure 5

Tumor cell proliferation analysis by Ki‐67 immunostaining. A, Ki‐67‐stained sections of H226 tumors at days 1, 3, and 7 after injection with 0 MBq (intact NZ‐12 only) and 3.70 MBq of ⁹⁰Y‐labeled NZ‐12. B, Quantification of proliferating (Ki‐67 positive) cells. Data are mean and SD. **P < .01, *P < .05

Figure 6

Podoplanin expression analysis in human mesothelioma specimens. A, Representative images of immunohistochemical staining with LpMab17. B, High magnification images of (A)

Figure 7

Therapeutic efficacy of external‐beam radiation with X‐rays in nude mice bearing H226 tumors. A, Tumor growth curves after irradiation with X‐rays. Data indicate mean and SD (n = 5). **P < .01 and *P < .05 vs 0 Gy. ⁺⁺ P < .01 and ⁺ P < .05, 25 Gy vs 50 Gy. B, Body weight changes after irradiation with X‐rays. Data indicate mean and SD (n = 5)