Expression of HMP/AN2, a melanoma associated antigen, in murine cerebral gliomas: potential for radioimmunotargeting

Abstract

Human melanoma proteoglycan (HMP), a melanoma-associated antigen, is expressed in both human melanomas and gliomas. We used HMP-specific monoclonal antibody (mAb) VT68.2 to investigate whether murine GL261 cerebral gliomas express the HMP homologue AN2 and to determine whether AN2 could be targeted for selective delivery of radiation in vivo. HMP-specific mAb VT68.2 stained murine GL261 glioma cells grown in culture and intracerebrally in syngeneic C57BL/6 mice. Positron emission tomography with radiolabeled mAb VT68.2 showed high-contrast, positive images of gliomas against a negative background. At 96 h after injection, glioma uptake of radiolabeled mAb VT68.2 was 10x greater than that of the isotype control mAb and 20x greater than that detected in normal cerebral tissue. Our results show murine GL261 cerebral gliomas express AN2 and HMP-specific mAb VT68.2 accumulates selectively and specifically at high concentration and is retained within murine cerebral gliomas. Thus, HMP is a potential target for antibody-mediated selective delivery of radiation to cerebral gliomas in vivo.

Fig. 1

Immunoreactivity of GL261 glioma cells. (A) Flow cytometry results showing relative saturation of binding sites on GL261 cells by mAbs. (a-c) Comparative binding with 0.5 μg of unlabelled VT68.2 (a), ¹²⁴I-mAb VT68.2 (HMP-specific) (b) and ¹²⁴I-mAb MF1-30 (isotype matched control) (c). (d-f) The binding with 0.25 μg (d), 2 μg (e) and 8 μg (f) of ¹²⁴I-mAb VT68.2. (B) Results of radioligand binding assay showing ¹²⁴I-mAb VT68.2 radioactivity (Filled circle) and ¹²⁴I-mAb MF11-30 radioactivity (empty circle) on GL261 cells as a function of mAb concentration. Triangles represent background counts obtained from blank tubes. Each symbol represents mean (±SD) of values for each dilution of mAb.

Fig. 2

(A-B) Hematoxylin stained GL261 glioma sections showing tumor and brain adjacent to tumor (BAT) at 10x (A) and 20x (B) magnifications. (C-D) Immunofluorescence microscopy of GL261 gliomas stained by HMP-specific mAb VT68.2 showing BAT (C) and central tumor (D). (E) Minimal glial fibrillary acidic protein (GFAP) reactivity of GL261 tumor. (F) Negative control M 11-30 staining of GL261 tumor and BAT.

Fig. 3

Sequential microPET images of two glioma-bearing mice injected with ¹²⁴I-mAb VT68.2 (A) and ¹²⁴I-mAb MF11-30 (B). High-contrast positive images of glioma (yellow arrow) reveal gradual accumulation of HMP-specific ¹²⁴I-mAb VT68.2. In contrast, no visible accumulation of isotype-control ¹²⁴I-mAb MF11-30 within glioma is found at any of the time points (red arrow). The gross appearance of the corresponding gliomas at autopsy is displayed in the column at far left. Mice were imaged in the prone position. The left side of the image represents the left side of the corresponding mouse brain. (C) Quantitative accumulation of ¹²⁴I-labeled mAb VT68.2 (Filled circle) and mAb MF11-30 (empty circles) into gliomas expressed as the percent injected dose per gram (%ID/g) of tissue over time. Each symbol represents the mean of values (±SD) obtained from 6 mice.

Fig. 4

Biodistribution of radiolabeled antibody obtained by ex vivo gamma counting of dissected tissues 96 hours after injection. Counts are displayed as the percent injected dose per gram (%ID/g) of tissue. (A) Accumulation of ¹²⁴I-mAb VT68.2 (Filled bar) is significantly greater than that of ¹²⁴I-mAb MF11-30 (empty bar) in glioma (*P<0.0005) and tumor-adjacent right cerebral hemisphere (RC) (†P < 0.001). Accumulation of ¹²⁴I-mAbs VT68.2 and MF11-30 are not different in normal left cerebral hemisphere (LC) (P = 0.18). (B) Accumulation of ¹²⁴I-mAb VT68.2 in different organs in glioma-bearing mice (n=6) and normal mice (n=6). Comparison of paired values did not reach statistical significance in any of the organs. Each bar represents mean (+SD) of values obtained from 6 mice in the corresponding group.