Citations

Abstract Gastrointestinal (GI) cancers are rising in people < 50 and remain major causes of cancer mortality despite therapeutic advances. Chimeric antigen receptor T (CART) cell therapy has transformed treatment of hematological cancers. However, its efficacy in GI cancers has been hindered by antigen heterogeneity and immunosuppressive cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME.) Here we aimed to 1) study CART-CAF interactions and 2) engineer CART cells capable of eliminating CAFs to overcome their inhibitory role and enhance their antitumor activity in GI cancers. We first analyzed primary CAFs from 20 GI cancer patients. CAFs showed high fibroblast activation protein (FAP) expression and significantly suppressed T cells proliferation (26-77%, p≤0.0001). We next generated CART cells capable of safely eliminating both GI cancer cells and CAFs. To target cancer cells, we developed a novel nanobody (VHH)-based CAR against Claudin18.2, a GI cancer specific target. To avoid off-tumor toxicity in healthy FAP+ tissues, we incorporated an inducible FAP-CAR using the synNotch platform, enabling conditional targeting upon engagement with Claudin18.2+ cells, restricting FAP targeting within the TME. VHH Claudin18.2 CART cells showed superior specificity and efficacy vs scFv-based CART cells (hu8E85 or zolbetuximab). In Claudin18.2+ SNU-C2B xenografts, tumors were 92mm3 (VHH) vs 48mm3 (huE85) vs 211mm3 (zolbetuximab) on day 23 post-CART, median survival undefined vs 17 days vs 23 days respectively; p=0005. In Claudin18.2+ LS411N xenografts, tumors were 315mm3 vs 106mm3 vs 382 mm3 respectively at 23 days post-CART, median survival undefined vs 19 days vs undefined respectively; p=0004. VHH Claudin18.2 CART cells showed no off-tumor effects, while hu8E85 CART induced significant toxicity. We next established a co-engraftment model. NSG mice received 1x106 SNU-C2B and 6x106 CAFs from a GI cancer patient. At 500 mm3 tumors, mice received 5x106 VHH Claudin18.2 or inducible Claudin18.2/FAP CART. Dual-targeting CART cells showed greater antitumor response (728mm3 vs 320mm3, p=0.0011 at day 20 post-CART), expansion (61 vs 2,350 CART/µl of blood, p=0.0037), and tumor infiltration (8.71 x 105 vs 1.03 x 106 CART/g of tumor, p<0.0001). This was also observed in gastric, colon, and pancreatic PDX models, with no observable off-tumor cytotoxic effects with inducible dual-targeting Claudin18.2/FAP CART. Our findings demonstrate that our novel VHH-based Claudin18.2 CART cells are highly specific and effective against GI malignancies, and that inducible Claudin18.2/FAP dual-targeting CART cells safely remodel the TME into an immune-permissive state. This approach represents a mechanistically innovative and translationally relevant strategy to overcome CAF-mediated immunosuppression, enhance CART cell efficacy, and improve clinical outcomes in refractory solid tumors. Citation Format: Omar L. Gutierrez Ruiz, Jennifer Feigin, Brooke Kimball, R. Leo Sakemura, Elizabeth L. Siegler, Dominic A. Skeele, Ateka Saleh, Claudia Manriquez Roman, Ismail Can, Olivia Sirpilla, Kun Yun, Carli M. Stewart, Mehrdad Hefazi, Yoh Yamaguchi, Lionel Aurelien Kankeu Fonkoua, Long K. Mai, Truc Huynh, Sophia Y. Goldberg, Grace E. DeFranco, Sara Foote, Ekene Johnkennedy Newton Ogbodo, Hong Xia, Michael Redig, Matthew L. Matthew, Tamiel Nichole Turley, Skyeler Klinge, John Alton Copland, Saad J. Kenderian. Optimized CART cell therapy to remodel the tumor microenvironment and eliminate GI malignancies [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 1525.

