{
  "abstract": "Background BCMA-targeted CAR T-cell therapy has demonstrated remarkable clinical efficacy in patients with relapsed or refractory multiple myeloma (MM). 1 2 Nevertheless, concerns remain regarding neurotoxicity, including Parkinsonism-like movement and cognitive disorders in patients receiving ciltacabtagene autoleucel (cilta-cel) and idecabtagene vicleucel (ide-cel).3 4 The underlying mechanism is not yet fully understood. In this study, we observed high tonic signaling in cilta-cel-like CAR T-cell products and interrogated associated biological implications. We developed a novel BCMA-targeted CAR T-cell, LTG3551, which demonstrates strong anti-myeloma activity while exhibiting a potentially reduced risk of off-target toxicity.Methods The In-house developed BCMA-targeted CAR, LTG3551, incorporates two VHH binders, CD8α hinge and transmembrane domains, and 4-1BB and CD3ζ signaling domains. Cilta-cel-like and ide-cel-like CARs were generated from publicly available sequences. The cilta-cel-like construct was deconstructed into two single binder CARs (VHH1 and VHH2) using the original framework. Anti-tumor efficacy of CAR T cells was evaluated by luciferase-based in vitro killing assays and in MM.1S xenograft models. Metabolic activity of CAR T cells was assessed via the Seahorse assay. Apoptotic makers (Annexin V and TRAIL) and T-cell exhaustion markers (PD-1, TIM-3, and LAG-3) were measured by flow cytometry. Cytokine levels from CAR T cells without co-culture with tumor cells was evaluated by MACSPlex. Potential microglia toxicity by BCMA-CAR T cells was determined by BCMA-CAR T-cell co-culture with HMC3 cells in a xCELLigence platform.Results LTG3551 demonstrated comparable anti-tumor efficacy to cilta-cel-like CAR in in vitro cytotoxicity assays and MM.1S xenograft model. Cilta-cel-like CAR T cells showed features of tonic signaling, including elevated expression of apoptotic markers, spontaneous cytokine release, and higher metabolic activity in the absence of antigen stimulation. Intriguingly, VHH2, but not VHH1, recapitulates these features independently, indicating VHH2 as the primary driver of tonic signaling within the bi-paratopic cilta-cel-like CAR. Moreover, cilta-cel-like CAR T cells exhibited significant off-target cytotoxicity against BCMA-negative HMC3 cells. In contrast, our novel CAR T-cell, LTG3551, exhibited low tonic signaling, compelling anti-tumor activity, and negligible off-target effects.Conclusions This study demonstrates that cilta-cel-like CAR T cells induce off-target cytotoxicity against the microglia cell line HMC3, which is likely mediated by the high level of tonic signaling. In contrast, the in-house developed BCMA-CAR T-cell, LTG3551, shows comparable anti-myeloma activity while minimizing off-target toxicity through reduced tonic signaling. These findings support LTG3551 as a potentially safer and effective therapeutic approach for the treatment of MM.References San-Miguel J, Dhakal B, Yong K, Spencer A, Anguille S, Mateos MV, Fernández de Larrea C, Martínez-López J, Moreau P, Touzeau C, Leleu X. cilta-cel or standard care in lenalidomide-refractory multiple myeloma. New England Journal of Medicine. 2023 Jul 27;389(4):335-47.Rodriguez-Otero P, Ailawadhi S, Arnulf B, Patel K, Cavo M, Nooka AK, Manier S, Callander N, Costa LJ, Vij R, Bahlis NJ. Ide-cel or standard regimens in relapsed and refractory multiple myeloma. New England Journal of Medicine. 2023 Mar 16;388(11):1002-14.Van Oekelen O, Aleman A, Upadhyaya B, Schnakenberg S, Madduri D, Gavane S, Teruya-Feldstein J, Crary JF, Fowkes ME, Stacy CB, Kim-Schulze S. Neurocognitive and hypokinetic movement disorder with features of parkinsonism after BCMA-targeting CAR-T cell therapy. Nature medicine. 2021 Dec;27(12):2099-103.Gust J. BCMA-CAR T-cell treatment–associated parkinsonism. Blood 2023 Oct 5;142(14):1181–3.",
  "authors": [
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Xu Wang"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Peirong Hu"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Brittany Steimle"
    },
    {
      "affiliations": [
        "Miltenyi Biotec B.V. and Co. KG, Bergisch Gladbach, Germany"
      ],
      "name": "Michael Wagner"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Saule Nurmukhambetova"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Genqing Liang"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Ngoc Tran"
    },
    {
      "affiliations": [
        "Miltenyi Biotec B.V. and Co. KG, Bergisch Gladbach, Germany"
      ],
      "name": "Jens Kopatz"
    },
    {
      "affiliations": [
        "Miltenyi Biotec B.V. and Co. KG, Bergisch Gladbach, Germany"
      ],
      "name": "Katrin Krischer"
    },
    {
      "affiliations": [
        "Miltenyi Biotec B.V. and Co. KG, Bergisch Gladbach, Germany"
      ],
      "name": "Ilia Kelmanson"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Kun Luo"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Wei Xiong"
    },
    {
      "affiliations": [
        "Miltenyi Biotec B.V. and Co. KG, Bergisch Gladbach, Germany"
      ],
      "name": "Janina Brauner"
    },
    {
      "affiliations": [
        "Miltenyi Biotec B.V. and Co. KG, Bergisch Gladbach, Germany"
      ],
      "name": "Olaf Hardt"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Alun J Carter"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Rimas J Orentas"
    }
  ],
  "title": "292 Optimized bi-paratopic BCMA-specific CAR T cells designed to minimize tonic signaling and off-target toxicity",
  "uid": "45dd4670-bddd-5cf5-82f3-510593241c0b"
}