Citations

Abstract Glioblastoma (GBM) remains the most lethal primary brain cancer due to its remarkable metabolic plasticity and therapeutic resistance. Here, we identify cholesterol dependency as a therapeutically exploitable vulnerability in GBM using two FDA-approved drugs: the H1 histamine antagonist clemastine and the retinoid X receptor agonist bexarotene. Combined treatment induces potent synergistic anti-tumor activity across patient-derived glioma models, suppressing proliferation, stemness, and survival at sub-IC50 concentrations. Mechanistically, this therapy disrupts cholesterol biosynthesis, transport, and homeostasis, triggering endoplasmic reticulum stress and activation of the unfolded protein response, ultimately leading to autophagy and apoptotic cell death. Orthotopic patient-derived glioma models recapitulate these mechanisms in vivo, where local intracranial administration significantly reduces tumor progression and prolongs survival using fourfold lower doses than systemic intraperitoneal delivery. Single-cell RNA sequencing revealed activation of regeneration and plasticity programs, accompanied by immune microenvironment remodeling and enhanced inflammatory signaling. Importantly, syngeneic models preserved immune cell composition, supporting future integration with immunotherapeutic strategies. Together, these findings establish cholesterol dysregulation–induced metabolic collapse as a promising therapeutic approach for GBM.

Background: A diagnosis of cancer creates a stressful situation owing to the uncertainties related to the thought of possible suffering from complications associated with treatment toxicities, the fear of not responding to the prescribed treatment, or death. The prevalence of mental health disorders is relatively higher in cancer patients vis-à-vis the general population. Methods: We performed a retrospective cohort study to evaluate the impact of mental health disorders on outcomes of relapsed and/or refractory diffuse large B-cell lymphoma (DLBCL) treated with chimeric antigen receptor T-cell (CAR T-cell) therapy using data from the TriNetX research network. Results: A total of 1587 patients met the inclusion criteria (858 with a mental health diagnosis and 729 without). After propensity score matching for age and gender, 1320 patients were included in the analysis (660 in each group). The 1-year OS was higher in patients without a mental health disorder (76.0% versus 69.4%, P = 0.004). Patients with mental health disorders had a higher incidence of cytokine release syndrome (all grades) compared with those without (35.5% versus 27.6%, P < 0.001) and immune effector cell-associated neurotoxicity syndrome (all grades) (16.8% versus 10.1%, P < 0.001). Conclusions: These findings emphasize the importance of thorough psychiatric evaluation and management in patients receiving CAR T-cell therapy. Patients with mental health disorders may need significant assistance with coordinating medical services to better adhere to required care management. Future studies are needed to help determine whether preventive and real-time mental health interventions can favorably impact outcomes of these patients.

Abstract Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of B-cell malignancies. Despite the clinical success of CD19-targeted approaches, relapsed/refractory (R/R) disease—often driven by antigen loss or immune escape—remains a major challenge. To overcome this, we developed a novel CAR-T cell therapy targeting BAFF-R, a receptor consistently expressed in chronic lymphocytic leukemia (CLL) and other B-cell malignancies. We developed a new anti-BAFF-R monoclonal antibody and engineered the MC10029 CAR construct using its scFv. Antigen-specific cytotoxicity was validated in both in vitro and in vivo models, including Nalm-6 (leukemia) and Z138 (lymphoma), and CD19-knockout tumor cells to model (R/R) disease. MC10029 CAR-T cells were generated from healthy donors and CLL patients, and their cytotoxicity was evaluated against MEC-1, a CLL cell line, as well as autologous CLL patient tumor samples. These studies demonstrated potent and specific antitumor efficacy. We transitioned to clinical-grade production, using GMP-compliant lentivirus to manufacture MC10029 CAR-T cells under regulatory guidelines. With FDA IND approval, we initiated a Phase 1 clinical trial to evaluate safety and dosing. Our first patient, treated with a low-dose, single infusion, achieved a complete metabolic response within 2 months. The therapy is well tolerated, with no severe advent events such as cytokine release syndrome or neurotoxicity, supporting both the safety and efficacy of the therapy. Conclusion: This study represents Mayo Clinic’s first homegrown CAR-T cell therapy, developed entirely in-house—from antibody discovery and CAR design to GMP manufacturing and clinical trial—marking a significant step toward personalized, next-generation immunotherapy for B-cell malignancies. Citation Format: Yan Luo, Shaohua Guo, Yaqing Qie, Martha E. Gadd, Tanya Hundal, Hemant S. Murthy, Mohamed A. Kharfan-Dabaja, Hong Qin. From gene to bedside: Mayo Clinic’s first-in-house BAFF-R CAR-T [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 3719.

