Targeting a novel surface antigen, ryk, with the antibody-drug conjugate, slv-404, is effective in richter transformation patient-derived xenografts

Abstract

Abstract RYK belongs to the receptor-like tyrosine kinase (RTK) family and plays a role in Wnt canonical and non-canonical pathways. The best-known member of the family is ROR1, which is selectively expressed by cancer cells, and which has been exploited to design therapeutic antibodies and antibody-drug conjugates, currently in clinical trials. RNA sequencing data showed that RYK is widely expressed in malignant B cells, including chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, Richter trasformation (RT) and Burkitt lymphoma, but is absent in normal mature lymphocytes. Correlation analysis with clinical features and prognostic disease factors revealed higher RYK expression in treatment-naïve CLL patients with a favorable cytogenetic profile. We then confirmed RYK expression in CLL primary samples and patient-derived xenograft (PDX) models of RT. qRT-PCR and immunoblotting, demonstrated robust, albeit variable, RYK expression in tumor cells, while healthy donor (HD) peripheral blood mononuclear cells (PBMCs) displayed markedly lower RYK expression. Flow cytometry studies confirmed strong RYK expression in CLL and RT samples, while normal B and NK cells exhibited low-to-moderate levels and T lymphocytes were negative. Subsequently, we evaluated the efficacy of SLV-404, a novel anti-RYK antibody-drug conjugate (ADC), composed of a chimeric antibody against the RYK extracellular domain, conjugated through a cleavable linker to eight molecules of deruxtecan (DXd), a topoisomerase I (TOP1) inhibitor. Importantly, TOP1, the target of DXd, was overexpressed in malignant B cells, while undetectable in healthy PBMCs, suggesting a favourable therapeutic window for SLV-404. SLV-404 efficacy was initially evaluated in vitro. RT-PDX cells exposed to 1.34 μM SLV-404 showed a time-dependent increase in apoptosis, correlating with TOP1 levels. Primary CLL cells showed limited sensitivity under basal conditions, but apoptosis increased after CpG/IL-15 stimulation, likely due to increased TOP1 expression. Importantly, SLV-404 did not induce apoptosis in healthy PBMCs or in their B, T, and NK subpopulations. SLV-404 effects were then evaluated in vivo in four RT-PDXs. RT cells were initially injected subcutaneously and left to engraft. Mice were randomized to vehicle or SLV-404 (10 mg/kg, intravenously (iv), once a week for 3 weeks) group. SLV-404 significantly extended animal lifespan of 61 days (P<.04), 40 days (P<.004), 68 days (P<.02), and 97 days (P<.01), respectively for RS1316, RS9737, IP867/17, and RS1050 model. We then evaluated the efficacy of RYK treatment in a systemic RT model, where leukemic cells administered iv. At the end of treatment schedule, disease spread was evaluated. Flow cytometry analysis of target organs showed that SLV-404 led to a strong decrease of RT cells to nearly undetectable levels in all compartments, with statistically significant differences in spleen (SP) and bone marrow (BM), across the PDXs. The RS1050 model showed the most significant reduction, with P<.0001 in peripheral blood, SP, and BM, as well as significant reductions in the liver (P<.01) and brain (P<.004). These findings were corroborated by immunohistochemical (IHC) analysis using an anti-human CD20 antibody to detect RS cells. While vehicle-treated mice exhibited high tumor burden in SP and BM, SLV-404 treatment led to complete disease eradication in SP across all models, consistent with flow cytometry data. However, residual disease remained detectable in BM. Consistent with these results, SLV-404 significantly extended survival in all models tested, with median life span doubled in the treated group (P<.004). Nevertheless, all animals eventually relapsed, consistent with residual disease observed in BM. These data support the evaluation of next generation anti-RYK ADCs utilizing a novel linker structure with enhanced tissue penetration, potency, and efficacy. These studies are currently underway. In summary, these results show that the novel antigen RYK is a suitable target for an ADC therapy. Moreover, we demonstrated the therapeutic potential of a novel RYK-based ADC for the treatment of RT, still representing a significant unmet clinical need.