Evaluation of Anti-LGR5 Antibodies by ImmunoPET for Imaging Colorectal Tumors and Development of Antibody–Drug Conjugates

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis. Recently, we showed that LGR5 is a promising target for antibody-drug conjugate (ADC) therapy. However, it is important to identify LGR5-positive tumors that would respond to ADC treatment. Prior to drug conjugation, we evaluated two different anti-LGR5 monoclonal antibodies (mAbs), 8F2 and 9G5, using ⁸⁹Zr-immunoPET to select the optimal mAb for ADC development and tumor imaging. Binding, specificity, and internalization were compared, and mAbs were prescreened as ADC candidates against colon cancer cells using secondary ADCs. Both mAbs demonstrated strong, specific binding in 293T-LGR5 cells but not 293T-vector cells. In DLD-1 colorectal cancer cells, which express high levels of LGR5, the mAbs rapidly internalized into lysosomes and promoted ADC-induced cytotoxicity, with 8F2 exhibiting slightly higher potency. No binding was detected in DLD-1-shLGR5 (LGR5 knockdown) cells. ⁸⁹Zr-DFO-LGR5 mAbs were generated and shown to retain high affinity and LGR5-dependent uptake in vitro. PET/CT imaging of DLD-1 tumors was performed 5 days postinjection of ⁸⁹Zr-DFO-LGR5 mAbs, and findings were consistent with biodistribution data, which showed significantly higher tumor uptake (%ID/g) for ⁸⁹Zr-DFO-8F2 (17.9 ± 2.2) compared to ⁸⁹Zr-DFO-9G5 (5.5 ± 1.2) and ⁸⁹Zr-DFO-IgG (3.8 ± 1.0). No significant uptake was observed in DLD-1-shLGR5 tumors. This study identifies 8F2 as the optimal candidate for ADC development and provides initial evidence that ⁸⁹Zr-DFO-LGR5 mAbs may be utilized to stratify tumors which would respond best to LGR5-targeted ADC therapy.