Biology subjects

Rodriguez-Diaz, S.

Publications and source records attributed to Rodriguez-Diaz, S..

3 recordsLinked to original sources

bioRxiv2026

Rational engineering of sdAb-based CAR T cells targeting BCMA enhances antitumor efficacy and persistence in Multiple Myeloma

BCMA-directed CAR T therapies have transformed the treatment of relapsed/refractory multiple myeloma (MM), yet durable responses remain limited by insufficient persistence and functional exhaustion. The contribution of antigen-binding domain properties to CAR T cell performance is not fully defined. Here, we report the rational discovery and engineering of BCMA-targeting single-domain antibodies (sdAbs) and their incorporation into second-generation CAR T cells. We generated sdAbs recognizing distinct BCMA epitopes with diverse binding kinetics. Comprehensive biophysical and functional characterization identified sdAb5 as a lead candidate with balanced binding kinetics and a non-overlapping epitope relative to clinically approved constructs. sdAb-based CAR T cells exhibited potent antigen-specific cytotoxicity, minimal tonic signaling, and preserved IL-2 production without excessive inflammatory activation. In xenograft models, sdAb5-based CAR T cells induced durable tumor control, enhanced persistence, and improved responses to tumor rechallenge compared with ide-cel and cilta-cel constructs. Notably, this superior functionality correlated with balanced binding kinetics rather than maximal affinity. Single-cell transcriptomic analyses corroborated these findings, revealing a restrained inflammatory activation in sdAb5-based CAR T cells with preservation of transcriptional plasticity. Collectively, these findings support antigen-binding optimization as a key determinant of CAR T-cell durability and identify sdAb5 as a candidate for next-generation BCMA-directed therapies in MM.

bioengineering↗

bioRxiv2026

Rapid and dynamic reprogramming within the tumor microenvironment drives EDA-CAR-T dysfunction and compromised therapeutic efficacy in solid tumors

BackgroundChimeric antigen receptor (CAR)-T cell therapies efficacy in solid tumors remains limited, largely due to the profoundly immunosuppressive tumor microenvironment (TME) which drives CAR-T cells to dysfunction and poor persistence. A comprehensive understanding of the dynamic interplay between CAR-T cells and the TME is therefore critical for the rational design of more effective CAR-T strategies for solid cancers. MethodsHere, we performed single-cell RNA sequencing of tumor samples from immunocompetent mice treated with stroma-targeting EDA-CAR-T cells, profiling CAR-T cell states and TME programs at the peak of antitumor response and during subsequent tumor progression. ResultsOur analysis revealed a marked temporal remodeling of EDA-CAR-T cells within the TME, where early antitumor efficacy is associated with concurrent expansion of cytotoxic effector CD8 CAR-T cells and activation of memory CD4 CAR-T subsets. Moreover, EDA-CAR-T cells effectively engaged the myeloid compartment, resulting in strengthened communication networks involving T cell activation. However, by tumor progression, EDA-CAR-T cells suffered a widespread transcriptional reprogramming towards dysfunction, characterized by loss of effector programs alongside induction of exhaustion and immunoregulatory pathways within the TME, including PD-L1/PD-L2 and TGF{beta} signaling, which impairs sustained immune responses. Notably, early CAR-T cell activation led to increased susceptibility to TME-mediated immunosuppression, revealing EDA-CAR-T-specific soluble galectin-mediated cell-to-cell interaction networks. ConclusionsTogether, this works offers a high-resolution view of CAR-T cell dynamics within the solid TME, uncovering cellular and molecular mechanisms of rapid functional decline and identifying regulatory pathways within the TME that can be exploited to improve CAR-T cell therapy efficacy in solid tumors. KEY MESSAGES OF THE ARTICLEO_ST_ABSWhat is already known on this topicC_ST_ABSThe determinants of CAR-T cell therapeutic efficacy in solid tumors remain poorly defined, largely due to the complexity of the immunosuppressive tumor microenvironment. In this effort, it is necessary to perform comprehensive and detailed mechanistic studies that capture CAR-T cell dynamics within the solid tumor microenvironment to understand treatment failure. What this study addsWe performed single-cell profiling of stroma-targeting EDA-CAR-T cells, revealing their dynamic reprogramming toward dysfunction within the solid tumor microenvironment. We dissected CAR-T cell states and their cell-to-cell interactions with the tumor microenvironment across response and tumor progression and identified mechanisms linking CAR-T cell functionality and therapeutic failure. How this study might affect research, practice or policyThis study provides comprehensive mechanistic insights from an immunocompetent model that can be leveraged to identify shared determinants of CAR-T cell functionality in solid tumors and potentially guide the rational development of improved CAR-T cell therapies.

genomics↗

bioRxiv2025

Single-Cell Multiomics Reveals Regulatory Mechanisms of CAR T Cell Persistence and Dysfunction in Multiple Myeloma

Understanding the mechanisms that drive chimeric antigen receptor (CAR) T cell function and persistence in multiple myeloma (MM) remains a critical challenge for improving therapeutic outcomes. In this study, we applied single-cell multiomics and gene regulatory network (GRN) analysis to characterize the transcriptional dynamics and clonal evolution of BCMA-targeted CAR T cells in longitudinally collected bone marrow (BM) and peripheral blood (PB) samples from MM patients. Our results revealed that CAR T cells infiltrating BM exhibited a more activated and exhausted phenotype compared to their PB counterparts, with key transcriptional regulators driving these changes. Dysregulation in the effector-to-memory transition led to an increased presence of terminally differentiated CAR T cells, correlating with poor persistence. Additionally, we identified a hyperexpanded CAR T clone in the BM of a patient with partial response, marked by elevated IL10 expression. Functional analyses demonstrated that stimulation of endogenous TCR enhanced IL10 production, potentially contributing to impaired CAR T cell proliferation and persistence. These findings uncover critical regulatory mechanisms influencing CAR T cell dynamics, offering new insights into improving CAR T cell persistence and therapeutic efficacy in MM and highlights potential molecular targets for optimizing CAR T cell therapy in patients with MM.

genomics↗