Treatment of multiple sclerosis with CD19-targeting CAR-T-cells

Treatment of multiple sclerosis with CD19-targeting CAR-T-cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication Treatment of multiple sclerosis with CD19-targeting CAR-T-cells Claudia Lengerke, Antje Giede-Jeppe, Malte Roerden, Anna Stanger, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9023358/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Three patients with refractory MS received CD19-targeting chimeric antigen receptor (CAR)-T-cells (UKT-CART19.1) after standard lymphodepletion and were followed-up for 3, 7 and respectively 12 months. All showed neuroinflammation on magnetic resonance imaging (MRI) and/or clinical deterioration prior to treatment. UKT-CART19.1 was well tolerated, stabilized clinical impairment, and reduced intrathecal antibody levels and inflammatory lesions on MRI imaging. Transient B-cell depletion was followed by reconstitution with predominately naïve B-cells. We propose that CD19-targeting CAR-T-cells are safe and effective in refractory MS warranting further exploration in clinical trials. Notably, this report provides first evidence of neuroimaging responses to CAR-T-cell therapy in MS. Health sciences/Neurology/Neurological disorders/Demyelinating diseases/Multiple sclerosis Biological sciences/Immunology/Autoimmunity Figures Figure 1 Figure 2 Main Multiple sclerosis(MS) is an autoimmune disorder of the central nervous system(CNS) caused by heterogeneous neuroinflammation and subsequent neurodegeneration 1 . Early MS activity is mainly driven by peripheral T-cell activation and recurring CNS infiltration by immune cells, where autoreactive B-cells portray key mediators 2 . With progression, disease activity shifts towards a CNS-intrinsic and likely compartmentalized neuroinflammation caused by the proliferation of CNS-residing immune cells 3 . Disease-modifying B-cell-depleting monoclonal antibodies show modest control of disease progression. This limited therapeutic efficacy is likely due to their poor capacity to cross the blood-brain-barrier 4,5 . Chimeric antigen receptor(CAR)-T-cells directed against the B-cell antigen CD19 have shown promising results in the treatment of B-cell-mediated autoimmune diseases 6-8 possibly reaching b cell niches in the periphery. Since CAR-T-cells also infiltrate the CNS, they may also represent a powerful treatment option for MS. Here, we explore the use of academically point-of-care manufactured CD19-CAR-T-cells(UKT-CART19.1) in the treatment of three MS-patients. Patient#1 was a 51-year-old male with a 33-year disease course and secondary-progressive MS(Fig.1A, upper panel). Clinically, he exhibited spastic tetraparesis with wheelchair dependance (EDSS-score 9 , for subscores and PROMs see supplemental material). Several lines of treatment had failed to alter the secondary-progressive trajectory with high risk to result in a fully bedridden state. Ocrelizumab as the most recent therapy had been discontinued 18 months prior to CAR-T-cell therapy due to a lack of clinical efficacy. Baseline MRI(prior to CAR-T-cell therapy) revealed multiple small supra- and infratentorial T2-hyperintensive inflammatory lesions as well as a prominent, widespread inflammatory lesion with contrast enhancement in the cervical myelon, which had been evident for many years, likely reflecting a smouldering lesion 10,11 (Fig.2F left panel). Patient#2 was a 40-year-old male with primary-progressive MS diagnosed at the age of 37 (Fig.1A, middle panel). Prior treatment with corticosteroids and ocrelizumab did not show a disease-modifiying effect and the patient experienced a progressive decline in walking ability. At baseline, the patient showed left-sided internuclear ophthalmoplegia, spastic hemiparesis(EDSS-score 5.0; TableS1), and a progressive loss in maximum walking distance(250 m before CAR-T-cell therapy). On MRI, a T2-hyperintensive inflammatory white matter lesion with a paramagnetic rim(Fig.2G, upper left panel), next to additional lesions in the thalamus and cervical myelon and inflammatory alterations of cranial nerves and radicular/spinal roots were noted. Ocrelizumab had been discontinued 5 months before CAR-T-cell therapy due to lack of efficacy. Patient#3 was a 34-year-old female with highly active relapsing-remitting MS diagnosed at the age of 27(Fig.1A, bottom panel). She had relapsed under treatment with glatiramer-acetate, dimethyl-fumarate and natalizumab. Ocrelizumab