{
  "abstract": "Background Ex vivo chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable clinical success in hematologic malignancies. Such transformative efficacy, however, has yet to be realized in solid tumors where CAR-T activity is hindered by poor trafficking and infiltration, the immunosuppressive tumor microenvironment (TME), and limited persistence. CAR-myeloid cells (CAR-M) can infiltrate and modulate the TME, enhancing T cell activity, but their short persistence limits clinical efficacy. Complex manufacturing, high cost, and need for lymphodepletion present additional obstacles that limit patient accessibility to ex vivo cell therapy. To address these challenges, we are pursuing an in vivo multicellular CAR therapy to enable durable and synergistic anti-tumor activity in solid tumors.Methods We developed a virus-like particle (VLP) platform using helper-dependent adenovirus harboring a 35-kilobase cargo capacity to accommodate a transgene encoding genetic regulatory elements driving a tumor-targeting CAR. The VLP selectively targets CD46 which is highly expressed on primitive hematopoietic stem cells (HSCs); mobilization of HSCs into the blood enables in situ transduction. Co-transduction of the therapeutic payload with a VLP harboring Sleeping Beauty transposase facilitates stable transgene integration in HSCs. To leverage the anti-tumor synergy of innate and adaptive immunity, we designed a VLP comprising concatenated myeloid- and lymphoid-restricted HER2 CARs to generate an HSC-derived, multiplexed CAR-M/NK/T cell therapy ( figure 1).Results In vitro transduction of primary human immune cells confirmed lineage specificity and functional CAR expression in myeloid, T, and NK cells. To evaluate in vivo activity, VLPs encoding a ubiquitous CAG-driven CAR or lineage-restricted CAR were administered to HSC-mobilized, immune competent mice expressing human CD46. While CAG-driven HER2 CAR was detected on all immune lineages, myeloid- and T/NK-specific promoters restricted CAR expression to the targeted lineages (figure 2A). At the study endpoint, bone marrow (BM) from CAR-expressing mice was transplanted to orthotopic EO771/huHER2 tumor-bearing mice. The multiplexed CAR-M/NK/T cell therapy mediated superior tumor suppression compared to single-lineage CAR alone or GFP control (figure 2B). Tumor control was coupled to intra-tumoral HER2 CAR-M activation and a significant increase in CAR-T and CAR-NK cell infiltration and activation (figure 2C-E).Conclusions These data establish robust proof of concept for in vivo multiplexed CAR-M/NK/T cell therapy via HSC engineering. HSCs comprise a self-renewing reservoir of CAR-M, NK, and T cells mediating synergistic and durable anti-tumor activity. This platform has the potential to overcome myriad therapeutic challenges in advanced solid tumors and provide an innovative off-the-shelf therapy for expanded patient access.Ethics Approval All animal studies were done under supervision and approval by IACUC.Abstract 302 Figure 1HSC engineering with VLP encoding lineage-specific HER2 CAR to generate CAR-M, NK, and T cells. The CAR payload is integrated in HSCs but only expressed when T/NK-restricted and myeloid (Myel)-restricted promoters are active in mature immune cellsAbstract 302 Figure 2In vivo HSC-derived multiplexed CAR-M/NK/T cell generation and tumor control. A) HER2 CAR expression in targeted lineages. B) Superior tumor suppression mediated by multiplexed CAR-M/NK/T cells. C) CD80 up-regulation in intra-tumoral CAR-M. Abundant infiltration of lineage-restricted CAR-NK (D) and CAR-T (E) in tumors",
  "authors": [
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Yiwen Zhao"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Chirayu Chokshi"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Richard Davidson"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Ying-Cing Lin"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Cristina Santoriello"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Dhruv Varshney"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Alvin Pratama"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Dennis Ramos Trinidad"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Charles Fox"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Alexey Seregin"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Adam Golding"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Chapman Wright"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Robert Peters"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Corinne Decker"
    },
    {
      "affiliations": [
        "SimnovaBio, Cambridge, MA, USA"
      ],
      "name": "Yinghua Wang"
    }
  ],
  "title": "302 In vivo HSC engineering with VLPs generates lineage-restricted, multiplexed CAR-M, NK, and T cells to cooperatively mediate robust and durable solid tumor control in pre-clinical models",
  "uid": "e03873df-79f2-513a-af2a-afa15f6f060a"
}