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

Abstract

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 Zr-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.Zr-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 Zr-DFO-LGR5 mAbs, and findings were consistent with biodistribution data, which showed significantly higher tumor uptake (%ID/g) forZr-DFO-8F2 (17.9 ± 2.2) compared to Zr-DFO-9G5 (5.5 ± 1.2) andZr-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 Zr-DFO-LGR5 mAbs may be utilized to stratify tumors which would respond best to LGR5-targeted ADC therapy.

Antibody–Drug Conjugates for Cancer Therapy

“…LGR5 is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis [ 181 ]. LGR5 overexpression was shown to correlate with poor clinical outcomes [ 182 ].…”

“…LGR5 overexpression was shown to correlate with poor clinical outcomes [ 182 ]. Two different anti-LGR5 monoclonal antibodies, 8F2 and 9G5, were evaluated using 89 Zr immunoPET to select the optimal mAb for ADC development and tumor imaging [ 181 ]. Two LGR5-targeting ADCs were generated by conjugating the anti-LGR5 mAb (anti-LGR5 mAb) to MMAE via a cleavable or non-cleavable chemical linker [ 183 ].…”

Interactions Between Tumor Biology and Targeted Nanoplatforms for Imaging Applications

“…The leucine‐rich repeat‐containing G‐protein coupled receptor 5 (LGR5) is considered to be a bona fide marker of CSCs. Researchers have used 89 Zr immunoPET to evaluate and select efficient anti‐LGR5 mAbs (8F2 and 9G5) for the development of Ab‐drug conjugates (ADCs), imaging, and monitoring of LGR5‐positive tumor response to therapy 318. They demonstrated that the 8F2‐based ADC was more effective for toxin delivery to LGR5‐positive tumors, and suggested 89 Zr‐labeled anti‐LGR5 mAbs could be used to stratify tumors, for best response to LGR5‐targeted ADC therapy.…”

Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion

“…The mouse variable heavy and light chain (VH and VL) sequences were amplified by PCR, cloned, and then sequenced. To produce a mouse-human chimera 10C7 mAb, the mouse VH and VL were subcloned into pCEP4 expression vectors containing the constant region of human IgG1 heavy chain (CH) and kappa light chain (CL) as previously described (54). Large-scale mAb production and purification was performed as reported earlier (31).…”

“…Images were acquired using confocal Leica TCS SP5 microscope with LAS AF Lite software. Whole cell-based binding assays were performed as previously described (54). Fluorescence intensity was quantified using Tecan Infinite M1000 plate reader.…”