Abstract 3901: Therapeutic targeting of LGR5-positive cancer stem cells
Authors: Mulero Maria Carmen , Rice Catherine , Jahdav Sukshala A. , Pi Willie , Lin Alexander , Nguyen Mindy , Tai Owen , Howell Stephen B. 
Published: March 2024

Abstract
Introduction: Destruction of cancer stem cells (CSC) is a major therapeutic goal. LGR5 marks active stem cells in many types of solid cancers. It has been very difficult to isolate good antibodies to native LGR5 and attempts to develop ADCs targeting LGR5 have not advanced. We took the alternative approach of using a natural LGR5 ligand to target monomethylauristatin (MMAE) to LGR5-expressing CSC. An Fc domain was fused to the LGR binding domains of RSPO1 and the sortase reaction was used to conjugate a Gly3-val/cit-PAB-MMAE linker at the C-terminal end to create FcF2-MMAE. We previously reported its efficacy in ovarian models here document its activity in other aggressive cancer models.
Methods: FcF2-MMAE was produced by transient transfection in HEK293E cells and purified by a sequential Ni-NTA and ion exchange chromatography. OVCAR8 ovarian cancer cells were molecularly engineered to stably express an empty vector (OVCAR8/EV) or LGR5 (OVCAR8/LGR5.6) at a 10-fold higher level. The sortase A enzyme was produced in E. coli. Flow cytometry was used to quantify cell surface LGR5 in live cells using an anti-LGR5 antibody.
Results: DEPMAP tools were used to identify cell types with high levels of WNT signaling that drives expression of LGR5. FCM confirmed LGR5 expression in colon LoVo, gastric AGS and neuroblastoma SKNAS cells. All three lines were very sensitive to FcF2-MMAE; GI50 values in 2D cultures were: LoVo 4.5, AGS 0.7 and SKNAS 8.6 nM. IC50 values for stem cell-derived spheroids grown from these lines were: 11.0, 3.0 and 1.70 nM, respectively. The MTD of FcF2-MMAE was found to be &gt;1.5 nmol/g in mice. The plasma half-life was 29.7 h. A q7dx4 IP dose schedule was found to be more effective than a q4dx4 IP schedule. FcF2-MMAE inhibited in vivo SC growth in all 3 models at a dose of 1 nmol/g q7dx4, producing cures in a fraction of AGS tumors and delaying SKNAS growth &gt;2-fold in the absence adverse events. In addition to MMAE, we showed that FcF2-His could be conjugated to the either deruxtecan or PNU159682; FcF2-His loaded with either one of these cytotoxins had low nM or sub nM cytotoxicity and retained the same high selectivity of FcF2-MMAE when tested in isogenic OVCAR8/EV and OVCAR8/LGR5 cells.
Conclusions: FcF2-MMAE has activity in xenograft models of very aggressive tumors driven by high levels of WNT signaling (LoVo, AGS, and SKNAS) and LGR5 expression. Efficacy was obtained at doses well below its MTD indicating a wide therapeutic window. The FcF2 core accommodates multiple types of warheads without loss of targeting selectivity. The ability to switch out one cytotoxin for another provides versatility in targeting different diseases. FcF2-MMAE has advantages over ASCs. Its small size relative to antibodies favors better tumor penetration. The binding domains of RSPO1 also target stem cells expressing LGR4 and LGR6 as well as LGR5, something that cannot be achieved with an ADC. These features support the further development of FcF2-MMAE as a stem cell-targeted therapeutic.
Citation Format: Maria Carmen Mulero, Catherine Rice, Sukshala A. Jahdav, Willie Pi, Alexander Lin, Mindy Nguyen, Owen Tai, Stephen B. Howell. Therapeutic targeting of LGR5-positive cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3901.