Effectiveness of 225Ac-Labeled Anti-EGFR Radioimmunoconjugate in EGFR-Positive Kirsten Rat Sarcoma Viral Oncogene and BRAF Mutant Colorectal Cancer Models

Eighty percent of colorectal cancers (CRCs) overexpress epidermal growth factor receptor (EGFR). Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 40% of CRCs and drive de novo resistance to anti-EGFR drugs. BRAF oncogene is mutated in 7%- 10% of CRCs, with even worse prognosis. We have evaluated the effectiveness of [Ac-225]Ac-macropa-nimotuzumab in KRAS mutant and in KRAS wild -type and BRAFV600E mutant EGFR-positive CRC cells in vitro and in vivo. Anti-CD20 [Ac-225]Ac-macropa-rituximab was developed and used as a nonspecific radioimmunoconjugate. Methods: Anti-EGFR antibody nimotuzumab was radiolabeled with Ac-225 via an 18 -membered macrocyclic chelator p-SCN-macropa. The immunoconjugate was characterized using flow cytometry, radioligand binding assay, and high-performance liquid chromatography, and internalization was studied using live -cell imaging. In vitro cytotoxicity was evaluated in 2 -dimensional monolayer EGFR-positive KRAS mutant DLD-1, SW620, and SNU-C2B; in KRAS wild -type and BRAFV600E mutant HT -29 CRC cell lines; and in 3 -dimensional spheroids. Dosimetry was studied in healthy mice. The in vivo efficacy of [Ac-225]Ac-macropa-nimotuzumab was evaluated in mice bearing DLD-1, SW620, and HT -29 xenografts after treatment with 3 doses of 13 kBq/dose administered 10 d apart. Results: In all cell lines, in vitro studies showed enhanced cytotoxicity of [Ac-225]Ac-macropa- nimotuzumab compared with nimotuzumab and controls. The inhibitory concentration of 50% in the DLD-1 cell line was 1.8 nM for [Ac-225]Ac-macropa-nimotuzumab versus 84.1 nM for nimotuzumab. Similarly, the inhibitory concentration of 50% was up to 79 -fold lower for [Ac-225]Ac-macropa-nimotuzumab than for nimotuzumab in KRAS mutant SNU-C2B and SW620 and in KRAS wild -type and BRAFV600E mutant HT -29 CRC cell lines. A similar trend was observed for 3 -dimensional spheroids. Internalization peaked 24-48 h after incubation and depended on EGFR expression. In the [Ac-225]Ac-macropa- nimotuzumab group, 3 of 7 mice bearing DLD-1 tumors had complete remission. Median survival was 40 and 34 d for mice treated with phosphate -buffered saline and [Ac-225]Ac-macropa-rituximab (control), respectively, whereas it was not reached for the [Ac-225]Ac-macropa- nimotuzumab group (>90 d). Similarly, median survival of mice bearing HT -29 xenografts was 16 and 12.5 d for those treated with [Ac-225]Ac- macropa-rituximab and phosphate -buffered saline, respectively, and was not reached for those treated with [Ac-225]Ac-macropa-nimotuzumab (>90 d). One of 7 mice bearing HT -29 xenografts and treated using [Ac-225]Ac-macropa-nimotuzumab had complete remission. Compared with untreated mice, [Ac-225]Ac-macropa-nimotuzumab more than doubled (16 vs. 41 d) the median survival of mice bearing SW620 xenografts. Conclusion: [Ac-225]Ac-macropa-nimotuzumab is effective against KRAS mutant and BRAFV600E mutant CRC models.