had to be discontinued due to recurrent severe infections requiring mechanical ventilation. At baseline, she showed spastic hemiparesis, a maximum walking distance of 600 m(EDSS-score 2.5; TableS1), and a predominantly cortical lesion burden with no definitive active inflammatory lesions on MRI. CAR-T-cell therapy was well tolerated in all patients. Patient#1 developed a moderate cytokine release syndrome(CRS; ASTCT grade II 12 ) and was treated with antipyretics and tocilizumab. Patient#2 and #3 developed a mild CRS(ASTCT gradeI) managed with antipyretics. No treatment-associated neurotoxicity(immune effector cell-associated neurotoxicity syndrome;ICANS) was observed in any patient. Hematotoxicity was moderate in all patients(Fig.1B). No late ICANS was observed during outpatient follow-up of 12, 7 and 3 months, respectively. CAR-T-cells showed robust in vivo expansion(Fig.1C) and were predominantely CD8-positive with an effector-memory phenotype(Fig.1D-E). Rapid B-cell depletion was noted in all patients following treatment(Fig.2A). IgG levels remained within the normal range in patient#1 and #2, whereas patient#3 had presented with pre-existing hypogammaglobulinemia and received prophylactic intravenous immunoglobulins after CAR-T-cell therapy(Fig.2B). No late toxicities or severe infections were noted during outpatient follow-up. Patient#2 developed a SARS-CoV-2 infection within 3 months after treatment, which was clinically mild and treated with nirmatrelvir/ritonavir. B-cell reconstitution was noted 3-4 months after CAR-T-cell infusion and recovering B-cells showed a predominantely naïve phenotype(Fig.2C-D). Intrathecal IgG levels were reduced in patient#1 and #2 after CAR-T-cell therapy(Fig.2E). In patient#3, intrathecal IgG levels were slightly increased after treatment following prophylactic intravenous immunoglobulin infusion(<7days prior to CSF sampling). At MS diagnosis, patients#1 and #2 showed type 3 oligoclonal band patterns on CSF/serum isoelectric focusing(IEF), indicating intrathecal IgG synthesis with bands in both CSF and serum and additional CSF-only bands 13 . Both patients subsequently shifted to stable type 4 patterns(identical oligoclonal bands in CSF/serum) before and after CAR-T-cell treatment, consistent with a systemic immune response without local intrathecal synthesis. Patient#3 showed a type 3 pattern at baseline, which cleared to type 1(no oligoclonal bands in CSF/serum) after CAR-T-cell treatment. During follow-up, no patient reported further deterioration of disease-associated symptoms and EDSS-scores remained either stable or improved slightly(Figure1A; TableS1). Patient#1 reported an improvement in mobilization(not reflected in EDSS change). Patient#2 reported a subjective improvement in cognitive abilities and concentration span and the self-reported walking distance increased from ~250m at baseline to more than 700m 7 months after treatment(EDSS4.0). Patient#3 reported overall stable symptoms during the 3-month follow-up period(EDSS2.0). Further neurological assessments including 9-hole-peg test, timed-25-ft walk, and symbol-digit-modalities test matched pre-treatment values(TableS2). Follow-up MRI 3 months after CAR-T-cell therapy demonstrated a regression of inflammatory lesions: In patient#1, the prominent inflammatory lesion in the cervical myelon showed lack of contrast enhancement following treatment, which remained stable at the lastest follow-up imaging 10 months after treatment(Fig.2F right panel). In patient#2, the white matter lesion with paramagnetic rim showed regression on follow-up imaging(Fig.2G), whereas cranial nerve and radicular contrast uptake persisted. We identified a reduction in T2-lesion volume in both cases. 3-month follow-up imaging for patient#3 showed no new T2- or contrast-enhancing lesions. PBVC values within all patients ranged from −0.86% to +0.37%, remaining within the expected physiological range according to published reference data14(Table S3). Therapeutic options for MS patients failing approved disease-modifying therapies remain limited. CD19-targeting CAR-T-cells are a promising novel treatment approach that may target disease-driving niches of autoreactive tissue-resident B-cells from compartmentalized CNS inflammation in progressive MS 15 . B-cell-directed CAR-T-cell therapy for MS is under investigation in early clinical trials 16 , but data on safety and efficacy of this treatment approach are limited to case