Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the field of immune therapy in treating relapsed and refractory B cell malignancies. Despite remarkable progress, CD19 CAR-T cell therapy is associated with major side effects, most significantly are cytokine-release syndrome and neurotoxicity. Of note, 40-60% of patients receiving CD19 CAR-T cells suffer from severe neurotoxicity termed immune cell-associated-neurotoxicity-syndrome (ICANS), which remains a major obstacle limiting their therapeutic efficacy. Several pre-clinical reports using xenograft mouse models have been published to address ICANS, however, they do not fully recapitulate the clinical scenario. Identifying and targeting the molecular mediators of neurotoxicity could help ameliorate ICANS induced by CD19 CAR-T cells.

In order to understand the molecular mechanisms leading to CD19 CAR-T cell-induced ICANS, we established syngeneic ICANS mouse models for B-cell malignancies including non-Hodgkin's Lymphoma (B-NHL) and Acute Lymphoblastic Leukemia (B-ALL). Micro-array and single-nuclei RNA sequencing analyses were performed to unveil key signaling pathways in microglia leading to ICANS pathophysiology. Cognitive impairment resulting from the transfer of CD19 CAR-T cells was assessed with behavior studies in murine models. Imaging mass cytometry was performed on autopsied ICANS and control patient cohorts to study the relevance of microglial activation in patients.

We previously demonstrated that microglial upregulation of tumor necrosis factor-alpha (TNFα) results in graft versus host disease of the central nervous system (Mathew and Vinnakota et al. JCI 2020). Similarly, our ICANS mouse models exhibit morphological and functional signs of microglial activation with increased expression of TNFα and GM-CSF along with endothelial damage in lymphoma-bearing mice that received CD19 CAR-T cells when compared to non-transduced controls. Behavioral studies revealed that mice receiving CD19 CAR-T cells exhibit cognitive deficits and increased anxiety. The behavior deficits were however rescued by microglia depletion indicating an important role for microglia in mediating CD19 CAR-T cells induced ICANS. Mechanistically, we identified the activation of p-38 mitogen-activated protein kinase (MAPK) in microglia isolated from mice receiving CD19 CAR-T cells. Furthermore, pharmacological inhibition of TGF-β-activated kinase-1 (TAK1), an upstream target for p-38 MAPK significantly reduced microglial activation, lowered microglia production of TNF and GM-CSF, and improved cognitive function in these mice. In line with these results, microglia-specific knockdown of TAK1 using Cx3cr1 creER:Tak1 fl/fl mice revealed reduced microglial activation and improved cognitive function when compared to littermate controls. Moreover, TAK1 inhibition combined with CD19 CAR-T cell therapy synergistically improved anti-lymphoma/leukemia effects leading to better survival in B-cell malignancy-bearing mice. Translocator-protein-positron-emission-tomography (TSPO-PET) on ICANS patients revealed signs of microglial activation. In addition, Imaging-mass cytometry analysis on post-mortem ICANS patient cohort revealed upregulation of myeloid cell markers further confirming the in vivo findings.

In summary, our results identify TAK1/p38 MAPK-pathway as a key mediator of CAR19 CAR-T cell-induced neurotoxicity. Targeting this axis diminished the neurotoxicity associated with this therapy. This study provides a rationale for testing TAK1-inhibition in a clinical trial for treating CD19 CAR-T cell-induced neurotoxicity.

Wäsch:Sanofi: Honoraria; BMS: Other: Travel support; Pfizer: Other: Travel support; Kite/Gilead: Other: Travel support; Janssen: Other: Travel support; Takeda: Honoraria; Pfizer: Honoraria; Kite/Gilead: Honoraria; Janssen: Honoraria; BMS/Celgene: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Sanofi: Research Funding; Janssen: Research Funding; Takeda: Consultancy; Sanofi: Consultancy; Pfizer: Consultancy; Kite/Gilead: Consultancy; Novartis: Consultancy; Janssen: Consultancy; BMS/Celgene: Consultancy; Amgen: Consultancy. Simonetta:Incyte: Other: speaker fees; Kite/Gilead: Other: speaker fees; Kite/Gilead: Consultancy; Incyte: Consultancy; BMS/Celgene: Consultancy; Astra Zeneca: Other: Travel support; Kite/Gilead: Other: Travel support; Novartis: Other: Travel support; Janssen: Other: Travel support; Neovii: Other: Travel support; BMS/Celgene: Research Funding; Kite/Gilead: Research Funding; Novartis: Research Funding. Fry:Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Blazar:BlueRock Therapeutics: Current Employment, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Current Employment, Research Funding. Kortüm:Pfizer: Honoraria; BMS: Honoraria; Takeda: Honoraria; GSK: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Rasche:Sanofi: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Skyline Dx: Research Funding; Pfizer: Consultancy, Honoraria; Roche: Honoraria; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Mackensen:Miltenyi Biomedicine, Ixaka, Novartis, BMS/Celgene, Gilead/KITE: Honoraria; Kyverna: Research Funding. Mackall:Adaptimmune: Consultancy; Lyell Immunopharma: Current equity holder in private company, Research Funding; Link Cell Therapies: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; CARGO: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Immatics: Consultancy; Mammoth: Consultancy, Current equity holder in private company. Ruella:NanoString: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Beckman Coulter: Research Funding; AbClon: Consultancy, Research Funding; Bayer: Consultancy; GlaxoSmithKline: Consultancy; viTToria biotherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Founder, Research Funding. Subklewe:Molecular Partners: Consultancy, Honoraria, Research Funding; AstraZeneca: Speakers Bureau; Seagen: Research Funding; AvenCell: Consultancy, Honoraria; Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; Ichnos Sciences: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Other: Travel Support, Speakers Bureau; Incyte Biosciences: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding; GSK: Speakers Bureau; LAWG: Speakers Bureau; Springer Healthcare: Speakers Bureau; AbbVie: Consultancy, Honoraria; Autolus: Consultancy, Honoraria; advesya (CanCell Therapeutics): Consultancy, Honoraria; Genmab US: Consultancy, Honoraria; Interius BioTherapeutics: Consultancy, Honoraria; Nektar Therapeutics: Consultancy, Honoraria; Orbital Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Scare: Consultancy, Honoraria. Gill:Kite Pharma: Consultancy; Carisma Therapeutics: Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: patents, Research Funding; Interius Biotherapeutics: Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Asher: Research Funding; Currus: Membership on an entity's Board of Directors or advisory committees; Inndura: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; NKILT: Membership on an entity's Board of Directors or advisory committees; Vor Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding. Zeiser:VectivBio: Consultancy; Sanofi: Consultancy, Honoraria; Medac: Honoraria; MNK: Consultancy, Honoraria; incyte: Consultancy, Honoraria; novartis: Consultancy, Honoraria, Research Funding.

2023
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