{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T05:59:24Z","timestamp":1790834364234,"version":"4.1.0"},"reference-count":21,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2020,7,20]],"date-time":"2020-07-20T00:00:00Z","timestamp":1595203200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Barbara und Wilfried Mohr-Stiftung","award":["DM1669\/100"],"award-info":[{"award-number":["DM1669\/100"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Immunotherapy with CD19-specific chimeric antigen receptor (CAR-) T cells has shown excellent efficacy in relapsed\/refractory B-cell cancers. The in vivo expansion and persistence of CAR-T cells after infusion are important response- and toxicity-determining variables, but diagnostic tools are largely missing. We showed previously for axi-cel that digital PCR (dPCR) is excellently suited to monitoring CAR-T cells in vivo. Here, we aimed to develop an analogous dPCR assay for tisa-cel. To do so, we cloned and sequenced the CAR construct from the lentiviral tisa-cel vector and designed primers and Black hole quencher (BHQ) probes complimentary to sequences present in the FMC63 scFv part of axi-cel (assay A), tisa-cel (T), and both constructs (U = \u201cuniversal\u201d). In conjunction with excellent specificity, all assays have a detection limit of one single CAR copy, corresponding to a sensitivity of approximately 1 in 5000 cells (0.02%) for 100 ng genomic DNA (for one vector copy per transduced cell). The new universal assay was first validated using patient samples previously quantified with the axi-cel-specific dPCR and thereafter applied to quantify and monitor adoptively transferred axi-cel and tisa-cel T cells in post-infusion samples (peripheral blood, bone marrow, liquor, and ascites). Actual CAR-T counts per \u00b5l were calculated, taking into account vector copy and peripheral blood mononuclear cell (PBMC) numbers, and showed very good correlation with flow cytometry results. We conclude that our novel dPCR assay is optimally suited to monitoring tisa-cel and axi-cel CAR-T cells in real-time in various body fluids.<\/jats:p>","DOI":"10.3390\/cancers12071970","type":"journal-article","created":{"date-parts":[[2020,7,21]],"date-time":"2020-07-21T06:38:55Z","timestamp":1595313535000},"page":"1970","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR"],"prefix":"10.3390","volume":"12","author":[{"given":"Anita","family":"Badbaran","sequence":"first","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre (UMC) Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina","family":"Berger","sequence":"additional","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre (UMC) Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9050-6766","authenticated-orcid":false,"given":"Kristoffer","family":"Riecken","sequence":"additional","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre (UMC) Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne","family":"Kruchen","sequence":"additional","affiliation":[{"name":"Paediatric Stem Cell Transplantation and Immunology, UMC Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Geffken","sequence":"additional","affiliation":[{"name":"Institute for Transfusion Medicine, UMC Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7477-6632","authenticated-orcid":false,"given":"Ingo","family":"M\u00fcller","sequence":"additional","affiliation":[{"name":"Paediatric Stem Cell Transplantation and Immunology, UMC Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolaus","family":"Kr\u00f6ger","sequence":"additional","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre (UMC) Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francis A.","family":"Ayuk","sequence":"additional","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre (UMC) Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9780-7211","authenticated-orcid":false,"given":"Boris","family":"Fehse","sequence":"additional","affiliation":[{"name":"Department of Stem Cell Transplantation, University Medical Centre (UMC) Hamburg-Eppendorf, 20246 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,7,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"10024","DOI":"10.1073\/pnas.86.24.10024","article-title":"Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity","volume":"86","author":"Gross","year":"1989","journal-title":"Proc. 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