studies 17 . Our case series treated with UKT-CART19.1 supports the favorable safety profile of CAR-T-cell therapy observed in the treatment of other autoimmune diseases 6-8 . Importantly, no neurotoxicity was observed in our patients despite active CNS inflammation at the time of treatment, and no severe infections were observed during follow-up. Treatment-induced B-cell depletion was transient, with naïve B-cells reconstituting 3-4 months after CAR-T infusion. Whether B-cell depletion following CAR-T cell therapy is qualitatively distinct from other B-cell-directed therapies currently remains unknown. The clearing from oligoclonal bands from the CSF in one patient may support a recently proposed curative potential of CAR-T-cell therapy for B-cell-driven autoimmune diseases 18 . In line with this notion, we observed no further clinical deterioration captured on EDSS or PROMs during the follow-up of up to 12 months. This long-term disease stabilization at the same time argues against a simple lymphodepleting chemotherapy-mediated effect, which would be expected to be transient 19 . While PROMs are pivotal to capture patient perspectives 20 , they are susceptible to placebo effects and have to be interpreted with caution 21 . Finally, follow-up MRI demonstrated a regression of inflammatory lesions after CAR-T cell therapy. Cranial nerve contrast enhancement noted in one MS-patient portrays an atypical finding, which is reported to occur in 3-8% of MS-patients 22,23 . Further CSF analysis ruled out infection. While our case series does not allow to draw definitive conclusions regarding efficacy, these results may indicate responses to treatment and to our knowledge, this is the first report of sustained reponses on neuroimaging following CAR-T-cell therapy for MS. Together, we observed a favorable safety profile and promising signs of clinical and neuroimaging efficacy with the use of UKT-CART19.1 in patients with MS. Our results align with recently published early reports showing favorable safety profiles for CAR-T cell therapy for MS using either CD19 and/or BCMA as target antigens 24-26 . Similar to our observations, B-cell depletion was transient, lasting for ~3 months, and reconstituion was charactierized by naïve B cells in these preliminary reports of small MS patient cohorts. Uncertainties remain whether these observations reflect a true immune reset and whether they are associated with durable remission. An investigator-initiated trial will be launched at our institution to further evaluate treatment with UKT-CART19.1 in patients with MS. Declarations "Treatment if multiple sclerosis with CD19-targeting CAR-T-cells", an approval has been obtained by the Ethics Committee of the University of Tübingen for the presented analyses, and all patients have provided their consent to participate and publish their clinical data. Acknowledgements The project was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to CL and WB (DFG grant 467578951 and 46757750), and to MR (MINT-Clinician Scientist program, DFG grant 493665037). We thank the personnel involved in the treatment of the patients, the GMP-manufacturing process of UKT-CART19.1 (especially Peter Lang, Daniel Atar, Christiane Braun, Marina Schmidt, Katrin Lutz), the leukapheresis, and the flow cytometry diagnostics. We thank Miltenyi Biotec for supplying CAR vectors. Author Contributions AGJ, MR, LH, CL, WB designed the research, CF and WB performed the treatment. LH led and WB supervised the CAR-T-cell production. AS performed flow cytometry analyses. BB led and analyzed imaging studies. AGJ, JS, MK and UZ identified patients for treatment and performed SCF analyses. MR, AGJ and CL wrote the manuscript draft, all authors edited the manuscript. Funding This project was partly funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – grants 467578951, 46757750) and the MINT-CS program (DFG grant 493665037). Competing Interests There are no competing interests related to this study. Data sharing statement The authors confirm that all data supporting the findings of this study are available within the article. Other data are not publicly available due to their containing of information that could compromise the privacy of involved patients. References Hemmer, B., Kerschensteiner, M. & Korn, T. Role of the innate and adaptive immune responses in the course of multiple sclerosis. Lancet Neurol 14, 406–419 (2015). Cencioni, M.T., Mattoscio, M., Magliozzi, R., Bar-Or, A. & Muraro, P.A. B