Bispecific CD33-TIM3 CAR T cells enhance specificity while maintaining efficacy against AML

Wang, Yingshuai (2025): Bispecific CD33-TIM3 CAR T cells enhance specificity while maintaining efficacy against AML. Dissertation, LMU München: Medizinische Fakultät

Abstract

Background: CD33, a specific antigen prevalent on myeloid cells, is found in 88% of samples from patients with acute myeloid leukemia (AML), highlighting its potential as an immunotherapeutic target. Despite numerous ongoing clinical trials targeting AML, challenges related to CAR T-cell specificity—termed "on-target off-leukemia"—continue to present significant hurdles. In response, this research focuses on the development of dual CAR T cells that concurrently target CD33 and TIM3. TIM3 has been identified on leukemic stem cells (LSCs) and is absent on normal hematopoietic cells. Additionally, the role of TIM3 in impeding immune regulation underscores its suitability as a secondary target in AML immunotherapy strategies. The objective of this investigation is to construct and evaluate dual CAR T cells targeting CD33 and TIM3 to enhance the specificity and maintain the anti-leukemic efficacy of these cells. Methods: This study initiated by producing anti-TIM3 antibodies using hybridoma technology. These antibodies were then validated for their specificity through enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS). DNA sequencing was performed of anti-TIM3 single-chain variable fragments (scFv) derived from these hybridomas. The structural interactions of CD33 and TIM3 scFvs with each antigen were modeled using AlphaFold2, referencing the TIM3 structure (PDB ID: 5F71) and CD33 structure (PDB ID: 6D48). The CD33 scFv was derived from gemtuzumab ozogamicin (clone hP67.6) and both incorporated along with costimulatory domains (CD28 or 4-1BB) into a pMP71 vector. A retroviral production system utilizing 293Vec-GALV and RD114 cells facilitated the production of the retroviruses. In vitro, the cytotoxicity of the CAR-T cells was tested against both wild-type and TIM3-transduced AML cell lines (THP-1 and OCI-AML3) through co-culture assays by multiparametric flow cytometry (MPFC). Cytokine release assays (CBA) were used to measure IFN-γ and IL-2 secretion, and cell-target avidity was analyzed using a Z-Movie analyzer. Off-target effect was monitored through colony-forming unit (CFU) assay on CD34+ cells from healthy donors after 14 days. For long-term efficacy assessments, CAR-T cells were periodically re-stimulated every four days by co-culturing with irradiated TIM3 expressing SKM-1 cells at a 1:1 effector-to-target (E:T) ratio over 24 days. During these intervals, analyses of CAR-T cell proliferation, checkpoint marker expression, and T cell subset differentiation were conducted via MPFC. Conclusion: This study successfully generated dual-target CAR T cells utilizing both "AND" and "OR" gating strategies to target CD33 and TIM3, enhancing their efficacy to AML cells in vitro. The findings demonstrated that these CAR T cells exhibit superior binding avidity and cytotoxic capabilities towards cells expressing both CD33 and TIM3 antigens, as opposed to targeting a single antigen. Notably, the split CAR T cell approach effectively eradicated CD33+TIM3+ cell lines and primary AML cells, while minimizing impact on healthy hematopoietic cells. The implications of these results suggest that dual CAR T cell configurations might serve as potential bridging therapies before hematopoietic stem cell transplantation. The split CAR T cell particularly offered a potential safer alternative, possibly obviating the need for allogeneic stem cell transplantation due to on-target-off-leukemia toxicity. These advancements have the potential to markedly alter the therapeutic landscape for AML, offering more precise, effective, and safer options for treatment.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: CD33, TIM-3, Dual CAR T cells, Acute Myeloid Leukemia, Split/Tandem/Compound CAR T cells
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 29. August 2025
• 1. Bericht­erstatter:in: Subklewe, Marion