Abstract 1806: Cymirafen: A protein-drug conjugate that target LGR4/LGR5/LGR6-positive cancer stem cells

Abstract INTRODUCTION: Cancer stem cells (CSC) are often drug-resistant and the source of recurrence; their destruction is a major therapeutic goal. LGR4/LGR5/LGR6 mark active stem cells and highly expressed in many solid cancers. Cymirafen consists of a human IgG1 Fc domain fused to the receptor-binding domains of RSPO1. The sortase reaction is used to site-specifically conjugate cleavable Gly3-val/cit-PAB-MMAE linkers to the C-terminal ends of the two chains through the formation of a highly stable peptide bond. We report here pre-IND studies that support the further development of cymirafen. METHODS: The protein component of cymirafen is constitutively produced by a stable CHO-K1 clone. Kd values were determined by ELISA using recombinant human and cynomolgus LGR5-His. In vitro growth rate inhibition assays used the CCK8 reagent. CDX models used SC implantation. Plasma cymirafen was measured by ELISA using antibodies to different RSPO1 epitopes for capture and detection. Free MMAE in plasma was determined by LC-MS/MS. RESULTS: Cymirafen binds to human and cynomolgus LGR5 with Kd of 0.27 and 0.38 nM, respectively. Its DAR is in the range 1.8-2.0. Across a panel of human cancer cell lines of multiple types, 76% had an IC50 of <10 nM, and in 36% IC50 was <5 nM. Against pairs of isogenic LGR5-low and LGR5-high OVCAR8 and HEK293 cells, cymirafen exhibited a 1.5 log differential IC50 in vitro. When grown as CDX, cymirafen was >2-fold more effective against OVCAR8/LGR5 than OVCAR8/EV. On a dose schedule of 85 mg/kg q7dx4, cymirafen was well tolerated in mice and active in CDX models of multiple types of cancer (colon, gastric, ovarian, neuroblastoma). Only 0.001, 0.002 and 0.013% of the MMAE was released from cymirafen in human, rat and cynomolgus plasma during 96 h at 37oC. MTD in mice is ∼128 mg/kg. A dose-ranging study in rats identified a single dose STD10 of 75 mg/kg. Serum chemistries disclosed no organ dysfunction. Adverse events were limited to dose-related neutropenia > thrombocytopenia with recovery by two weeks at all doses. Initial and terminal plasma half-lives were 0.8 and 21.5 h; PK was close to linear over the range of 30 - 75 mg/kg. In SKNAS CDX tumors, cymirafen peaked at 4 h and remained above the IC50 value for this tumor for ∼24 h. CONCLUSIONS: Cymirafen targets tumors with increased levels of LGR4, LGR5 and potentially LGR6, and co-receptors RNF43 and ZNRF3; an ADC can target only one receptor at a time. This favors greater control of drug resistance and recurrence. Unlike MMAE ADCs with long half-lives and cumulative toxicity, cymirafen delivers MMAE to tumors at high concentrations over a shorter period of time. It has a much higher normalized MMAE content than MMAE-containing ADCs. This permits administration of much higher doses on a well-tolerated weekly schedule. These features distinguish cymirafen from all MMAE ADCs and support its further development as a stem cell-targeted therapeutic. Citation Format: Maria Carmen Mulero Roig, Sukshala A. Jadhav, Willie Pi, Owen Tai, Stephen B. Howell. Cymirafen: A protein-drug conjugate that target LGR4/LGR5/LGR6-positive cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1806.