cells in multiple sclerosis - from targeted depletion to immune reconstitution therapies. Nat Rev Neurol 17, 399–414 (2021). Magliozzi, R., et al. A Gradient of neuronal loss and meningeal inflammation in multiple sclerosis. Ann Neurol 68, 477–493 (2010). Correale, J., Halfon, M.J., Jack, D., Rubstein, A. & Villa, A. Acting centrally or peripherally: A renewed interest in the central nervous system penetration of disease-modifying drugs in multiple sclerosis. Mult Scler Relat Disord 56, 103264 (2021). Cencioni, M.T., Mattoscio, M., Magliozzi, R., Bar-Or, A. & Muraro, P.A. B cells in multiple sclerosis — from targeted depletion to immune reconstitution therapies. Nature Reviews Neurology 17, 399–414 (2021). Zulfiqar, F., et al. Outcomes with chimeric antigen receptor T-cell therapy in Rheumatological disorders: A systematic review. Transpl Immunol 87, 102137 (2024). Pecher, A.C., et al. CD19-Targeting CAR T Cells for Myositis and Interstitial Lung Disease Associated With Antisynthetase Syndrome. Jama 329, 2154–2162 (2023). Zhang, Y., et al. Anti-BCMA/CD19 CAR T cell therapy in patients with refractory generalized myasthenia gravis: a single-arm, phase 1 trial. eClinicalMedicine 90(2025). Kurtzke, J.F. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 33, 1444–1452 (1983). Haase, S. & Linker, R.A. Inflammation in multiple sclerosis. Ther Adv Neurol Disord 14, 17562864211007687 (2021). further references – see supplemental section Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalMaterialfin.docx Supplemental Material Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9023358","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":600838636,"identity":"0d0c3189-4b5c-473e-b2fe-357a50a8c13b","order_by":0,"name":"Claudia 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19:40:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9023358/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9023358/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104887206,"identity":"0b6bab38-2eb5-4594-b882-b0b419521ecd","added_by":"auto","created_at":"2026-03-18 10:11:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":158656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSafety of CAR-T-cell therapy in three patients with MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Overview of clinical disease course including onset of main symptoms, EDSS-scores and MS specific treatments. (B)Time-course display of absolute neutrophil counts (ANC), hemoglobin levels and platelets in the peripheral blood showing moderate hematotoxicity associated with CAR-T-cell therapy in all patients. (C)CAR-T-cell expansion following infusion, depicted as absolute cell numbers and as percentage of lymphocytes in the peripheral blood. (D)Flow cytometry-based analysis of CAR-T-cell subsets on day 10 and day 35±3 after infusion. (E) Flow cytometry-based analysis of CD8\u003csup\u003e+\u003c/sup\u003e CAR-T-cell phenotypes on day 10±2 after infusion, depicting subsets with different T-cell differentiation, percentage of activated(CD38\u003csup\u003e+\u003c/sup\u003eHLA-DR\u003csup\u003e+\u003c/sup\u003e) T-cells and exemplary flow plots of CAR-T-cell phenotyping.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-9023358/v1/1789439710b1723db153426e.png"},{"id":104887208,"identity":"a6f37d4a-4b00-4624-a45f-f210acee9d00","added_by":"auto","created_at":"2026-03-18 10:11:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1173880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eB-cell and neuroimaging changes induced by CAR-T-cell therapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B)Time-course display of (A)absolute B-cell counts and (B)IgG levels in the peripheral blood. (C)Flow cytometry-based analysis of B-cell subsets in the periphreral blood before and after CAR-T-cell therapy. (D)Flow cytometry plots of B-cell phenotyping for patient #1 before and after therapy. (E)IgG levels in cerebro-spinal fluid before and after CAR-T-cell therapy in mg/l(left panel) and \u0026nbsp;CSF-to-serum ration(right panel). (F)Neuroimaging of patient #1 before and 10 months after CAR-T-cell therapy. After therapy, the contrast enhancement of the cervical myelon lesion is no longer visible, and lesion size has decreased. (G)Neuroimaging of patient #2 before and 3 months after CAR-T-cell therapy. Upper panel: Decreased visibility of paramagnetic rim after therapy. Lower panel: Aligned 3D T2-FLAIR images before(left panel) and after therapy(middle panel), showing a decrease in lesion volume. The lesion regression becomes evident as a hypointense ring in the subtraction map(right panel). (F-G)Pathologic alterations indicated by arrows.