Sakemura, Leo

Abstract BACKGROUND Glioblastoma (GBM) is a universally lethal brain cancer with limited durable responses to immunotherapy, largely due to its immunosuppressive tumor microenvironment (TME). Ephrin receptor A3 (EphA3), a receptor tyrosine kinase minimally expressed in normal tissue, is enriched on GBM cells, glioma stem cells (GSCs), and M2-like tumor-associated macrophages (TAMs). We hypothesize that dual targeting of TAMs and tumor cells by EphA3-CAR-T is a viable treatment strategy. METHODS We developed EphA3-directed second-generation CAR-T cells (EphA3-CAR-T) incorporating the single-chain variable fragment of ifabotuzumab, an anti-EphA3 monoclonal antibody, achieving >90% transduction efficiency. We evaluated antitumor efficacy in vitro; the functional activities of EphA3-CAR-T on GSCs were assessed via selective cytotoxicity, T cell proliferation, degranulation assays, and depletion of M2-like macrophages. In vivo, 1 × 10⁶ EphA3⁺ U251 MG cells and/or 1 × 10⁶ ex vivo–polarized M2-like macrophages were implanted in the flanks of NOD-SCID-γ⁻/⁻ mice. Comparative studies with EGFRvIII-directed CAR-T cells were also performed. RESULTS Immunohistochemistry confirmed EphA3 expression across both tumor and immune compartments in patient-derived specimens. EphA3-CAR-T cells demonstrated potent, selective cytotoxicity (p<0.001), T cell proliferation (p<0.001), and CD107α degranulation (p<0.001) against EphA3⁺ GBM and GSCs, while also reversing TAM-mediated immunosuppression via depletion of ex vivo–polarized M2-like macrophages (p<0.05), compared to other validated CAR-Ts. In vivo, EphA3-CAR-T significantly reduced tumor volume (p<0.001) and improved survival (p<0.01), in the presence of TAMs. In contrast, EGFRvIII CAR-T cells were inactivated under the same TME conditions. EphA3-CAR-T cells remained active in models co-engrafted with macrophages, suggesting preserved function within the immunosuppressive TME. CONCLUSIONS EphA3-CAR-T therapy addresses a major barrier to CAR-T success in GBM: TME-mediated immunosuppression. This tumor- and TME-targeting approach demonstrated robust preclinical activity. These findings support the clinical translation of EphA3-CAR-T cells as a next-generation immunotherapy for GBM.

Abstract BACKGROUND In GBM, effective chimeric antigen receptor T-cell (CART) therapy remains a challenge; immunosuppressive components within the tumor microenvironment (TME) inhibit CART cells. EphA3 is a receptor tyrosine kinase with low expression in adult tissues but is highly expressed in tumor neovasculature and tumor stromal cells in GBM and other solid tumors. METHODS We performed RNA sequencing and Immunohistochemical staining for of EphA3 in a clinically annotated GBM PDX biobank and M01 M1 and M2 polarized macrophages. We then investigated the suppressive capability of M2 macrophage on EphA3-CART in an SNB-19 GBM cell co-culture. We tested the antiantitumor potential of EphA3-CART against luciferase+ SNB-19 GBM cells in vivo with flank and orthotopically engrafted NOD-SCID-ɣ-/- (NSG) mice. Engraftment was confirmed by bioluminescent imaging (BLI) and vernier caliber (flank) for 7-14 days post-injection. Mice were randomized to be treated with EphA3-CART or untransduced T-cells (UTD). Subsequently tumor size and mouse survival was monitored to determine the antitumor effect. RESULTS EphA3 was highly expressed on GBM PDX models as well as M2 macrophages. Compared to M0 and M1 macrophages, M2 macrophages significantly suppressed EphA3-CART However, despite the suppressive activity of the M2 macrophage, EphA3-CART exhibited significant antitumor activity . In both flank and orthotopically implanted mouse models, there was an observed reduction in tumor volume with EphA3-CART treated model compared to the UTD counterpart. EphA3-CART treatment resulted in significantly improved survival of the treated models compared to UTD models (hazard ratio = 0.053 **p = 0.005). CONCLUSION We demonstrate that EphA3 is expressed on GBM cells as well as on M2 macrophages. EphA3 CART is able to overcome and ameliorate the effect of a major component of the tumor microenvironment (TME), M2 macrophages. EphA3 CART appears effective in flank and orthotopic mouse models of GBM.

