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Fine-tuning affinity and spacer design enhances T cell potency in DLL3 and BCMA CAR T cells
Journal article   Open access   Peer reviewed

Fine-tuning affinity and spacer design enhances T cell potency in DLL3 and BCMA CAR T cells

Nicholas Mazzanti, Ninkka Tamot, Andrea Francese, Jinquan Luo, M Jack Borrok, Julie Rossillo, Joseph Plummer, Gauri Anand Patwardhan, Chi Shing Sum, Michael Ports, …
mAbs, v 18(1), 2602989
2026
DOI: https://doi.org/10.1080/19420862.2025.2602989
PMID: 41383027
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1080/19420862.2025.2602989View
Published, Version of Record (VoR) Open

Abstract

B-Cell Maturation Antigen - immunology Cell Line, Tumor Humans Immunotherapy, Adoptive - methods Intracellular Signaling Peptides and Proteins - immunology Membrane Proteins - immunology Receptors, Chimeric Antigen - genetics Receptors, Chimeric Antigen - immunology T-Lymphocytes - immunology
Chimeric antigen receptor (CAR)-modified T cells have garnered substantial attention due to their clinical success, culminating in six Food and Drug Administration-approved therapies for hematological malignancies. Notably, CD19-specific CAR T cell therapies have achieved remarkable clinical efficacy in treating B-cell malignancies, but these profound and durable responses are not observed in CAR T therapies targeting other indications, particularly solid tumors. Key design elements of CAR constructs - namely, antigen binding affinity and spacer length - play critical roles in determining T cell effector function and overall therapeutic effectiveness. Refining CAR designs may enhance T cell functionality, extend clinical application, and potentially apply CAR T cell therapies across a wider array of malignancies. In this study, affinity variant and spacer variant CARs targeting BCMA and DLL3 tumor antigens were evaluated using measurements of antigen-binding properties and effector function. Each panel of CARs spanned 2-3 logs of antigen binding affinity (BCMA: 181 pM KD to 74 nM KD, DLL3: 417 pM to 407 nM). Additionally, CAR T cells were challenged with tumor spheroids composed of BCMA H929 and DLL3 SHP77 tumor cells. We show that for both tumor models, higher affinity CARs (KD stronger than approximately 100 nM) paired with an intermediate length spacer (IgG1 Fc, CH2-CH3, 230AA) elicited the strongest levels of tumor killing, CAR T cell expansion, and proinflammatory cytokine production. These CARs displayed the strongest cellular affinity when measured in a conjugation assay, suggesting a relationship between cellular affinity and T cell functional performance. This study highlights the critical role of CAR design in enhancing T cell functionality, demonstrating that high-affinity CARs combined with intermediate-length spacers yield superior performance in targeting BCMA and DLL3 antigens. This study provides a framework for rational CAR design, informing strategies to broaden the clinical utility of CAR T-cell therapies beyond hematologic cancers.

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Collaboration types
Industry collaboration
Domestic collaboration
Web of Science research areas
Medicine, Research & Experimental
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