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-9023358/v1/3274cd5c1d509db64890059d.png"},{"id":104887637,"identity":"0757c950-66bf-44b2-89e2-f51d2c581ed5","added_by":"auto","created_at":"2026-03-18 10:12:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1596023,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9023358/v1/ac56dc2e-9ba1-43ab-af58-49ea99e1097f.pdf"},{"id":104887248,"identity":"378dc351-b7fd-4342-a6b2-9f7a645f2db4","added_by":"auto","created_at":"2026-03-18 10:11:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":418052,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"SupplementalMaterialfin.docx","url":"https://assets-eu.researchsquare.com/files/rs-9023358/v1/99acb377efeb4bd3fa78966d.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Treatment of multiple sclerosis with CD19-targeting CAR-T-cells","fulltext":[{"header":"Main","content":"\u003cp\u003eMultiple sclerosis(MS) is an autoimmune disorder of the central nervous system(CNS) caused by heterogeneous neuroinflammation and subsequent neurodegeneration\u003csup\u003e1\u003c/sup\u003e. Early MS activity is mainly driven by peripheral T-cell activation and recurring CNS infiltration by immune cells, where autoreactive B-cells portray key mediators\u003csup\u003e2\u003c/sup\u003e. With progression, disease activity shifts towards a CNS-intrinsic and likely compartmentalized neuroinflammation caused by the proliferation of CNS-residing immune cells\u003csup\u003e3\u003c/sup\u003e. Disease-modifying B-cell-depleting monoclonal antibodies show modest control of disease progression. This limited therapeutic efficacy is likely due to their poor capacity to cross the blood-brain-barrier\u003csup\u003e4,5\u003c/sup\u003e. Chimeric antigen receptor(CAR)-T-cells directed against the B-cell antigen CD19 have shown promising results in the treatment of B-cell-mediated autoimmune diseases\u003csup\u003e6-8\u003c/sup\u003e possibly reaching b cell niches in the periphery. Since CAR-T-cells also infiltrate the CNS, they may also represent a powerful treatment option for MS. Here, we explore the use of academically point-of-care manufactured CD19-CAR-T-cells(UKT-CART19.1) in the treatment of three MS-patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePatient#1 was a 51-year-old male with a 33-year disease course and secondary-progressive MS(Fig.1A, upper panel). Clinically, he exhibited spastic tetraparesis with wheelchair dependance (EDSS-score\u003csup\u003e9\u003c/sup\u003e, for subscores and PROMs see supplemental material). Several lines of treatment had failed to alter the secondary-progressive trajectory with high risk to result in a fully bedridden state. Ocrelizumab as the most recent therapy had been discontinued 18 months prior to CAR-T-cell therapy due to a lack of clinical efficacy. Baseline MRI(prior to CAR-T-cell therapy) revealed multiple small supra- and infratentorial T2-hyperintensive inflammatory lesions as well as a prominent, widespread inflammatory lesion with contrast enhancement in the cervical myelon, which had been evident for many years, likely reflecting a smouldering lesion\u003csup\u003e10,11\u003c/sup\u003e (Fig.2F left panel). Patient#2 was a 40-year-old male with primary-progressive MS diagnosed at the age of 37 (Fig.1A, middle panel). Prior treatment with corticosteroids and ocrelizumab did not show a disease-modifiying effect and the patient experienced a progressive decline in walking ability. At baseline, the patient showed left-sided internuclear ophthalmoplegia, spastic hemiparesis(EDSS-score 5.0; TableS1), and a progressive loss in maximum walking distance(250 m before CAR-T-cell therapy). On MRI, a T2-hyperintensive inflammatory white matter lesion with a paramagnetic rim(Fig.2G, upper left panel), next to additional lesions in the thalamus and cervical myelon and inflammatory alterations of cranial nerves and radicular/spinal roots were noted. Ocrelizumab had been discontinued 5 months before CAR-T-cell therapy due to lack of efficacy. Patient#3 was a 34-year-old female with highly active relapsing-remitting MS diagnosed at the age of 27(Fig.1A, bottom panel). She had relapsed under treatment with glatiramer-acetate, dimethyl-fumarate and natalizumab. Ocrelizumab had to be discontinued due to recurrent severe infections requiring mechanical ventilation. At baseline, she showed spastic hemiparesis, a maximum walking distance of 600 m(EDSS-score 2.5; TableS1), and a predominantly cortical lesion burden with no definitive active inflammatory lesions on MRI.