Gadd, Martha E.

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.

While acknowledging that newer therapies have improved survival rates in chronic lymphocytic leukemia (CLL), patients with high-risk disease features are at an increased risk of treatment failure. Allogeneic hematopoietic cell transplantation (allo-HCT) was traditionally offered as front-line consolidation in high-risk CLL; however, with the emergence of targeted therapies like Bruton tyrosine kinase (BTK) and B-cell lymphoma 2 (BCL-2) inhibitors, the role of allo-HCT has been relegated to later stages of the disease. Patients with relapsed/refractory (R/R) CLL who have failed both BTK and BCL-2 inhibitors represent a therapeutic challenge owing to a poor prognosis. Chimeric antigen receptor T-cell (CAR T) therapies targeting CD19 have improved response rates and overall survival in various types of R/R B-cell non-Hodgkin lymphomas. For CLL, no approved CAR T-cell therapies are yet available. Emerging data appear to show a therapeutic benefit of CAR T-cell therapy in patients with R/R CLL, even after failing an allo-HCT.

e19501 Background: Relapsed and/or refractory (R/R) disease is observed in patients who receive CD19 targeting chimeric antigen receptor (CAR)-T cells to treat B-cell lymphoid leukemias and lymphomas. We generated a novel CAR, MC10029 that targets B-cell activating factor receptor (BAFF-R) to address this unmet medical need. Methods: We engineered a second-generation BAFF-R CAR (MC10029 CAR). The construct includes the single chain variable fragment (scFv) of our newly developed BAFF-R antibody with CD28 costimulatory and CD3ζ signaling domains. We confirmed MC10029 CAR-T cell antigen-specific activation with in vitro degranulation assays and a direct cytolytic assay. We used BAFF-R positive leukemia and lymphoma cell lines in both in vitro and in vivo models to further characterize MC10029 CAR-T cells, and finally, we enriched tumor primary cells from chronic lymphocytic leukemia (CLL) patients as a target for MC10029 CAR-T cells. In preparation for our Phase I clinical trial, we have manufactured three batches of CAR-T cells using a MC10029-encoding lentiviral vector produced under GMP conditions. The production batches of MC10029 CAR-T cells were evaluated for product quality with Viability, Identity and Potency as well as Safety from adventitious viral agents. Antigen specific cytotoxicity of the MC10029 CAR-T cells was confirmed using our standard degranulation assay. Results: MC10029 CAR-T cells elicited in vitro antigen-specific cytotoxicity against Nalm-6 cells, Z138 cells, MEC-1 cells, and a CD19 knock out Nalm-6 model designed to mimic CD19-negative R/R disease. We demonstrated in vivo antitumor effects of MC10029 CAR-T cells in NSG mice challenged with these same cell lines. We showed cytotoxicity of MC10029 CAR-T cells against CLL patients’ tumors. We also showed our quality control results to highlight our clinical trial trajectory. Conclusions: Novel MC10029 CAR-T cells showed broad utility against BAFF-R positive B-cell lymphoid malignancies in a series of in vitro, in vivo, and ex vivo models. With these robust data, we have moved forward to generate clinical grade MC10029 CAR-T cells using cGMP manufacturing and quality protocols in preparation of a MC10029 CAR-T cell Phase I clinical trial.

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults with CD19-targeting CAR T-cell therapy showing an excellent initial curative response. However, 10-20% of patients who receive anti-C19 CAR T-cell treatment experience relapse due to CD19 antigen loss. B-cell activating factor receptor (BAFF-R) that is expressed on the surface of normal and neoplastic B cells mediates the survival of mature peripheral B cells along with the B-cell receptor. Since B-CLL is characterized by apoptotic disruption and subsequent accumulation of mature B cells, BAFF-R is a logical, novel target for CAR T-cell therapy directed against B-CLL, especially if CD19 antigen loss occurs. In this study, we first manufactured BAFF-R CAR T-cells by infecting healthy donor Tnaïve/memory (Tn/mem) cells with BAFF-R CAR lentivirus before evaluating the antitumor activity of BAFF-R CAR T-cells on wild type MEC-1 (a B-CLL cell line), modified MEC-1, and primary cell lines. Antigen-specific cytotoxicity and activation of the BAFF-R CAR T-cells or control CD19 CAR T-cells were determined upon incubation with B-CLL cell lines and patient-derived primary B-CLL by measuring degranulation and IFN-γ release, respectively. BAFF-R CAR T-cells demonstrated substantial cytotoxicity against MEC-1, CD19-deficient MEC-1, and primary B-CLL cell lines; CD19 CAR T-cells showed no effect on CD19-deficient MEC-1 cells but did show effects with primary B-CLL and MEC-1 cells. Similarly, the release of IFN-γ was greater when BAFF-R CAR T-cells were co-cultured with CD19-deficient MEC-1 or primary B-CLL compared to CD19 CAR T-cells co-cultured with CD19-deficient MEC-1. In summary, BAFF-R CAR T-cells exert significant antitumor properties not only on patient-derived primary B-CLL cells but also on a CD19-deficient B-CLL cell line. We, therefore, assert BAFF-R-targeting CAR T-cells to be a novel therapeutic avenue for patients newly diagnosed with B-CLL and patients experiencing relapsed B-CLL after anti-CD19 therapy. Citation Format: Yan Luo, Kaihin Lui, Tommy T. To, Andrew liu, Farah Yassine, Mohamed A. Kharfan Dabaja, Martha E. Gadd, Hong Qin. Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2855.