In silico design of degron tags for precise modulation of CAR T-cell activity using clinically utilized drugs

Abstract

Abstract Introduction Over the past decade, Chimeric Antigen Receptor (CAR) T-cell therapies have transformed the treatment landscape for hematologic malignancies, demonstrating remarkable efficacy in conditions such as B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM). However, immune effector cell-associated hematotoxicity (ICAHT) has emerged as a distinct and frequent complication, characterized by early or late-onset cytopenias that are often prolonged. These cytopenias are associated with high infection risk, transfusion dependency, and non-relapse mortality. Once CAR T cells are administered, their intrinsic function can no longer be directly controlled, leaving clinicians reliant on adjunctive pharmacological agents to modulate their effects. Degron tags have been developed to enable small molecule-medaited targeted protein degradation, offering a potential strategy for precise control of cellular therapies. While auxin-based or dTAG degrons have shown promise in preclinical models, challenges such as the large size of the tags, difficulties in genome editing, and the high cost of small molecules for in vivo degradation remain significant barriers. This study aimed to design and validate novel degron tags with improved efficiency and applicability for CAR T-cell modulation utilizing IMiD compounds in clinical use. Methods Computational approaches were utilized to design proteins capable of binding to the E3 ubiquitin ligase complex CRL4CRBN in a compound-dependent manner. The degron scaffolds were subsequently screened using next-generation sequencing (NGS) assays to identify candidate sequences. These sequences were cloned into a reporter cell line and tested against their native peptide counterparts to determine IC50 values for protein degradation. Additionally, selected degron candidates were evaluated in an in vitro CD19-CAR T-cell killing assay to assess their ability to modulate CAR T-cell function in a dose-dependent manner. Results A total of 1,214 peptide sequences were computationally designed for targeted protein degradation and subsequently screened using next-generation sequencing (NGS). From this, 12 unique degron sequences were identified and selected for further evaluation. Among these, 3 novel degrons demonstrated significantly lower IC50 values for protein degradation compared to the native peptide when tested with third- and next-generation IMiDs, indicating enhanced potency. Functional assays revealed that the designed degrons significantly inhibited CAR T-cell-induced tumor cell death in a concentration-dependent manner, correlating with CAR protein degradation. Notably, Mezigomide and Avadomide provided the most stable inhibition at the lowest concentrations. Additionally, we observed that specific degrons exhibited selective inhibition of CAR function depending on the IMiD used, with the degree of inhibition correlating with the extent of protein degradation. These findings underscore the potential of our degrons to precisely regulate CAR T-cell activity, mitigating excessive cytotoxicity while maintaining therapeutic efficacy. ConclusionThis study presents a novel approach to modulating CAR T-cell function through the development of highly efficient degron tags. By enabling precise control of cellular therapies, these degrons address a critical need for managing ICAHT and optimizing therapeutic outcomes. Future investigations will focus on in vivo validation and the potential translation of this technology to clinical applications, paving the way for safer and more controllable cellular therapies.