\u003c/p\u003e\n\u003cp\u003eCAR-T-cell therapy was well tolerated in all patients. Patient#1 developed a moderate cytokine release syndrome(CRS; ASTCT grade II\u003csup\u003e12\u003c/sup\u003e) and was treated with antipyretics and tocilizumab. Patient#2 and #3 developed a mild CRS(ASTCT gradeI) managed with antipyretics. No treatment-associated neurotoxicity(immune effector cell-associated neurotoxicity syndrome;ICANS) was observed in any patient. Hematotoxicity was moderate in all patients(Fig.1B). No late ICANS was observed during outpatient follow-up of 12, 7 and 3 months, respectively. CAR-T-cells showed robust in vivo expansion(Fig.1C) and were predominantely CD8-positive with an effector-memory phenotype(Fig.1D-E). Rapid B-cell depletion was noted in all patients following treatment(Fig.2A). IgG levels remained within the normal range in patient#1 and #2, whereas patient#3 had presented with pre-existing hypogammaglobulinemia and received prophylactic intravenous immunoglobulins after CAR-T-cell therapy(Fig.2B). No late toxicities or severe infections were noted during outpatient follow-up. Patient#2 developed a SARS-CoV-2 infection within 3 months after treatment, which was clinically mild and treated with nirmatrelvir/ritonavir. B-cell reconstitution was noted 3-4 months after CAR-T-cell infusion and recovering B-cells showed a predominantely na\u0026iuml;ve phenotype(Fig.2C-D). Intrathecal IgG levels were reduced in patient#1 and #2 after CAR-T-cell therapy(Fig.2E). In patient#3, intrathecal IgG levels were slightly increased after treatment following prophylactic intravenous immunoglobulin infusion(\u0026lt;7days prior to CSF sampling).\u003c/p\u003e\n\u003cp\u003eAt MS diagnosis, patients#1 and #2 showed type 3 oligoclonal band patterns on CSF/serum isoelectric focusing(IEF), indicating intrathecal IgG synthesis with bands in both CSF and serum and additional CSF-only bands\u003csup\u003e13\u003c/sup\u003e. Both patients subsequently shifted to stable type 4 patterns(identical oligoclonal bands in CSF/serum) before and after CAR-T-cell treatment, consistent with a systemic immune response without local intrathecal synthesis. Patient#3 showed a type 3 pattern at baseline, which cleared to type 1(no oligoclonal bands in CSF/serum) after CAR-T-cell treatment.\u003c/p\u003e\n\u003cp\u003eDuring follow-up, no patient reported further deterioration of disease-associated symptoms and EDSS-scores remained either stable or improved slightly(Figure1A; TableS1). Patient#1 reported an improvement in mobilization(not reflected in EDSS change). Patient#2 reported a subjective improvement in cognitive abilities and concentration span and the self-reported walking distance increased from ~250m at baseline to more than 700m 7 months after treatment(EDSS4.0). Patient#3 reported overall stable symptoms during the 3-month follow-up period(EDSS2.0). Further neurological assessments including 9-hole-peg test, timed-25-ft walk, \u0026nbsp;and symbol-digit-modalities test matched pre-treatment values(TableS2). Follow-up MRI 3 months after CAR-T-cell therapy demonstrated a regression of inflammatory lesions: In patient#1, the prominent inflammatory lesion in the cervical myelon showed lack of contrast enhancement following treatment, which remained stable at the lastest follow-up imaging 10 months after treatment(Fig.2F right panel). In patient#2, the white matter lesion with paramagnetic rim showed regression on follow-up imaging(Fig.2G), whereas cranial nerve and radicular contrast uptake persisted. We identified a reduction in T2-lesion volume in both cases. 