Ayers-Ringler, Jennifer

Abstract Background: Diffuse pleural mesothelioma (DPM) is a highly aggressive malignancy with limited treatment options and poor prognosis. Although immune checkpoint inhibitors have improved outcomes, particularly for non-epithelioid subtypes, there remains no approved targeted therapy for mesothelioma. EGFR and MET are receptor tyrosine kinases frequently overexpressed in DPM, suggesting susceptibility to targeted antibody therapy. Amivantamab is a bispecific EGFR/MET antibody, originally developed for EGFR-mutant lung cancer, that blocks growth signals and engaging immune mechanisms. Given the high levels of EGFR and MET expression in mesothelioma, we evaluated the therapeutic potential of amivantamab in preclinical models. Methods: EGFR and MET transcript levels in DPM were analyzed using TCGA. Eight human mesothelioma cell lines were assessed by Western blotting and flow cytometry. Amivantamab binding, internalization, and signaling inhibition were examined using flow cytometry, confocal microscopy, and ligand-stimulated phosphorylation assays. Cytotoxicity and immune-effector activity were quantified using PBMCs, NK cells, and monocytes from healthy donors, along with an ADCC reporter assay. In vivo efficacy was tested in a DPM patient-derived xenograft (PDX) model in NOD/SCID-γ (NSG) mice supplemented with human NK cells and treated with amivantamab or control IgG. Results: TCGA analysis showed frequent EGFR/MET co-expression in DPM. Mesothelioma cell lines expressed higher EGFR and MET levels compared with mesothelial cells. Amivantamab strongly bound both receptors, induced receptor internalization, and inhibited EGF- and HGF-stimulated phosphorylation of EGFR, MET, and downstream AKT/ERK signaling. Direct cytotoxicity was minimal; however, the addition of PBMCs or NK cells resulted in robust, dose-dependent tumor cell killing consistent with antibody-dependent cellular cytotoxicity (ADCC). Reporter assays demonstrated 7-11-fold Fcγ receptor activation in tumor cells compared with ∼2-fold activation in non-malignant mesothelial cells. Co-culture with monocytes revealed amivantamab-induced trogocytosis, evidenced by loss of EGFR and MET from tumor-cell membranes. In the DPM PDX model, amivantamab produced 89.1% tumor-growth inhibition (TGI) relative to controls (P < 0.0001), with no overt toxicity observed in treated mice. Conclusions: Dual EGFR/MET targeting with amivantamab produced potent antitumor effects in mesothelioma through coordinated receptor-signaling blockade, ADCC, and monocyte-mediated trogocytosis. These results support amivantamab as a promising targeted therapeutic strategy for EGFR/MET-positive DPM, particularly epithelioid subtypes that currently derive limited benefit from immunotherapy. Citation Format: Shinichiro Suzuki, Kaushal Parikh, Ezequiel Tolosa, Kuan-Li Wu, Jennifer Ayers-Ringler, Lin Yang, Katherine E. Smith, Farhad Kosari, Aaron S. Mansfield, . Enhancing antibody-dependent cellular cytotoxicityin mesothelioma through concurrent EGFR and MET targeting withamivantamab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5547.

Abstract Background: Diffuse pleural mesothelioma (DPM) is a rare and aggressive cancer arising from mesothelial cells of the pleura. Over 2, 500 new cases are diagnosed annually in the United States, mainly attributable to asbestos exposure. The prognosis is poor, and while nivolumab (PD-1 inhibitor) and ipilimumab (CTLA-4 inhibitor) have been approved for first-line treatment of unresectable DPM, the three-year survival rate is only 23%. DPM tumors express high levels of epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition (MET), but targeted therapies like erlotinib (EGFR tyrosine kinase inhibitors) have shown no clinical benefit. We hypothesized that amivantamab, a bispecific antibody that targets both EGFR and MET, may have activity against DPM through antibody-dependent cellular cytotoxicity (ADCC). Objective: The purpose of this study was to assess the potential of amivantamab to mediate ADCC in tissue culture experiments with DPM cell lines. Methods: We assessed DPM for the expression of EGFR and MET using available data from bulk RNA sequencing. We also examined protein expressions of EGFR and MET by Western blot analysis in five epithelial (EM-Meso, H2452, H2461, Mero-41 and Mero-95), three sarcomatoid (H2373, H2596 and ONE58), and one normal mesothelial (MeT-5A) cell lines. Co-culture experiments were performed with the DPM and normal cell lines and NK cells in the presence of amivantamab to evaluate ADCC. Results: We identified high levels of EGFR and MET expression in both epithelial and sarcomatoid cases of DPM from 183 patients. This finding was corroborated by Western blot analysis of all eight DPM cell lines. In vitro experiments demonstrated that amivantamab induced dose-dependent ADCC in DPM cell lines, with robust NK cell-mediated ADCC observed across all tested DPM lines. Notably, sarcomatoid-type DPM cells were more sensitive to amivantamab-induced ADCC than epithelial-type cells. These findings suggest that amivantamab may offer therapeutic benefits for DPM tumors with high EGFR and MET expression. Conclusion: This study provides preclinical evidence supporting the potential of amivantamab as a novel therapeutic option for DPM, especially in sarcomatoid tumors. Ongoing in vivo studies including in patient-derived xenograft (PDX) models will further evaluate the efficacy of amivantamab in DPM. Citation Format: Shinichiro Suzuki, Lin Yang, Jennifer Ayers-Ringler, Ezequiel J. Tolosa, Kuan-Li Wu, Janet L. Schaefer-Klein, Farhad Kosari, Aaron S. Mansfield. Antitumor activity of amivantamab in diffuse pleural mesothelioma [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 7331.

