Abstract 6015: Unveiling RYK: A breakthrough tumor-associated antigen in lymphoma treatment

Abstract This work explores expression and therapeutic potential of RYK, a ROR-family receptor tyrosine kinase (RTK) member, in chronic lymphocytic leukemia (CLL) and Richter syndrome (RS). RYK belongs to a family of conserved proteins acting as alternative receptors/co-receptors of Wnt ligands. While the best-known member of the family, ROR1, has been targeted with various therapeutic modalities, little is known for RYK. RNA-sequencing data shows that RYK is expressed in CLL, RS, diffuse large B-cell lymphoma, and Burkitt’s lymphoma primary cells and malignant B cell lines of different developmental stages, as also confirmed both at mRNA and protein levels. Like ROR1, RYK expression was restricted to cancer cells, with undetectable levels in B, T, or NK cells, obtained from healthy donors. Given this pattern of expression, an antibody drug conjugate (ADC) against RYK, SLV-404, was generated, consisting of a chimeric antibody targeting the extracellular portion of the receptor, a cleavable linker, and a cytotoxic agent, Dxd, an inhibitor of the topoisomerase I (TOP1) with a target drug-to-antibody ratio (DAR) of eight. Expression data showed that TOP1 is mainly expressed in cancer cells and tumor cells are uniquely sensitive to its inhibition.We then investigated in vitro apoptotic responses following SLV-404 treatment. After 72/96 hours of exposure, a marked apoptotic induction was observed by conventional flow cytometry assays (Annexin V/PI staining) and at the biochemical level by Western blot analysis of PARP1/Caspase 3 cleavage, XIAP reduction, and histone H2A.X phosphorylation. SLV-404 effects were then evaluated in vivo exploiting four established RS patient-derived xenograft models (RS-PDX) and a cell line-derived xenograft model, U-RT1. RS cells were initially injected subcutaneously and left to engraft. Mice were then randomly assigned to two different groups: vehicle (phosphate-buffered saline) and SLV-404 (10 mg/kg), all treated by intravenous administration (iv), once a week for 3 weeks in total. SLV-404 significantly extended animal lifespan of 61 days (P<.04), 36 days (P<.02), 68 days (P<.02), 97 days (P<.01) and 47 days (P<.01), respectively for RS1316, RS9737, IP867/17, RS1050 and U-RT1.We then moved to a systemic model of RS, with leukemic cells injected iv and left to engraft 14 days before administering treatment once a week for 3 weeks in total. Disease spread was then evaluated by flow cytometry analysis of target organs, using tumor-specific human markers. Preliminary data confirm that treatment with SLV-404 resulted in dramatic decreases in RS cells to almost undetectable levels in all compartments, reaching statistical significance in spleen and bone marrow (P<.0001).Together, these results suggest that RYK is a novel tumor-associated antigen, making it a suitable ADC target. Moreover, we demonstrate the therapeutic potential of a novel RYK-based ADC for the treatment of RS. Citation Format: Matilde Micillo, Beatrice Pirajno, Giulia Omezzolli, Amelia Fascì, Nahal Nabelsi, Francesco E. Vallone, Victor Navarro-Tableros, Elisabetta Bolli, Samir Mouhissine, Riccardo Moia, Candida Vitale, Marta Coscia, Gianluca Gaidano, Brian J. Lannutti, Katti A. Jessen, Tiziana Vaisitti, Silvia Deaglio. Unveiling RYK: A breakthrough tumor-associated antigen in lymphoma treatment [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 6015.