3-month follow-up imaging for patient#3 showed no new T2- or contrast-enhancing lesions. PBVC values within all patients ranged from \u0026minus;0.86% to +0.37%, remaining within the expected physiological range according to published reference data14(Table S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherapeutic options for MS patients failing approved disease-modifying therapies remain limited. CD19-targeting CAR-T-cells are a promising novel treatment approach that may target disease-driving niches of autoreactive tissue-resident B-cells from compartmentalized CNS inflammation in progressive MS\u003csup\u003e15\u003c/sup\u003e. B-cell-directed CAR-T-cell therapy for MS is under investigation in early clinical trials\u003csup\u003e16\u003c/sup\u003e, but data on safety and efficacy of this treatment approach are limited to case studies\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur case series treated with UKT-CART19.1 supports the favorable safety profile of CAR-T-cell therapy observed in the treatment of other autoimmune diseases\u003csup\u003e6-8\u003c/sup\u003e. Importantly, no neurotoxicity was observed in our patients despite active CNS inflammation at the time of treatment, and no severe infections were observed during follow-up. Treatment-induced B-cell depletion was transient, with na\u0026iuml;ve B-cells reconstituting 3-4 months after CAR-T infusion. Whether B-cell depletion following CAR-T cell therapy is qualitatively distinct from other B-cell-directed therapies currently remains unknown. The clearing from oligoclonal bands from the CSF in one patient may support a recently proposed curative potential of CAR-T-cell therapy for B-cell-driven autoimmune diseases\u003csup\u003e18\u003c/sup\u003e. In line with this notion, we observed no further clinical deterioration captured on EDSS or PROMs during the follow-up of up to 12 months. This long-term disease stabilization at the same time argues against a simple lymphodepleting chemotherapy-mediated effect, which would be expected to be transient\u003csup\u003e19\u003c/sup\u003e. While PROMs are pivotal to capture patient perspectives\u003csup\u003e20\u003c/sup\u003e, they are susceptible to placebo effects and have to be interpreted with caution\u003csup\u003e21\u003c/sup\u003e. Finally, follow-up MRI demonstrated a regression of inflammatory lesions after CAR-T cell therapy. \u0026nbsp;Cranial nerve contrast enhancement noted in one MS-patient portrays an atypical finding, which is reported to occur in 3-8% of MS-patients\u003csup\u003e22,23\u003c/sup\u003e. Further CSF analysis ruled out infection. While our case series does not allow to draw definitive conclusions regarding efficacy, these results may indicate responses to treatment and to our knowledge, this is the first report of sustained reponses on neuroimaging following CAR-T-cell therapy for MS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, we observed a favorable safety profile and promising signs of clinical and neuroimaging efficacy with the use of UKT-CART19.1 in patients with MS. Our results align with recently published early reports showing favorable safety profiles for CAR-T cell therapy for MS using either CD19 and/or BCMA as target antigens\u003csup\u003e24-26\u003c/sup\u003e. Similar to our observations, B-cell depletion was transient, lasting for ~3 months, and reconstituion was charactierized by na\u0026iuml;ve B cells in these preliminary reports of small MS patient cohorts. Uncertainties remain whether these observations reflect a true immune reset and whether they are associated with durable remission. An investigator-initiated trial will be launched at our institution to further evaluate treatment with UKT-CART19.1 in patients with MS."},{"header":"Declarations","content":"\u003cp\u003e\"Treatment if multiple sclerosis with CD19-targeting CAR-T-cells\", an approval has been obtained by the Ethics Committee of the University of Tübingen for the presented analyses, and all patients have provided their consent to participate and publish their clinical data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to CL and WB (DFG grant 467578951 and 46757750), and to MR (MINT-Clinician Scientist program, DFG grant 493665037). We thank the personnel involved in the treatment of the patients, the GMP-manufacturing process of UKT-CART19.1 (especially Peter Lang, Daniel Atar, Christiane Braun, Marina Schmidt, Katrin Lutz), the leukapheresis, and the flow cytometry diagnostics. We thank Miltenyi Biotec for supplying CAR vectors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAGJ, MR, LH, CL, WB designed the research, CF and WB performed the treatment. LH led and WB supervised the CAR-T-cell production. AS performed flow cytometry analyses. BB led and analyzed imaging studies. AGJ, JS, MK and UZ identified patients for treatment and performed SCF analyses. MR, AGJ and CL wrote the manuscript draft, all authors edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was partly funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – grants\u0026nbsp;467578951, 46757750) and the MINT-CS program (DFG grant 493665037).