Effectiveness of ²²⁵Ac-Labeled Anti-EGFR Radioimmunoconjugate in EGFR-Positive Kirsten Rat Sarcoma Viral Oncogene and...

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 [²²⁵Ac]Ac-macropa-nimotuzumab in KRAS mutant and in KRAS wild-type and BRAF^V600E mutant EGFR-positive CRC cells in vitro and in vivo. Anti-CD20 [²²⁵Ac]Ac-macropa-rituximab was developed and used as a nonspecific radioimmunoconjugate. Methods: Anti-EGFR antibody nimotuzumab was radiolabeled with ²²⁵Ac 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 BRAF^V600E mutant HT-29 CRC cell lines; and in 3-dimensional spheroids. Dosimetry was studied in healthy mice. The in vivo efficacy of [²²⁵Ac]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 [²²⁵Ac]Ac-macropa-nimotuzumab compared with nimotuzumab and controls. The inhibitory concentration of 50% in the DLD-1 cell line was 1.8 nM for [²²⁵Ac]Ac-macropa-nimotuzumab versus 84.1 nM for nimotuzumab. Similarly, the inhibitory concentration of 50% was up to 79-fold lower for [²²⁵Ac]Ac-macropa-nimotuzumab than for nimotuzumab in KRAS mutant SNU-C2B and SW620 and in KRAS wild-type and BRAF^V600E 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 [²²⁵Ac]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 [²²⁵Ac]Ac-macropa-rituximab (control), respectively, whereas it was not reached for the [²²⁵Ac]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 [²²⁵Ac]Ac-macropa-rituximab and phosphate-buffered saline, respectively, and was not reached for those treated with [²²⁵Ac]Ac-macropa-nimotuzumab (>90 d). One of 7 mice bearing HT-29 xenografts and treated using [²²⁵Ac]Ac-macropa-nimotuzumab had complete remission. Compared with untreated mice, [²²⁵Ac]Ac-macropa-nimotuzumab more than doubled (16 vs. 41 d) the median survival of mice bearing SW620 xenografts. Conclusion: [²²⁵Ac]Ac-macropa-nimotuzumab is effective against KRAS mutant and BRAF^V600E mutant CRC models.