Avidity and functional characterization of CD19/BCMA dual-targeting CAR-T cell designs in NHL

CD19-directed CAR-T therapy has achieved remarkable clinical outcomes in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Nevertheless, a significant proportion of patients develop primary resistance or relapse, often associated with CD19 antigen loss or downregulation. Given the co-expression of CD19 and BCMA in NHL, we hypothesized that dual antigen targeting could improve therapeutic durability and mitigate antigen escape. To address this, we developed a panel of CD19/BCMA dual-targeting CAR-T cells building on our academic platforms targeting CD19 (varnimcabtagene autoleucel, ARI0001) and BCMA (cesnicabtagene autoleucel, ARI0002h). Multiple strategies were explored, including pooled mono-targeted products, co-transduction with two lentiviral vectors, bicistronic constructs, and tandem/loop CAR designs incorporating dual binding domains within a single receptor. Functional activity and avidity were assessed across varying antigen expression conditions. We found that dual CAR-T cells generated through co-transduction consistently showed superior performance relative to single-target CD19 CAR-T cells and other dual-targeting formats, particularly in models with low CD19 expression. A first-in-human phase I clinical trial (CARTDBG-01; NCT06097455) is currently underway to evaluate the safety and efficacy of ARI0003 in NHL.

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Related publication: Bachiller, M., Barceló-Genestar, N., Rodriguez-Garcia, A., Alserawan, L., Dobaño-López, C., Giménez-Alejandre, M., ... & Guedan, S. (2025). ARI0003: Co-transduced CD19/BCMA dual-targeting CAR-T cells for the treatment of non-Hodgkin lymphoma. Molecular Therapy, 33(1), 317-335. https://doi.org/10.1016/j.ymthe.2024.11.028

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90% of immuno-oncology drugs entering Phase I clinical trials fail, due to a poor predictive value of current preclinical assays. To solve this, scientists and drug developers are turning to Cell Avidity, the total binding strength between cells, to better predict therapeutic efficacy.​

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Chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment, but its efficacy remains limited in solid tumors due to antigen heterogeneity, an immunosuppressive microenvironment, and the glycocalyx barrier. The glycocalyx, composed of dense glycoproteins such as MUC1, is markedly expanded in cancers, where it impedes immune cell access and antigen engagement, thereby reducing therapeutic efficacy. In most adenocarcinomas, the Tn antigen, comprising N-acetylgalactosamine linked to serine or threonine, is overexpressed. Tn-MUC1, a truncated form of MUC1 decorated with Tn antigen, is frequently overexpressed in pancreatic cancer. Here, we incorporate a non-signaling glyco-bridge binder recognizing Tn-MUC1 into mesothelin-directed CAR-T cells. This bridge enhances tumor recognition and cytotoxicity by increasing avidity and facilitating CAR activation in a density- and affinity-dependent manner. To directly validate these effects at the cell interaction level, we used Lumicks z-Movi to quantify CAR-T binding strength to tumor targets. CAR-T cells equipped with the Tn-MUC1 glyco-bridge exhibited higher cell avidity toward Tn-MUC1-expressing tumor cells compared to a CD19 bridge control. To broaden its applicability, we design a tandem Helix pomatia agglutinin (HPA) lectin-based bridge that recognizes Tn antigens across cancer types. CAR-T cells with the HPA-bridge exhibit superior cytotoxicity in pancreatic cancer models.

Chimeric antigen receptor (CAR)-T cell therapies showed remarkable efficacies in treating otherwise intractable cancers. However, current clinically approved CAR-T therapies are limited by low antigen sensitivity, impeding their efficacy against cancers with low antigen expression. Here, to address this issue, we engineered CARs targeting CD19, CD22 and HER2 by including intrinsically disordered regions (IDRs) that promote signaling condensation. We discovered that the CAR fused with an IDR from FUS, EWS or TAF15 promoted the formation of CAR-T conjugation with cancer targets, the mechanical strength of CAR-T synapses and membrane-proximal signaling, which led to an increased release of cytotoxic factors and a higher killing activity toward low-antigen-expressing cancer cells in vitro. Moreover, the IDR CAR-T displayed improved antitumor effects in both blood cancer and solid tumor models. No elevated tonic signaling was observed. Together, our work demonstrates IDRs as a new toolset for improving CAR-T function through inducing biomolecular condensation.

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This webinar is in Chinese. 此次线上研讨会为中文讲座

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