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy remains synthesizing a focused set of heterobifunctional compounds and screening them for target protein degradation.

Background Aneurysm treatments are crucial to minimize the rupture risk. The underlying molecular processes mediating cellular remodeling, endothelialization, and aneurysm healing following endovascular treatment are poorly understood. The current study aims to explore circulating miRNA as a treatment and outcome-associated biomarkers in patients undergoing endovascular treatment. Methods Patients undergoing endovascular interventions for unruptured intracranial aneurysms, using either flow diverter placement or coil embolization, were enrolled. Blood samples were collected before the intervention and during a follow-up period between 6 and 18 months. Total mRNA/miRNA was isolated from plasma, followed by RNA-seq analysis. Gene Ontology analysis was used to identify pathways linked to altered miRNA expression. Results Twenty-three patients participated, with 13 (56.5%) undergoing flow diversion and 10 (43.5%) coil embolization. The median follow-up sample collection time was 10.70 months (SEM ± 1.32). No significant differences in angiographic occlusion were noted between intervention groups. Differentially expressed miRNAs were not identified between groups at baseline. However, at follow-up, 39 miRNAs were upregulated and 41 were downregulated, independent of intervention. Notably, three miRNAs (miR-4746-5p, miR-4685-3p, and miR-490-3p) were downregulated in the flow diversion group compared to the coil embolization group. Bioinformatics analysis revealed associations with upregulated fluid shear stress, p53, adherens junction pathways, along with downregulated apoptosis pathways. Conclusions This study suggests that fluid shear stress and apoptosis may influence aneurysm healing or thromboembolic events in flow diverter-treated patients. Further research is warranted to elucidate the functional significance of these findings in treatment outcomes, providing valuable insights for improved patient care in intracranial aneurysm management.

2565 Background: Mesothelin (MSLN) is overexpressed in mesothelioma and other solid tumors making it the focus of multiple targeted therapies, including antibody drug conjugates (ADCs), T cell receptor (TCR) fusion constructs, and chimeric antigen receptor T-cell (CAR-T) products. However, the clinical efficacy of these treatments remains limited. We hypothesize that soluble MSLN (sMSLN) in the plasma binds to MSLN-targeted therapies before reaching the tumor. Our study aimed to evaluate the effects of sMSLN on a MSLN-targeting antibody, anetumab, and explore methods to reduce sMSLN. Methods: Exploratory analysis in NCT03126630: sMSLN testing was performed on blood samples from participants enrolled in NCT03126630 at the Molecular Targets Core Lab, National Cancer Institute. The median was used to categorize high and low levels of sMSLN. Survival was estimated with the Kaplan-Meier method and compared between groups with the log rank test. Anetumab immunoprecipitation: Anetumab was covalently coupled to Dynabeads at 5 µg per mg of beads to immunoprecipitate MSLN from two plasma samples. Assays were performed in duplicate. Therapeutics plasma exchange (TPE):Whole blood samples were collected before and after one plasma volume of TPE with albumin as the replacement fluid in patients undergoing routine TPE for various medical conditions, such as autoimmune diseases and hyperviscosity syndromes. Plasma levels of sMSLN were measured with an ELISA assay in matched pre- and post-TPE plasma samples. Results: We obtained sMSLN levels from 40 patients enrolled in NCT03126630. For patients treated with both anetumab ravtansine and pembrolizumab, median progression free survival (PFS) was shorter in the high sMSLN group (5 months) vs the low sMSLN group (12 months). For patients treated with pembrolizumab alone, PFS was similar for patients with high and low sMSLN (4 vs 3.4 months). Anetumab significantly reduced the concentration of sMSLN in plasma samples as detected by MSLN ELISA (p<0.05), demonstrating that sMSLN can bind to and sequester anetumab. Next, we evaluated TPE as a mechanism to reduce sMSLN. We obtained pre- and post-TPE plasma samples from 15 patients undergoing routine TPE for various medical conditions. TPE consistently reduced sMSLN (p<0.05) with an average decrease of 43.6% or 15.4 ng/mL. Conclusions: We found that high sMSLN levels are associated with shorter PFS for anetumab ravtansine, but not pembrolizumab. Additionally, anetumab binds to sMSLN in the plasma, which suggests that sMSLN can sequester anti-MSLN antibodies and may limit the efficacy of MSLN-targeted therapies. High levels of sMSLN could potentially be used as a biomarker to select which patients should not receive MSLN-targeting therapies. Furthermore, our results indicate that sMSLN reduction is feasible with TPE. Decreasing sMSLN could restore the activity of MSLN-targeted therapies.