Abstract Several allogeneic CAR T-cell therapies are in clinical trials to evaluate safety, many of which rely on CRISPR/Cas-based genome editing. However, the Cas enzyme’s random repair mechanism elevates the risk of undesired, off-target effects, posing a challenge for safe allogeneic CAR T-cell generation. To mitigate the risk of therapy-related oncogenesis, we sought to pursue an alternative safe strategy to manufacture allogeneic CAR T-cells distinct from the preexisting ones in the clinic. We have developed a CRISPR RNA (crRNA) targeting T-cell receptor beta constant (TRBC) gene, which in conjunction with AsCas12a Ultra enzyme, causes site-specific editing by eliciting a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, which mitigates off-targeting risk. Targeted amplicon sequencing was employed to evaluate off-targeting and repair mechanisms. Graft versus host alloreactivity as well as CAR T persistence was evaluated using immunocompromised mice. In vivo tumor challenge study was conducted for over 3 months. Engraftment confirmation was done using immunohistochemistry and flow cytometry. Interestingly while evaluating the edited T-cells, we sequestered a unique T-cell population that showed TCR disruption yet remained CD3-positive and did not cause in vivo alloreactivity. These CD3-Retained, Allogeneically Functioning T-cells (CRAFT-cells) showed a similar growth rate as unedited T-cells and were then used as a platform to produce CD19-targeted CAR T-cells and BAFF-R-targeted CAR T-cells. When compared to the standard CD3-disrupted allogeneic CAR T-cells, CRAFT CAR T-cells displayed an equally robust cytotoxicity while possessing a safer genomic profile. CRAFT CAR T-cells could function as effector cells for bispecific T-cell engager (BiTE) and induce CD3-dependent cytotoxicity against tumor cells. Pertinently, CRAFT CAR T-cells as opposed to CD3-disrupted CAR T-cells, were present in mouse xenografts without alloreactivity. Having the onset of MMEJ-directed repair and minimal off-targeting, positions this CRAFT crRNA to generate safer allogeneic CAR T-cells with an overall reduced genotoxic profile in a clinical setting. This unique CRAFT CAR T-cell population displays in vivo persistence. Additionally, CRAFT CAR T can be combined with BiTE therapy, offering a promising and potentially more durable alternative to preexisting ex vivo allogeneic CAR T-cell therapies. Citation Format: Tanya Hundal, Yan Luo, Yaqing Qie, Martha E. Gadd, Shaohua Guo, Mohamed A. Kharfan-Dabaja, Hong Qin. CD3-positive allogeneic CAR T-cells: A novel platform with persistence, BiTE-capture, and no alloreactivity [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 4283.

Immunosuppression within the tumor microenvironment (TME) poses a significant challenge to the efficacy of Chimeric Antigen Receptor (CAR) T-cell therapy in solid tumors, including glioblastoma (GBM), the most common and deadly primary brain tumor. Tumor and TME-residing immunosuppressive cells can hijack program death 1(PD1) pathway to evade antitumor T-cell immunity by upregulating its ligand, PD-L1. Thus, targeting PD-L1 emerges as a promising therapeutic strategy to simultaneously eliminate tumor cells and immunosuppressive components within the TME. We developed a novel PD-L1-targeting CAR T-cell therapy, MC9999, and investigated its therapeutic effects against GBM. The antigen-specific cytotoxicity of MC9999 CAR T-cells was firmly validated using a LN229 GBM cell line and its PD-L1-deficient variant, as well as patient-derived brain tumor-initiating cells (BTICs). Furthermore, in vivo therapeutic antitumor effects were confirmed in orthotopic GBM models through intracranial administration of MC9999 CAR T-cells. To pave the way to the translation of MC9999 CAR T-cell therapy to patient bedside, we established a unique experimental system using patient-specific CAR T-cells against their own tumor and immunosuppressive cells. High-quality, patient-specific MC9999 CAR T-cells were manufactured using T cells isolated from peripheral blood, while paired autologous BTICs were generated to serve as target cells. Autologous immunosuppressive cells include monocyte-derived M2 macrophages from patient’s peripheral blood and tumor-associated macrophages (TAMs) isolated from patient’s tumor. Data from multiple patients’ samples consistently demonstrated that these patient-specific MC9999 CAR T-cells elicited robust cytotoxicity against autologous GBM tumor and TME-residing immunosuppressive cells, supporting our hypothesis that targeting PD-L1 effectively address both tumor and its TME. Immunohistochemistry (IHC) staining of our GBM patients’ tissues showed that PD-L1 expression was restricted to tumor cells and macrophages /microglia within the tumor, with no expression detected in neurons, astrocytes, oligodendrocytes, or blood vessels. These findings suggest that MC9999 CAR T-cells can be safely delivered to the brain without compromising normal cell populations. Ongoing studies are underway to further validate the safety profile of MC9999 CAR T-cell therapy. Collectively, the data obtained from this preclinical study support the effectiveness, feasibility and safety of local administration of MC9999 CAR T-cells as a promising new approach for GBM treatment. These encouraging results have motivated us to prepare an Investigational New Drug application to initiate a phase I clinical trial. Citation Format: Yan Luo, Yaqing Qie, Maria J. Ulloa-Navas, Jesus E. Sanchez-Garavito, Vanessa K. Jones, Martha E. Gadd, Mieu M. Brooks, Tanya Hundal, Andrew D. Brim, Isas K. Vazquez-Rosario, Haidong Dong, Alfredo Quinones-Hinojosa, Hong Qin. Intracranial delivery of MC9999 CAR T-cell therapy: a novel strategy to simultaneously eliminate GBM tumors and immunosuppressive components in the tumor microenvironment [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 4812.