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no competing interests related to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sharing statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all data supporting the findings of this study are available within the article. Other data are not publicly available due to their containing of information that could compromise the privacy of involved patients.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eHemmer, B., Kerschensteiner, M. \u0026amp; Korn, T. Role of the innate and adaptive immune responses in the course of multiple sclerosis. Lancet Neurol 14, 406\u0026ndash;419 (2015).\u003c/li\u003e\n\u003cli\u003eCencioni, M.T., Mattoscio, M., Magliozzi, R., Bar-Or, A. \u0026amp; Muraro, P.A. B cells in multiple sclerosis - from targeted depletion to immune reconstitution therapies. Nat Rev Neurol 17, 399\u0026ndash;414 (2021).\u003c/li\u003e\n\u003cli\u003eMagliozzi, R., et al. A Gradient of neuronal loss and meningeal inflammation in multiple sclerosis. Ann Neurol 68, 477\u0026ndash;493 (2010).\u003c/li\u003e\n\u003cli\u003eCorreale, J., Halfon, M.J., Jack, D., Rubstein, A. \u0026amp; Villa, A. Acting centrally or peripherally: A renewed interest in the central nervous system penetration of disease-modifying drugs in multiple sclerosis. Mult Scler Relat Disord 56, 103264 (2021).\u003c/li\u003e\n\u003cli\u003eCencioni, M.T., Mattoscio, M., Magliozzi, R., Bar-Or, A. \u0026amp; Muraro, P.A. B cells in multiple sclerosis \u0026mdash; from targeted depletion to immune reconstitution therapies. Nature Reviews Neurology 17, 399\u0026ndash;414 (2021).\u003c/li\u003e\n\u003cli\u003eZulfiqar, F., et al. Outcomes with chimeric antigen receptor T-cell therapy in Rheumatological disorders: A systematic review. Transpl Immunol 87, 102137 (2024).\u003c/li\u003e\n\u003cli\u003ePecher, A.C., et al. CD19-Targeting CAR T Cells for Myositis and Interstitial Lung Disease Associated With Antisynthetase Syndrome. Jama 329, 2154\u0026ndash;2162 (2023).\u003c/li\u003e\n\u003cli\u003eZhang, Y., et al. Anti-BCMA/CD19 CAR T cell therapy in patients with refractory generalized myasthenia gravis: a single-arm, phase 1 trial. eClinicalMedicine 90(2025).\u003c/li\u003e\n\u003cli\u003eKurtzke, J.F. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 33, 1444\u0026ndash;1452 (1983).\u003c/li\u003e\n\u003cli\u003eHaase, S. \u0026amp; Linker, R.A. Inflammation in multiple sclerosis. Ther Adv Neurol Disord 14, 17562864211007687 (2021). \u003c/li\u003e\n\u003cli\u003efurther references \u0026ndash; see supplemental section\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9023358/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9023358/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Three patients with refractory MS received CD19-targeting chimeric antigen receptor (CAR)-T-cells (UKT-CART19.1) after standard lymphodepletion and were followed-up for 3, 7 and respectively 12 months. All showed neuroinflammation on magnetic resonance imaging (MRI) and/or clinical deterioration prior to treatment. UKT-CART19.1 was well tolerated, stabilized clinical impairment, and reduced intrathecal antibody levels and inflammatory lesions on MRI imaging. Transient B-cell depletion was followed by reconstitution with predominately naïve B-cells. We propose that CD19-targeting CAR-T-cells are safe and effective in refractory MS warranting further exploration in clinical trials. Notably, this report provides first evidence of neuroimaging responses to CAR-T-cell therapy in MS.","manuscriptTitle":"Treatment of multiple sclerosis with CD19-targeting CAR-T-cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 10:08:14","doi":"10.21203/rs.3.rs-9023358/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"82eb339a-8d4c-4f5b-9544-67a8f2ab1717","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63942174,"name":"Health sciences/Neurology/Neurological disorders/Demyelinating diseases/Multiple sclerosis"},{"id":63942175,"name":"Biological sciences/Immunology/Autoimmunity"}],"tags":[],"updatedAt":"2026-03-18T10:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 10:08:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9023358","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9023358","identity":"rs-9023358","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}