Abstract Antibody-drug conjugates (ADCs) are increasingly being used in clinic, and many novel ADCs are being tested in clinical trials. Most ADCs act by delivering cytotoxic payloads into target cells. The activity of most ADCs is predicated upon internalization to release the cytotoxic payloads intracellularly to act on the malignant cells. The degree of ADC internalization is incomplete or absent for many cell surface targets, limiting the activity of ADCs. PROTACs are heterobifunctional molecules that result in the polyubiquitylation of their targets by inducing proximity with E3 ligases. The proteasome then recognizes the poly-ubiquitin chains and degrades these targets. We hypothesized that PROTACs that act on the same targets as ADCs could improve the internalization and efficacy of these ADCs. Using multiple cell lines derived from breast, lung or pancreatic cancers that express the oncogenic cell surface proteins EGFR (ERBB1), HER2 (ERBB2) or MET (HGF), we measured antibody internalization with live cell imaging. Antibodies were labeled with a pH sensitive reagent that fluoresces red (640 nm) upon exposure to acidic environments. Fluorescence was measured over time to create an area under the curve which was compared between the cell lines that were treated with and without PROTACs. Antibody internalization was observed in all cell lines that expressed the targets recognized by the antibodies. The addition of PROTACs targeting the same oncogenic cell surface proteins as the antibodies significantly increased antibody internalization beyond that observed without PROTACs. Internalization of antibodies was confirmed by measuring intracellular immunoglobulins by Western blot. Antibody internalization with PROTACs was dose-dependent, increased over time and was blocked by either the clatharin inhibitor Dyngo4a or the proteasome inhibitor MG132. In the case of HER2 for which there are FDA-approved anti-HER2 ADCs, the cytotoxicity of trastuzumab emtansine (TDM1) significantly increased with the addition of a HER2-targeting PROTAC across a range of doses. PROTACs that target the same oncogenic cell surface proteins as antibodies improve antibody internalization and ADC cytotoxicity, in a process dependent upon clatharin and the proteasome. This novel application of PROTACs may impact the use of ADCs, and provides a rationale to combine these agents in clinical trials. Citation Format: Ezequiel J. Tolosa, Lin Yang, Jennifer Ayers-Ringler, Jayapal Mallareddy, Janet Schaefer-Klein, Farhad Kosari, Amar Natarajan, Aaron S. Mansfield. Proteolysis targeting chimera (PROTAC)-driven internalization of antibodies targeting oncogenic cell surface receptors [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 4518.

Background Flow diverters (FDs) are an effective treatment for intracranial aneurysms, though not free from hemorrhagic complications. A previous study demonstrated increased vascular contractility after FD-implantation as a potential mechanism of distal complications. Our study aimed to investigate whether L-arginine medication affects vascular contractility following FD deployment in a rabbit model. Methods FDs were implanted in the aorta of normal rabbits (+FD, n = 10), with sham-operated aorta as controls (n = 5). L-Arginine was given in the drinking water (2.25% L-arginine hydrochloride) of half of the +FD animals (+FD/+Arg). Force contraction vascular contractility studies were performed on the aortic rings proximal and distal to the FD using an organ bath. Total eNOS, eNOS(pS1177), eNOS(pT495), COX-2, and S100A4 were quantified by western analysis on total protein lysates from aortic segments, normalizing to GAPDH. Results Mean vascular contractility was 53% higher in distal relative to proximal aortic segments (P = 0.0038) in +FD animals, but were not significantly different in +FD/+Arg animals, or in sham-operated controls. The +FD animals expressed significantly reduced levels of eNOS(pS1177) than sham-operated controls (P = 0.0335), while both the +FD and +FD/+Arg groups had reduced levels of eNOS(pT495) relative to sham-operated controls (P = 0.0331 and P = 0.0311, respectively). Conclusion These results suggest that L-arginine medication reduces distal vascular contractility after FD treatment via nitric oxide production and thus might mitigate risk for downstream complications.