A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor

Abstract

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with DeltaEGFR, a deletion mutation lacking exons 2-7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6(DeltaEGFR) (mouse fibroblast line NR6 transfected with DeltaEGFR). mAb 806 with selective reactivity for NR6(DeltaEGFR) in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-DeltaEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with DeltaEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without DeltaEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to DeltaEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that DeltaEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.

Figure 1

Hematoxylin/eosin (A–C) and immunohistochemical (D–O) (avidin–biotin-complex technique, diaminobenzidine tetrahydrochloride chromogen) staining of tumor xenografts U87MG_ΔEGFR (A, D, G, and J), squamous cell carcinoma cell line A431 (B, E, H, and K), and parental (nontransfected) U87MG cell line (C, F, I, and L) with 528 (D–F), 806 (G–I), DH8.3 (J–L). (D, G, and J) Intense staining of U87MG_ΔEGFR with all three antibodies. (E) Intense staining of A431 with 528. (H) Less intense staining of A431 with 806. (K) Negative staining of A431 with DH8.3. (F) Weak focal staining of parental U87MG with 528. (I and L) No staining of parental U87MG with 806 or DH8.3.

Figure 2

Hematoxylin/eosin (A–C) and immunohistochemical (D–L) (avidin–biotin-complex technique, diaminobenzidine tetrahydrochloride chromogen) staining of three glioblastoma specimens pretyped for presence of wtEGFR (A, D, G, and J), amplified EGFR (B, E, H, and K), or amplified EGFR and ΔEGFR (C, F, I, and L) with 528 (D–F), 806 (G–I), or DH8.3 (J–L). 528 staining of the three glioblastomas (D–F), 806 staining of glioblastoma having EGFR amplification without ΔEGFR (H) or with ΔEGFR (I), and DH8.3 staining of glioblastoma with amplified EGFR and ΔEGFR (L) are shown.

Figure 3

Immunohistochemical staining of normal tissues (A–F) and non-CNS tumors (G–O) with 528 (A, D, G, J, and M), 806 (B, E, H, K, and N), or DH8.3 (C, F, I, L, and O). Intense immunoreactivity of 528 with normal liver (A) and skin (D) is shown. (B) No 806 staining of liver. (E) Patchy focal staining of basal cells in epidermis of one skin (specimens from four individuals shown). No DH8.3 staining of liver (C) or skin (F). Squamous cell carcinoma of the head and neck (G–I) with intense immunoreactivity of 528 (G) and with 806 (H) but no staining with DH8.3 (I); serous-papillary ovarian carcinoma (J–L), staining with 528 (J) but not with 806 (K) or DH8.3 (L); invasive ductal carcinoma of the breast (M–O) with intense 528 staining (M) but no staining with 806 (N) or DH8.3 (O).

Figure 4

Immunohistochemical staining of glioblastomas with 528 (A, D, G, J, and M), 806 (B, E, H, K, and N), and DH8.3 (C, F, I, L, and O). (A–C) Case 26, amplified EGFR/ΔEGFR: 528- (A), 806- (B), and DH8.3-positive (C). (D–F) Case 27, no EGFR amplification/no ΔEGFR: 528- (D), 806- (E), and DH8.3-negative (F). (G–I) Case 6, amplified EGFR/no ΔEGFR: 528-positive (G), 806-positive (H), and DH8.3-negative (I). (J–L) Case 44, no EGFR amplification/no ΔEGFR: 528-positive (I), 806-negative (K), and DH8.3-negative (L). (M–O) Case 35, amplified EGFR/ΔEGFR: 528-positive (M), 806-negative (N), and DH8.3-positive (O).