Therapeutic efficacy of ²²⁵Ac-labeled nimotuzumab in a mouse model of EGFR positive KRAS-mutant metastatic colorectal cancer (mCRC)

Background: Metastatic colorectal cancer (mCRC) is the second leading cause of death from cancer with a 5-year survival rate of < 10% (stage IV), likely because over 45% of CRC patients have metastatic disease at initial diagnosis. About 80% of CRC patients overexpress EGFR. Mutations in the KRAS oncogene (present in 40% of mCRC) lead to constitutive over-activation of EGFR and drive de novo resistance to anti-EGFR drugs. In EGFR-positive mCRC patients with wild-type KRAS, the addition of anti-EGFR antibodies (e.g. cetuximab and panitumumab) to chemotherapy results in small albeit significant improvements in survival; there is no observed benefit in patients with KRAS mutations. Here, we propose to target KRAS-mutant EGFR positive mCRC using ²²⁵Ac-labeled nimotuzumab, a humanized anti-EGFR monoclonal antibody with affinity-optimized binding characteristics.

Methods: We synthesized eight-membered macrocyclic bifunctional chelator p-Bz-SCN-macropa for conjugation with nimotuzumab and rituximab (control), followed by radiolabeling with ²²⁵Ac for alpha particle therapy. The radiochemical yield of 225Ac-nimotuzumab and ²²⁵Ac-ritxumab was >95%. The radioimmunoconjugates were developed and characterized by flow cytometry, immunoreactivity, radioligand binding assays, HPLC and internalization rate (live-cell imaging). We have evaluated the in vitro cytotoxicity in EGFR positive KRAS mutant colorectal cancer cell lines DLD-1, HT-29, CCL-227 and CCL-250 with different levels of EGFR density. We have also evaluated the in vivo efficacy of ²²⁵Ac-nimotuzumab in mice bearing DLD-1-iRFP-702 tumors and monitored tumor growth using near infrared (NIR) imaging. Mice were treated with three doses of 350 nCi/dose administered 10 days apart. In vivo study endpoint was tumor volume >= 1500 mm3.

Results: Flow cytometry showed nearly > 95% binding to the cells. In all three EGFR positive colorectal cell lines (DLD-1, CCL-227, CCL-250) in vitro studies showed enhanced cytotoxicity of ²²⁵Ac-nimotuzumab compared with controls. HT29 had very low EGFR density/cell. IC50 in DLD-1 cell line: ²²⁵Ac-nimotuzumab (2.8 nM) < 225Ac-ritxumab (4.7 nM) < nimotuzumab (65 nM) < rituximab (305 nM). Similar trend was observed in the other KRAS-mutant mCRC cell lines. ²²⁵Ac-nimotuzumab prolonged the survival of mice bearing DLD-1 tumors compared with control antibody or non-treated controls. Median survival of DLD-1-iRFP-702 tumor bearing mice was 40 days and 34 days for non-treated and control ²²⁵Ac-rituximab treated mice, respectively, while median survival in ²²⁵Ac-nimotuzumab group had not been reached > 90 days since the start of treatment. There were no treatment related toxicities.

Conclusions: ²²⁵Ac-nimotuzumab shows very promising outcomes in KRAS-mutant mCRC models and warrants further investigation. Additional studies in other xenograft models are ongoing. Funding: This work is funded by the Canadian Institute of Health Research (CIHR) grant # 408132