1cc4f778ad692af3af94e1cd1bb0d452 270661.pdf e855eb715ac003f758988097907ecedff64487d8 270661.pdf dcc9d0035fad85a32ace1ab54be2528e60f0b7fa40201a7dca3a0a846cb32666 270661.pdf Title: IL-15 Enhances the Persistence and Function of BCMA-Targeting CAR-T Cells Compared to IL-2 or IL-15/IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation Subject: Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the treatment of B-lymphoid malignancies. For multiple myeloma (MM), B-cell maturation antigen (BCMA)-targeted CAR-T cells have achieved outstanding complete response rates, but unfortunately, patients often relapse within a year of receiving the therapy. Increased persistence and reduced dysfunction are crucial features that enhance the durability of CAR-T cell responses. One of the factors that influence CAR-T cell in vivo longevity and loss of function, but which has not yet been extensively studied for BCMA-directed CAR-T cells, are the cytokines used during their production. We here compared the impact of IL-2, IL-15 and a combination of IL-15/IL-7 on the phenotype and function of ARI2h, an academic BCMA-directed CAR-T cell that is currently being administered to MM patients. For this study, flow cytometry, in vitro cytotoxicity assays and analysis of cytokine release were performed. In addition, ARI2h cells expanded with IL-2, IL-15, or IL-15/IL-7 were injected into MM tumor-bearing mice to assess their in vivo efficacy. We demonstrated that each of the cytokine conditions was suitable for the expansion of ARI2h cells, with clear in vitro activity. Strikingly, however, IL-15-produced ARI2h cells had improved in vivo efficacy and persistence. When explored further, it was found that IL-15 drove a less-differentiated ARI2h phenotype, ameliorated parameters related to CAR-T cell dysfunction, and lowered the release of cytokines potentially involved in cytokine release syndrome and MM progression. Moreover, we observed that IL-15 was less potent in inducing T cell senescence and DNA damage accumulation, both of which may contribute to an unfavorable CAR-T cell phenotype. These findings show the superiority of IL-15 to IL-2 and IL-15/IL-7 in the quality of anti-BCMA CAR-T cells, particularly their efficacy and persistence, and as such, could improve the duration of responses if applied to the clinical production of CAR-T cells for patients. Keywords: CAR-T cells; multiple myeloma; IL-2; IL-15; IL-7; BCMA; senescence Author: Anthony M. Battram, Mireia Bachiller, Victor Lopez, Carlos Fernández de Larrea, Alvaro Urbano-Ispizua and Beatriz Martín-Antonio Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Jul 15 15:18:25 2021 CEST ModDate: Fri Jul 16 08:52:04 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 20 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5780653 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SSNIUI+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 RSNLSG+VnURWPalladioL Type 1 Custom yes yes no 15 0 NTXEAX+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 JCDENK+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 QARIDO+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 LCJEVJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 37 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 59 0 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CAR and Expand Well in Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: ARI2hIL-15 Functional Responses Are Indistinguishable from Those Exhibited by ARI2hIL-2 or ARI2hIL-15/IL-7 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: ARI2hIL-15 Has a Superior In Vivo Function Than ARI2hIL-15/IL-7 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: IL-15-Cultured CAR-T Cells Have a More Differentiated Phenotype When Additionally Expanded with IL-7 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: ARI2hIL-15 Cells Have Reduced Expression of LAG-3 and Augmented Levels of CD27 Compared to ARI2hIL-2 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: ARI2hIL-15 Cells Have Reduced DNA Damage Compared to ARI2hIL-2 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: ARI2hIL-15 Cells Secrete Lower Levels of Cytokines That Are Related to CAR-T Cell Toxicities or MM Progression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Cell Culture and T Cell Transduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Analysing CAR-T Cell Ribosomal S6 Protein and p38 MAPK Phosphorylation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Degranulation Assay and Granzyme B Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: SA Gal/Mitochondrial Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: In Vitro Cytotoxicity Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Long-Term Challenges Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Analysis of Cytokine Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: In Vivo Murine Experiments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Info: Title: IL-15 Enhances the Persistence and Function of BCMA-Targeting CAR-T Cells Compared to IL-2 or IL-15/IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation Author: Anthony M. Battram, Mireia Bachiller, Victor Lopez, Carlos Fernández de Larrea, Alvaro Urbano-Ispizua and Beatriz Martín-Antonio Subject: Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the treatment of B-lymphoid malignancies. For multiple myeloma (MM), B-cell maturation antigen (BCMA)-targeted CAR-T cells have achieved outstanding complete response rates, but unfortunately, patients often relapse within a year of receiving the therapy. Increased persistence and reduced dysfunction are crucial features that enhance the durability of CAR-T cell responses. One of the factors that influence CAR-T cell in vivo longevity and loss of function, but which has not yet been extensively studied for BCMA-directed CAR-T cells, are the cytokines used during their production. We here compared the impact of IL-2, IL-15 and a combination of IL-15/IL-7 on the phenotype and function of ARI2h, an academic BCMA-directed CAR-T cell that is currently being administered to MM patients. For this study, flow cytometry, in vitro cytotoxicity assays and analysis of cytokine release were performed. In addition, ARI2h cells expanded with IL-2, IL-15, or IL-15/IL-7 were injected into MM tumor-bearing mice to assess their in vivo efficacy. We demonstrated that each of the cytokine conditions was suitable for the expansion of ARI2h cells, with clear in vitro activity. Strikingly, however, IL-15-produced ARI2h cells had improved in vivo efficacy and persistence. When explored further, it was found that IL-15 drove a less-differentiated ARI2h phenotype, ameliorated parameters related to CAR-T cell dysfunction, and lowered the release of cytokines potentially involved in cytokine release syndrome and MM progression. Moreover, we observed that IL-15 was less potent in inducing T cell senescence and DNA damage accumulation, both of which may contribute to an unfavorable CAR-T cell phenotype. These findings show the superiority of IL-15 to IL-2 and IL-15/IL-7 in the quality of anti-BCMA CAR-T cells, particularly their efficacy and persistence, and as such, could improve the duration of responses if applied to the clinical production of CAR-T cells for patients. 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