{
  "abstract": "Background Preclinical studies in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) suggest that early-harvested CAR T cells retain a phenotype and demonstrate enhanced antitumor activity at lower doses. 1 Despite these observations, the distinct contributions of CD4+ and CD8+ CAR T cells to therapeutic efficacy remain not well understood.2 3 In this research study, we explore a potential role of CD4+ on CAR T-cell function and potency. This research study utilizes T cells from healthy donors to investigate fundamental aspects of T cell biology and may not be representative of clinical or commercial processes.Methods We compared CAR T cells that were made by different process timelines, assessing efficacy and changes in vitro and in a Nalm6 in vivo model. To investigate underlying mechanisms, we further employed high-dimensional spectral cytometry and single-cell sequencing.Results The CD19-CAR T made by the shortened process exhibited greater in vivo peak expansion and antitumor efficacy at lower doses compared to those of the standard process. The CAR T cells made by the shortened process exhibited higher oxygen consumption rates and a memory cell-like metabolic profile. In vitro, these CAR-T cells also demonstrated increased levels of CD4+ T cells with sustained survival and expansion following sequential Nalm6 challenge, whereas the cells made by the standard process exhibited a higher ratio of CD8+ T. Both CD4 and CD8 CAR T maintain more juvenile cell phenotype in the cells made by the shortened process. Further analysis on the post-tumor challenge shows no significant differences in CD8+ T cell memory phenotypes. Notably, we identified a unique CD4+ T cell cluster in the cells made by the shortened process post-tumor challenge, enriched for markers of memory cell-like phenotype (CD127, CD62L, and CD27). Single-cell RNA sequencing confirmed enrichment of a memory cell-like CD4+ CAR T cell population with elevated TCF7, IL7R, and ID3 expression in the shortened process. While the CAR T cells made by both processes exhibited comparable tumor cell killing overall, isolated CD4+ T cells from the cells made by shortened process exhibited increased cytotoxicity compared to those made by the standard process. Cytokine profiling showed a more responsive effector cell-like CD4+ CAR-T phenotype in the shorten process, with higher GZMB and IL-2 expression.Conclusions These findings underscore the potential that an appropriately developed and dosed shorter process product may preserve CD4 + T cell memory formation capability, supporting the potential development of new CAR T therapies with enhanced persistence, potency, and clinical durability.References Ghassemi S et al. Reducing ex vivo culture improves the antileukemic activity of chimeric antigen receptor (CAR) T cells. Cancer Immunol Res. 2018 Sep;6(9):1100-1109. doi: 10.1158/2326-6066.CIR-17-0405.Wang D et al. Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JCI Insight. 2018 May 17;3(10):e99048. doi: 10.1172/jci.insight.99048.Melenhorst JJ, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature. 2022 Feb;602(7897):503-509. doi: 10.1038/s41586-021-04390-6. Epub 2022 Feb 2. Erratum in: Nature. 2022 Dec;612(7941):E22. doi: 10.1038/s41586-022-05376-8.",
  "authors": [
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Hsing-Chuan Tsai"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Albert Millian"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Gabriela Balderrama-Gutierrez"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Maoyu Peng"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Tiffany Jehng"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Lei Huang"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Chad M Williams"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Stephanie Chen"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Tarinee Huang"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Melody Geragosian"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Waleed Haso"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Nebi Wondyfraw"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Sunanda Kumar"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Sophie Viaud"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Qi Cai"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "David Barrett"
    }
  ],
  "title": "284 Rapid manufacturing reveals memory cell-like CD4 CAR T cells as key drivers of sustained anti-tumor activity",
  "uid": "f47c8fd7-b9a9-5cf4-91fb-9018bb444957"
}