AbstractThe challenge of disease relapsed/refractory (R/R) remains a therapeutic hurdle in chimeric antigen receptor (CAR) T‐cell therapy, especially for hematological diseases, with chronic lymphocytic leukemia (CLL) being particularly resistant to CD19 CAR T cells. Currently, there is no approved CAR T‐cell therapy for CLL patients. In this study, we aimed to address this unmet medical need by choosing the B‐cell activating factor receptor (BAFF‐R) as a promising target for CAR design against CLL. BAFF‐R is essential for B‐cell survival and is consistently expressed on CLL tumors. Our research discovered that BAFF‐R CAR T‐cell therapy exerted the cytotoxic effects on both CLL cell lines and primary B cells derived from CLL patients. In addition, the CAR T cells exhibited cytotoxicity against CD19‐knockout CLL cells that are resistant to CD19 CAR T therapy. Furthermore, we were able to generate BAFF‐R CAR T cells from small blood samples collected from CLL patients and then demonstrated the cytotoxic effects of these patient‐derived CAR T cells against autologous tumor cells. Given these promising results, BAFF‐R CAR T‐cell therapy has the potential to meet the long‐standing need for an effective treatment on CLL patients.

Abstract Despite its success in treating blood cancers, chimeric antigen receptor (CAR) T-cell therapy has not yielded anticipated clinical outcomes in patients with solid tumors. This discrepancy is primarily attributed to the lack of dependable antigens and the immunosuppressive nature of the tumor microenvironment (TME). A pivotal evasion mechanism employed by tumor to counter antitumor immunity involves the upregulation of programmed death ligand 1 (PD-L1), which interacts with PD1 on T cells, deactivating effector T cells. A variety of immunosuppressive cells within the TME employ similar mechanisms to inhibit T-cell functions. These findings provide a rationale for targeting PD-L1 in both tumors and the immunosuppressive cells. Using a humanized anti-PD-L1 monoclonal antibody, we engineered a second-generation PD-L1 CAR, namely MC9999. MC9999 CAR T-cells exhibited antigen-specific cytotoxicity in vitro and elicited potent in vivo antitumor effects against PD-L1-expressing MDA-MD-232 breast cancer cells. The effectiveness of MC9999 CAR T-cells was confirmed across three other solid tumor models: non-small cell lung cancer, melanoma and glioblastoma (GBM). Notably, intravenous administration of MC9999 CAR T-cells eradicated intracranially established LN229 GBM tumors, suggesting the potential to penetrate the blood-brain barrier. In addition to targeting tumor cells, MC9999 CAR T-cells exhibited cytotoxicity against three immunosuppressive cell models, including HMC3 microglia cells, M2 macrophages, and importantly, primary tumor-associated macrophages (TAMs) isolated from GBM. Furthermore, we engineered patient-derived MC9999 CAR T-cells and demonstrated their effectiveness against two primary tumor cell lines derived from GBM tumors and HMC3 cells. Collectively, these proof-of-principle study findings substantiate the viability of targeting PD-L1 against both tumors and their immunosuppressive microenvironment. This prototype MC9999 CAR lays the groundwork for further development of clinically applicable CAR T-cell therapy against solid tumors. Citation Format: Yaqing Qie, Emiliano Sanchez Garavito, Maria J. Ulloa Navas, Tanya Hundal, Alfredo Quinones-Hinojosa, Hong Qin, Yan Luo. Weaponizing CAR T-cells to attack PD-L1 presenting cells in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB071.