MET-targeting antibody (emibetuzumab) and kinase inhibitor (merestinib) as single agent or in combination in a cancer model bearing MET exon 14 skipping

Abstract

Purpose Approximately 3% of lung cancer bears mutations leading to MET exon 14 skipping, an oncogenic driver which is further evidenced by case reports of patient response to MET kinase inhibitor treatment. Approximately 15% of tumors harboring MET exon14 skipping have concurrent MET amplification. Experimental Design Merestinib is a type II MET kinase inhibitor. Emibetuzumab, a bivalent anti-MET antibody, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET exon14 skipping and MET amplification. Results Merestinib inhibited Hs746t cell proliferation (IC50=34 nM) and totally eliminated pMET at 100nM. Emibetuzumab showed little anti-proliferative activity against Hs746t cells (IC50>100nM), did not reduce pMET, and slightly reduced cell surface MET. In the Hs746t xenograft model, dose dependent differences in durability of response were seen with merestinib including durable tumor regression (91.8%) at 12 mg/kg qd. Emibetuzumab treatment (10mg/kg qw) provided transient tumor regression (37.7%), but tumors re-grew while on treatment. Concurrent combination of merestinib (6 mg/kg qd) and emibetuzumab resulted in 85% tumor regression, while a sequential combination (initiating merestinib first) resulted in longer duration of treatment response. Conclusions Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). As a type II MET kinase inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or to patients who progress on type I MET inhibitor treatment. Data also support clinical evaluation of the sequential combination of merestinib with emibetuzumab when patients progress on single agent merestinib.

Keywords: Emibetuzumab; LY2801653; LY2875358; MET antibody; MET exon 14 skipping; MET kinase inhibitor; Merestinib.

Conflict of interest statement

Conflict of interest

All authors are full time employees of Eli Lilly and Company.

Ethical approval

All animal studies were performed in accordance with the International Association for Assessment and Accreditation of Laboratory Animal Care institutional guidelines. All in vivo experimental protocols were approved by the Eli Lilly and Company Animal Care and Use Committee.

This article does not contain any studies with human participants performed by any of the authors.

Figures

Fig. 1

In vitro effect of MET inhibitors on expression of total MET, pMET (Y1234/1235), and pMET (Y1003). a Western blot analysis of Hs746t (exon 14 skipping and MET amplification) and MKN45 (MET amplification) cells treated for 24 h with merestinib, PF04217903, or emibetuzumab at indicated concentrations. Blots were probed with antibodies to total MET, phospho-MET (Y1234/1235), and phospho-MET (Y1003). b Western blot analysis of Hs746t cells treated for 24 h with single agent MET inhibitors (emibetuzumab, merestinib, or PF04217903) or hIgG4 at the indicated concentrations or combination of emibetuzumab with either merestinib or PF04217903. Blots were probed with antibodies to total MET, and phospho-MET (Y1003)

Fig. 2

MET kinase inhibitors but not emibetuzumab inhibited MET signaling pathway in Hs746t cells in vitro by western blot analysis. Hs746t cells were treated for 24 h with 133 nM (20 μg/ml) or 667 nM (100 μg/ml) emibetuzumab or MET kinase inhibitors (merestinib, PF04217903, crizotinib, cabozantinib) at 15.6, 62.5, 250 or 1000 nM

Fig. 3

In vivo single agent anti-tumor effect of merestinib, emibetuzumab, and crizotinib in the Hs746t xenograft model. Approximately 5 million cells were implanted in female athymic nude mice (n = 7 per group). Vehicle (), merestinib at 6 mg/kg (suboptimal dose – insufficient target coverage for 24 h) () or 12 mg/kg (optimal dose) () was dosed once daily orally, emibetuzumab at 10 mg/kg () was dosed once weekly intraperitoneally (IP), or crizotinib at 25 mg/kg () was dosed once daily orally. Dosing of inhibitors began on day 11, when tumors were of average size of 150 mm3, and continued for 21 days. The waterfall plot shows the response of individual animals to treatment on day 32. Missing bars are due to animals removed early from the study because of tumor burden. In the waterfall plot, the three horizontal dotted lines represent the three cut points for: Progressive Disease as ∆T/C > 10%; Stable Disease as ∆T/C ≤ 10% to regression <−50%; Partial Response as regression ≥ − 50% and tumor volume > 14 mm3; Complete Response as tumor volume ≤ 14 mm3. Statistical analysis comparing tumor volumes of treatment groups to vehicle control group () was performed on Day 32, where at least half of the animals (4/7) in the vehicle group still remained on study. Dosing for merestinib (12 mg/kg) and crizotinib continued to Day 64 and tumor growth monitored for another 5 weeks post-treatment (data not shown)

Fig. 4

In vivo anti-tumor effect of the combination of merestinib and emibetuzumab in the Hs746t xenograft model. a Approximately 5 million cells were implanted in female athymic nude mice (n = 7 per group). Dosing of inhibitors began on day 14, when tumors reached average size 350 mm3, and continued for 28 days. As single agent, merestinib at 6 mg/kg (suboptimal dose – insufficient target coverage for 24 h) () was dosed once daily orally, and emibetuzumab at 10 mg/kg () was dosed once weekly intraperitoneally (IP). In combination, emibetuzumab at 10 mg/kg was dosed once weekly intraperitoneally (IP) concurrently with either merestinib at 6 mg/kg (suboptimal dose) () or merestinib at 12 mg/kg (optimal dose) () once daily orally. The waterfall plot shows the response of individual animals to treatment on day 27, when tumor volume analysis in comparison to the vehicle control group was performed. Missing bars are due to animals removed early from the study because of tumor burden. In the waterfall plot, the three horizontal dotted lines represent the three cut points for: Progressive Disease as ∆T/C > 10%; Stable Disease as ∆T/C ≤ 10% to regression <−50%; Partial Response as regression ≥ − 50% and tumor volume > 14 mm3; Complete Response as tumor volume ≤ 14 mm3. b Comparison of sequential and concurrent combination of merestinib (6 mg/kg once daily) with emibetuzumab (10 mg/kg once weekly). Approximately 5 million cells were implanted subcutaneously into the hind flank of female athymic nude mice (6 per group). Compound dosing began on day 21 when tumors reached an average size of 300–400 mm3. In the concurrent combination (), emibetuzumab was dosed for 7 cycles, with merestinib given for 50 days (dosing period is shown with the blue color double-arrowed horizontal bar). In the sequential combination () (dosing period and the sequence of the combination are shown with the red color double-arrowed horizontal bar), merestinib treatment was initially started as a single agent (Day 21), and upon tumor regression and regrowth to tumor size of 500 mm3, emibetuzumab was then added on for 6 cycles starting on Day 37. The last day of dosing was Day 70 and Day 78 for concurrent and sequential treatment group, respectively, and tumor growth was monitored post-treatment