Abstract Background Chimeric antigen receptor T cell (CART) therapy has revolutionized treatment for hematologic malignancies. However, durable remission following anti-CD19 CART therapy (CART19) remains limited to 40–50% of patients, often due to tumor relapse and CART dysfunction (PMID:31501612). We previously demonstrated that tumor-derived extracellular vesicles (EVs) from patients with chronic lymphocytic leukemia (CLL) can directly impair CART function (PMID:33388419). In this study, we aimed to identify tumor-derived EV microRNA (miRNA) cargo associated with clinical CART failure in B cell malignancies and define how they mechanistically drive CART cell dysfunction. Methods To achieve these aims, we profiled tumor-derived EV miRNA cargo using small RNA sequencing (RNAseq) in two independent patient cohorts. Cohort 1 compared plasma-derived EVs from untreated CLL patients to those from healthy donor EVs to identify miRNAs enriched in CLL (n=23). Cohort 2 included patients with diffuse large B cell lymphoma (DLBCL) treated with axicabtagene ciloleucel (CART19, n=12). Here, plasma EV miRNAs were analyzed at baseline (pre-CART treatment) and 1-month post-CART treatment and compared between CART responders (complete remission at 6 months) and nonresponders (no response at 1 month). Enriched miRNAs from both cohorts were identified following miRNA count normalization and differential expression analysis using adjusted p values of ≤0.05. Functional studies were conducted by transfecting CART19-28ζ cells with synthetic miRNA mimics or scrambled controls. CART cells were also generated with stable overexpression of miRNA via lentiviral shRNA delivery. CART function was assessed via in vitro killing, proliferation, apoptosis assays, RNAseq, and in vivo tumor xenograft models. Results In Cohort 1, we identified several miRNAs including miR-125a to be significantly enriched in CLL-derived EVs relative to healthy controls. Similarly, in Cohort 2, miRNA-125a was also identified among the miRNAs enriched in nonresponders vs. responders to CART treatment; Notably, miR-125a was significantly enriched in nonresponders at baseline (pre-CART treatment) and 1-month post-CART treatment (p=0.0177, p=0.0196). To define the functional effects of miR-125a, we delivered synthetic mimics or stable shRNA constructs encoding miR-125a into CART cells. Delivery of miR-125a was confirmed by RT-qPCR (p<0.0001 for mimic vs. scramble, p=0.0069 for shRNA vs. scramble). Here, miR-125a delivery and overexpression impaired CART cytotoxicity/killing of target tumor cells (~25% reduction, p= 0.0039), decreased antigen-specific proliferation (~47% reduction, p=0.0002), and increased apoptosis following antigen stimulation (1.8-fold increase, p=0.0002) as compared to scramble controls in vitro. We next studied how miR-125a impacts CART functions in vivo. Here, tumor-bearing mice treated with miR-125a–overexpressing CART cells (CART sh125a) exhibited accelerated tumor progression (4.1-fold increase, p=0.0019) and significantly reduced survival as compared to mice treated with scramble control CART treatment (CART shScr) (20% vs 80% survival at day 35, p=0.0064), underscoring the dramatic detrimental impact of miR-125a on therapeutic efficacy. To uncover changes in CART gene expression induced by miR-125a delivery, RNAseq was also performed across stimulated CART sh125a vs. CART shScr cells. CART sh125a cells demonstrated broad transcriptional changes, with gene set enrichment of E2F targets, G2/M checkpoint, and apoptosis pathways (FDR p<0.0001, p<0.0001, and p=0.006) further validating the role of miR-125a in disrupting CART cell cycle control, proliferation, and survival. Conclusion In summary, our study identifies miR-125a as a tumor-derived EV cargo that drives CART dysfunction in two separate B cell malignancies (CLL and DLBCL). miR-125a was enriched in both CLL EVs and nonresponders to CART19 therapy at baseline prior and post CART treatment. Functional studies demonstrate that miR-125a delivery and overexpression directly impair CART cytotoxicity and proliferation, increase CART apoptosis, reduce therapeutic efficacy in tumor xenograft models, and disrupt key pathways related to CART cell cycle regulation and survival. These findings position miR-125a as both a key mechanistic driver of CART failure and a potential predictive biomarker for treatment response, while highlighting it as a possible therapeutic target to improve CART outcomes in the clinic.

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 In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

Abstract The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)–cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)–cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

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.

Abstract Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.