Abstract Thyroid cancer is the most common endocrine cancer in the US, and its incidence is rising. Most thyroid cancer deaths are attributed to treatment-refractory, metastatic tumors. Thyroid stimulating hormone receptor (TSRH) expression is largely limited to the thyroid gland and is abundantly expressed on thyroid tumor cells, making TSRH a compelling target for advanced thyroid cancer diagnostics and therapeutics. Therefore, we developed a novel TSHR-targeted chimeric antigen receptor (CAR) T cell therapy to treat aggressive thyroid cancers. TSHR-CAR constructs were cloned into a lentiviral CAR construct containing 4-1BB and CD3ζ. First, we demonstrated potent TSHR-CART antigen-specific anti-tumor activity in vitro. Then, NOD-SCID-γ-/- (NSG) mice were inoculated subcutaneously with TSHR+ tumor cells and randomized by tumor volume to treatment with TSHR-CART cells or control Untransduced T cells (UTD). Treatment with TSHR-CART cells resulted in dose-dependent antitumor activity and prolonged survival. De-differentiated anaplastic thyroid cancers (ATC) downregulate TSHR. Our TSHR immunohistochemistry results corroborated these findings and displayed minimal TSHR protein expression, precluding successful TSHR-CART treatment. We therefore sought to sensitize these tumors with MAPK inhibitors, as a strategy to upregulate TSHR expression in patients with metastatic thyroid cancer. TSHR expression was upregulated in patient-derived xenograft (PDX) ATC models after one week of daily administration of the MAPK inhibitors (p=0.0024). After confirming that MAPK inhibition does not dampen TSHR-CART effector functions, we tested sequential and combination therapy of TSHR-CART with MEK and BRAF inhibition in vivo. NSG mice were engrafted with ATC BRAF-mutant PDX tumors and randomized by tumor volume to daily oral treatment with placebo or trametinib (MEK inhibitor) plus dabrafenib (BRAF inhibitor). One week later, mice received either UTD or TSHR-CART. Mice conditioned with trametinib plus dabrafenib (p=0.0018) and subsequently treated with TSHR-CART showed superior antitumor activity. However, the improved antitumor activity in this setting was transient. We therefore tested the durability of TSHR upregulation following MEK/BRAF inhibition and demonstrated that TSHR upregulation lasts less than 48-72 hours after discontinuation. Finally, we tested the combination of TSHR CART cells with MEK/BRAF inhibitors in ATC BRAF-mutant PDX tumors. Here, combining TSHR-CART cells with MEK/BRAF inhibitors result in durable control of the tumors. Collectively, our findings indicate that MEK/BRAF inhibition of de-differentiated thyroid cancers upregulated TSHR expression and enhanced TSHR-CART antitumor activity. This work represents a viable strategy to improve outcomes of patients with aggressive, metastatic thyroid cancers. Citation Format: Claudia Manriquez Roman, Kendall J. Schick, Justyna J. Gleba, Truc N. Huynh, Elizabeth L. Siegler, James L. Miller, Aylin Alasonyalilar Demirer, Matthew L. Pawlush, Ahmet Biligili, Long K. Mai, Erin Tapper, Leo R. Sakemura, Michelle J. Cox, Carli M. Stewart, Ismail Can, Ekene J. Ogbodo, Gaofeng Cui, Georges Mer, Gloria R. Olivier, Yushi Qiu, Robert C. Smallridge, Zubair C. Abba, Han W. Tun, John A. Copland, Saad S. Kenderian. Addition of MAPK inhibitors to prime and sensitize poorly differentiated thyroid cancers as a strategy to improve TSHR-CART cell therapy antitumor activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5074.