Engineering and characterisation of chimeric monoclonal antibody 806 (ch806) for targeted immunotherapy of tumours expressing de2-7 EGFR or amplified EGFR

Abstract

We report the generation of a chimeric monoclonal antibody (ch806) with specificity for an epitope on the epidermal growth factor receptor (EGFR) that is different from that targeted by all other anti-EGFR therapies. Ch806 antibody is reactive to both de2-7 and overexpressed wild-type (wt) EGFR but not native EGFR expressed in normal tissues at physiological levels. Ch806 was stably expressed in CHO (DHFR -/-) cells and purified for subsequent characterisation and validated for use in preliminary immunotherapy investigations. Ch806 retained the antigen binding specificity and affinity of the murine parental antibody. Furthermore, ch806 displayed enhanced antibody-dependent cellular cytotoxicity against target cells expressing the 806 antigen in the presence of human effector cells. Ch806 was successfully radiolabelled with both iodine-125 and indium-111 without loss of antigen binding affinity or specificity. The radioimmunoconjugates were stable in the presence of human serum at 37 degrees C for up to 9 days and displayed a terminal half-life (T(1/2beta)) of approximately 78 h in nude mice. Biodistribution studies undertaken in BALB/c nude mice bearing de2-7 EGFR-expressing or amplified EGFR-expressing xenografts revealed that (125)I-labelled ch806 failed to display any significant tumour retention. However, specific and prolonged tumour localisation of (111)In-labelled ch806 was demonstrated with uptake of 31%ID g(-1) and a tumour to blood ratio of 5 : 1 observed at 7 days postinjection. In vivo therapy studies with ch806 demonstrated significant antitumour effects on established de2-7 EGFR xenografts in BALB/c nude mice compared to control, and both murine 806 and the anti-EGFR 528 antibodies. These results support a potential therapeutic role of ch806 in the treatment of suitable EGFR-expressing tumours, and warrants further investigation of the potential of ch806 as a therapeutic agent.

Figure 1

FACS analysis of (A) A431 adenocarcinoma cell line (amplified EGFR) and (B) parental and (C) transfected U87MG glioma cell lines stably expressing wt (U87MG.wtEGFR) or (D) mutant EGFR (U87MG.de2-7). Cells were incubated with mAb806 (–), ch806 (- -) followed by Alexa488-labelled anti-mouse Ig. The plots represent fluorescence intensity on the abscissa and cell number per fluorescence channel on the ordinate. The negative control (irrelevant antibody) fluorescence is plotted on each panel (black line).

Figure 2

Immune effector function of mAb 806 (○), chimeric IgGl antibody ch806 (•) and Isotype control cG250 (▾). (A) Complement-dependent cytotoxicity activity of 0–10 μg ml⁻¹ antibody on target U87MG.de2-7 cells. Antibody-dependent cellular cytotoxity on target (B) A431 cells and (C) U87MG.de2-7 cells at effector-to-target cell ratio of 50 : 1 and antibody concentrations ranging from 0 to 10 μg ml⁻¹. Mean (bars;±s.d.) percent cytotoxicity of triplicate determinations are presented. Representative results from three separate experiments are shown.

Figure 3

The biodistribution over 7 days of ¹¹¹In-CHX-A″-DTPA- and ¹²⁵I-labelled ch806 in (A) U87MG.de2-7 xenografts, (B) A431 xenografts, (C) FaDu xenografts and serum from BALB/c nude mice. Results are expressed as mean (bars; ±s.d.) percent injected dose per gram (%ID g⁻¹) for each time point, (n=3–5 mice). The data are presented as: ¹¹¹In-serum (▪); ¹¹¹In-tumour (▴), ¹²⁵I-serum (•), and ¹²⁵I-tumour (▾).

Figure 4

Normal tissue biodistribution of radiolabelled ch806 over 7 days in BALB/c nude mice bearing tumour xenografts (n=5). Results of the biodistribution of (A) ¹¹¹In-CHX-A″-DTPA-ch806 and (B) ¹²⁵I-ch806 are presented for each tissue expressed as mean (bars;±s.d.) percent injected dose per gram (%ID g⁻¹) values.

Figure 5

Antitumour effect of ch806 on U87MG.de2-7 glioma xenograft growth rates. U87MG.de2-7 cells (3 × 10⁶) were injected s.c. into both flanks of 4–6-week-old BALB/c nude mice at day 0. Antibody treatment commenced on day 7 post-tumour-cell inoculation and consisted of six i.p. injections over 2 weeks of 1 mg ch806 (▴), 1 mg mAb 806 (•), 1 mg 528 anti-EGFR (▾), or vehicle control (▪) on days 7, 9, 11, 14, 16 and 18 (arrows). Mean tumour volume (bars;±s.e.) for each treatment group (n=5) is presented. ^*P=0.04, mAb 806 vs control, day 30. ^**P=0.001, ch806 vs control, day 30. ^***P=0.014, ch806 vs mAb 806, day 30; P=0.025, ch806 vs 528, day 30.