Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

Abstract

Purpose: Cetuximab and panitumumab have an effective therapeutic response in a subset of RAS Wild-Type (WT) metastatic colorectal cancers (mCRCs). Despite molecular-driven selection, all patients do not respond to epidermal growth factor receptor (EGFR) inhibitors and the onset of secondary resistance limits their clinical benefit.

Experimental design: We tested, in vitro and in vivo, the effect of SYM004, a 1:1 mixture of two recombinant human-mouse chimeric monoclonal antibodies (mAbs) directed against non-overlapping epitopes of the EGFR, on CRC models with acquired resistance to cetuximab.

Results: SYM004 showed a potent growth inhibitory effect in CRC cell lines with acquired resistance to cetuximab and MET activation. SYM004 treatment determined a significant induction of apoptosis and a strong inhibition of MET, AKT and MAPK phosphorilation in these resistant models. The data may further suggest SYM004 -driven induced internalization and degradation of the antibody-receptor complex, which prevents cross-interaction between EGFR and MET even in the presence of TGFα. Moreover, in vivo xenograft studies demonstrated that SYM004 has stronger antitumor activity than cetuximab in CRC models. Importantly, in the current work we observed a response to therapy in all cetuximab resistant tumors mice treated with SYM004. More importantly, four out of seven mice continue to respond to SYM004 after 30 weeks of treatment underling the prolonged effect of the drug.

Conclusion: These results suggest that the treatment with SYM004 could be a strategy to overcome acquired resistance to first generation of anti-EGFR therapies in mCRC as a result of MET activation.

Keywords: MET; SYM004; acquired resistance; cetuximab; metastatic colorectal cancer.

Figure 1. Effects of cetuximab or SYM004 treatment on cell proliferation in a panel of human CRC cell lines

(A-B) Cells were treated with different concentrations of cetuximab (range, 0.001 to 10 μg/ml) and SYM004 (range, 0.001 to 10 μg/ml) for 96 hours and evaluated for proliferation by MTT staining, as described in Materials and Methods. (C) The IC₅₀ was determined by interpolation from the dose-response curves. Results represent the median of three separate experiments, each performed in quadruplicate.

Figure 2. Effects of cetuximab or SYM004 in induction of apoptosis in a panel of human colorectal cancer cell lines

(A-B) Cetuximab-sensitive and resistant CRC cells were treated for 48 hours with the 5 μg/ml of cetuximab or SYM004. Apoptosis was evaluated with Annexin V staining, as described in Materials and Methods and the rate of apoptosis was expressed as a percentage of the total cells counted.

Figure 3. Effects of cetuximab or SYM004 on EGFR-dependent intracellular signaling in a panel of human colorectal cancer cell lines

Cells were treated with cetuximab and SYM004 at the indicated doses for 24 hrs. Total cell protein extracts (50μg) were subjected to immunoblotting with the indicated antibodies, as described in Materials and Methods. Anti-tubulin antibody was used for normalization of protein extract content. Experiments were repeated three times.

Figure 4. Effects of cetuximab or SYM004 in human colorectal cancer cell lines with acquired resistance to cetuximab such as MET activation and ERBB2 amplification

(A) Western blot analysis of protein expression in GEO, SW48, GEO-CR and SW48-CR cells treated with cetuximab (5 μg/ml) and SYM004 (5 μg/ml) was performed. Total cell protein extracts were subjected to immuneblotting with the indicated antibodies, as described in Materials and Methods. (B) Two mg of GEO cell or of GEO-CR cell protein extracts were immune-precipitated with a specific anti-MET antibody and then were immune-blotted with a specific anti-EGFR antibody, as described in Materials and Methods. (C) Western blot analysis of protein expression in SW48, SW48-CR, SW48H2 and SKBR3 was performed. Total cell protein extracts were subjected to immuneblotting with the indicated antibodies, as described in Materials and Methods. (D) HER2 gene amplified SW48 cells line (SW48H2) are exposed to different concentration of cetuximab (range, 0.001 to 10 μg/ml) and SYM004 (range, 0.001 to 10 μg/ml) for 96 hours and evaluated for proliferation by MTT staining, as described in Materials and Methods.

Figure 5. Effects of cetuximab or SYM004 on SW48 tumor xenografts

(A-B) Mice were injected subcutaneously in the right flank with SW48 cells as described in the Materials and Methods. After two weeks (average tumor size 200-300 mm³) mice were treated intraperitoneally with: PBS control, cetuximab (1 mg twice a week), SYM004 (50 mg/kg twice a week). The treatment was continued for 30 weeks. Each group consisted of 10 mice. Tumor volumes were measured three times a week. Animals were sacrificed when tumors achieved 2.000 mm³ in size. Abbreviations: CTR, control; A, median tumor volume (mm³); B, alive mice/total mice; C, number of mice without clinical evidence of progression.

Figure 6. Effect of SYM004 treatment after progression to Cetuximab therapy in SW48 tumor xenografts

(A-B) SW48 cells were injected s.c. into the right flank of seven nude mice. After two weeks mice were treated with Cetuximab (1 mg twice a week) by i.p. injection. Treatment was continued until disease progression. The black arrows indicate the time of progression to cetuximab. At progression phase mice were assigned to SYM004 treatment (50 mg/Kg twice a week) by i.p. injection. The treatment was continued until 30 weeks. At week 30 four out of seven mice were still responding to SYM004 (as indicated by double asterisk). Abbreviations: PD, progression disease; PR, partial response; SD, stable disease.